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- Describe how certain viruses may contribute to the development of tumors by altering proto-oncogenes or tumor-suppressor genes.
- Name 3 viruses that have been implicated in human cancers.
Five viruses, hepatitis B virus (HBV), hepatitis C virus (HCV), human papilloma virus (HPV), Epstein-Barr virus (EBV), and human T-lymphotropic virus type I (HTLV-I) are thought to contribute to over 15% of the world's cancers. Up to 80% of these human viral-associated cancers are cervical cancer (associated with HPV) and liver cancer (associated with HBV and HCV).
The hepatitis B virus (HBV) is a DNA virus that may potentially cause chronic hepatitis in those infected. There is a strong link between chronic infection with HBV and hepatocellular carcinoma, which typically appears after 30-50 years of chronic liver damage and liver cell replacement. Chronic carriers of HBV have a 300 times greater risk of eventually developing liver cancer. Around 90% of individuals infected at birth and 10% of individuals infected as adults become chronic carriers of HBV. There are about one million chronic carriers of HBV in the US. Worldwide, HBV is responsible for 60% of all liver cancer cases.
The hepatitis C virus (HCV) is a RNA virus that may also cause chronic hepatitis in those infected. As with HBV, there is a strong link between chronic infection with HCV and liver cancer, typically appearing after 30-50 years of chronic liver damage and liver cell replacement. Around 85% of individuals infected with HCV become chronic carriers and there are approximately four million chronic carriers of HCV in the US. Worldwide, HCV is responsible for 22 % of all liver cancer cases.
The human papilloma viruses (HPV) are responsible for warts. While warts are generally considered as benign tumors, some sexually-transmitted strains of HPV (HPV-16 and 18 are definitely carcinogenic in humans; HPV-31 and 33 are probably carcinogenic), have been implicated in cervical and vulvar cancer, rectal cancer, and squamous cell carcinoma of the penis. In these tumor cells the viral DNA is usually found integrated in host cell chromosomes. In the US, HPVs are associated with 82% of the deaths due to cervical cancer each year, as well as a million precancerous lesions.
The Epstein-Barr virus (EBV), a herpes virus, normally causes benign proliferations such as infectious mononucleosis and hairy leukoplakia of the tongue. However, it can contribute to non-Hodgkin's lymphoma in AIDS patients and post-transplantation lymphoproliferative diseases, appears to be an essential factor for posterior nasopharyngeal cancer in some individuals, can be a co-factor for Burkitt's lymphoma, and contributes to smooth-muscle tumors in immunosuppressed children.
The retrovirus human T-lymphotropic virus type I (HTLV-I) can induce a rare adult T-lymphocyte leukemia-lymphoma.
As mentioned previously, the development of tumors is a multistep process depending on the accumulation of mutations altering a number of genes. The altered genes then function collectively to cause malignant growth.
Viruses can play a role in cancer development both indirectly and directly, however. Indirectly, the viruses may induce immunosuppression so that cancer cells are not removed by immune responses, as in the case of HIV/AIDS, or they may cause long term damage to tissues resulting in large scale cell regeneration which increases the chances of natural mutation in proto-oncogenes and tumor suppressor genes, as in the case of HBV and HCV. Directly, by integrating into the host cell's chromosomes, some viruses may alter the normal function of the proto-oncogenes and tumor suppressor genes, as is seen with HPV and HBV.
However, most virus-associated cancers have long latency periods of several decades and only a small percentage of the people infected with the virus actually develop the cancer. This indicates other factors promoting changes in cellular genes are also involved. For example, in the case of cervical cancer and HPV, two variants of a tumor suppressor gene known as p53 are known. One form of the p53 gene produces a suppressor protein that is much more susceptible to degradation by an oncoprotein called E6 which is produced by carcinogenic strains of HPV.
Name the three most common viruses associated with cancer in the US and state the cancers with which they are associated.
Medscape article on infections associated with organisms mentioned in this Learning Object. Registration to access this website is free.
- Hepatitis B
- Hepatitis C
- Human Papilloma Virus
- Infectious Mononucleosis
- Human T-Cell Lymphotropic Viruses
- Hepatic Carcinoma
- Cervical Cancer
- Viruses are responsible for about 15% of the world’s cancers.
- Up to 80% of these human viral-associated cancers are cervical cancer (associated with human papilloma virus or HPV) and liver cancer (associated with the hepatitis B virus or HBV and the hepatitis C virus or HCV).
- The Epstein-Barr virus (EBV) and human T-lymphotropic virus type I (HTLV-I) also increase the risk of certain cancers.
- The development of tumors is a multistep process depending on the accumulation of mutations altering a number of genes.
- Most virus-associated cancers have long latency periods of several decades and only a small percentage of the people infected with the virus actually develop the cancer. This indicates other factors promoting changes in cellular genes are also involved.
The roles of IFN gamma in protection against tumor development and cancer immunoediting
Interferon-gamma (IFN gamma) is a cytokine that plays physiologically important roles in promoting innate and adaptive immune responses. The absence of IFN gamma production or cellular responsiveness in humans and experimental animals significantly predisposes the host to microbial infection, a result that validates the physiologic importance of this cytokine in preventing infectious disease. Recently, an additional role for IFN gamma in preventing development of primary and transplanted tumors has been identified. Although there now appears to be a consensus that IFN gamma promotes host responses to tumors, the mechanisms by which this cytokine achieves its effects remain unclear. In this review, we briefly discuss key issues of the molecular cell biology of IFN gamma and its receptor that are most relevant to IFN gamma-dependent anti-tumor effects and then focus on the data implicating IFN gamma as a critical immune system component that regulates tumor development. Potential mechanisms underlying IFN gamma's anti-tumor effects are discussed and a preliminary integrative model of IFN gamma's actions on tumors is proposed. Finally, the capacity of IFN gamma and lymphocytes to not only provide protection against tumor development but also to sculpt the immunogenic phenotype of tumors that develop in an immunocompetent host is presented and introduced as a "cancer immunoediting" process.
Interferons: Meaning, Production and Applications
Interferons are natural glycoproteins produced by virus-infected eukaryotic cells which protect host cells from virus infection. They were discovered by Isaacs and Lindenmann in 1957 in course of a study of the effect of UV-inactivated influenza virus on chick chorioallantoic membrane kept in an artificial medium.
They observed that the infected membrane produced a soluble substance in the medium which could inhibit the multiplication of active influenza virus inoculated in fresh chick chorioallantoic membranes. The substance was called interferon because it interfered with intra­cellular multiplication of viruses.
Later observations confirmed that such host-produced antiviral substances were common to many viruses. Viral interference is a phenomenon observed when multiplication of one virus is inhibited by another virus. For instance, when influenza-A virus is inoculated into the allantoic cavity of an embryonated egg followed after 24 hr by influenza-B virus, the multiplication of influenza-B virus is partly or completely inhibited. The reason why influenza-B virus cannot multiply is that the influenza-A virus infected cells produce interferon which partly or totally inhibits multiplication of B virus. The interferon also protects cells from influenza A virus.
Characteristics of Interferons:
An outstanding feature of interferons is that they are host-cell-specific and not virus-specific. This means that interferons produced by mouse or chicken will not protect human cells against the same virus which induced interferon in the experimental animals. On the other hand, an interferon produced by a virus X in an animal will protect the animal also from other viruses.
This is because interferons do not interact directly with the viruses. But they induce the virus infected cells to synthesize antiviral proteins which inhibit viral multiplication. These proteins have a wide inhibitory spectrum. As a result, not only the interferon-inducing virus, but others are also inhibited.
The reason why interferon produced by one species does not protect another species is that the same virus produces different interferons in different species. It has been observed that interferons produced by different host species following infection by the same virus differ in molecular weight as well as in other properties, like isoelectric point etc. Not only different species produce different interferons, different tissues of the same animal produce different interferons.
All types of interferons are proteins having a comparatively low molecular weight ranging between 15,000 to 40,000 Daltons. Hence, they are non-dialyzable and destroyed by proteolytic enzymes. Interferons are fairly stable at low pH (pH2) and can withstand moderate temperature being stable at 37°C for an hour or so. They are produced in minute amounts by the infected cells as a longer precursor having 23 amino acid residues more than the mature molecule.
Human interferons are of three main types. These are called alpha interferons (α-IFN), beta-interferons (β-IFN) and gamma-interferons (γ-IFN). Alpha-interferon contains many subtypes. The total subtypes exceed 20 in number.
It is produced by the B-lymphocytes, monocytes and macrophages. β-IFN is produced by the fibroblasts in the connective tissues. γ-IFN is synthesized by the T-lymphocytes after they are activated by antigens. α-IFN has been shown to be coded by as many as 20 distinct chromosomal genes, indicating thereby that the subtypes of this interferon represent a family of closely related proteins.
β-IFN appears to be a glycoprotein. It is coded by a single human gene. All the genes of α-IFN and β-IFN are located on the short arm of human chromosome 9. α-IFN proteins are all 166 amino acid long (except one). They are non-glycosylated and the proteins are monomeric. The single β-IFN protein is also 166 amino acid long and a glycoprotein. It is dimeric.
Production of Interferons:
Interferons are produced by living animal cells, both in vivo as well as cultured cells. Interferon production and its antiviral activity require expression of cellular genes, and these functions are blocked by inhibitors of transcription and translation. Thus, virus-infected host cells fail to produce interferon in presence of actinomycin D, an inhibitor of eukaryotic RNA polymerase. When the inhibitor is added after 2 hr of infection, interferon production is not inhibited, suggesting that transcription is completed by that time.
Interferon production starts after initiation of viral maturation and continues for 20 to 50 hr after that. Then the production stops, due to formation of a repressor which presumably is formed or activated only when the interferon concentration in the producing cell exceeds a certain threshold concentration. Most of the interferon is transported from the producing cell to other neighbouring cells.
The substance in a virus that is responsible for interferon synthesis by the host cell is known as interferon inducer. The nature of this substance was identified by Merigan (1970) as double-stranded RNA. The activity seems to reside in polyribonucleotide’s with a high helical content. The double- stranded RNA viruses — like reoviruses — can act as interferon inducer without replication. Single- stranded RNA viruses can act as inducers only after replication when they form double-stranded replicative intermediates. DNA-viruses can also induce interferons, presumably due to overlapping transcription of viral DNA as observed in case of vaccinia vinus (Fig. 6.39).
Fungal viruses which have mostly double-stranded RNA genomes are also efficient inducers of interferons. Some synthetic polymers containing riboinosinic acid, ribocytidylic acid (Poly I: C) as well as those containing riboadenylic acid and ribouridylic acid (Poly A: U) are also good inducers. All interferon inducers are characterized by high molecular weight, high density of anionic groups and resistance to enzymatic degradation. DNA and DNA-RNA hybrids have been found to be ineffective as interferon inducers.
The induction of interferon synthesis concerns α- and β-interferon’s which belong to a single class, called Type I. Gamma-interferon belongs to a separate class, called Type II. The human Υ-interferon is the single representative of its type. The gene coding the y-interferon protein is located on the long arm of chromosome 12. The gene has three introns, while the genes of α- and β- interferons are without any introns. Gamma-interferon (human) has 146 amino acids and is an N-glycosylated tetrameric protein. It is induced by antigenic stimulation of T-lymphocytes.
In presence of the inducer which is viral ds-RNA, the α- and β-interferon genes of the host chromosome(s) are activated to produce interferon m-RNAs. Those are then translated intoα- and β- interferon proteins. These proteins at first accumulate in the producing cell and eventually leave the cell to reach neighbouring host cells.
As the interferon concentration in the producing cell rises above a threshold level, it activates another gene of the producing cell which codes for a repressor protein which feeds back and stops further synthesis of interferon. As a result, virus-infected cells generally produce only small quantities of interferons.
The interferon molecules that leave the producing cell reach the neighbouring uninfected host cells and interact with the cell membrane or nuclear membrane receptors of these cells. Thereby these cells are induced to synthesise antiviral proteins. These antiviral proteins are the actual agents that provides protection to these host cells against viral infection.
Mechanism of Action of Interferons:
Type I interferons include α-IFN and β-IFN. These interferons do not interact with the viruses directly causing their inhibition, but they induce the formation of antiviral proteins which are activated to inhibit viral multiplications. These interferon-regulated proteins (IRPs) act presumably by blocking synthesis of the macromolecular components necessary for viral multiplication.
A general scheme for mechanism of action of type I interferons is shown in Fig. 6.40:
Several interferon regulated host proteins (IRPs) have been identified, though all of them have not been fully characterized. Among the better known of these proteins are a protein kinase and an enzyme catalyzing the formation of a short polymer of adenylic acid, the 2′, 5′-oligoadenylate synthetase (2′-5′ A synthetase).
The protein kinase is induced by Type I interferons. It has to be activated by ds-RNA. The activated kinase catalyses phosphorylation of initiation factor (el F-2) thereby causing inhibition of protein synthesis (Fig. 6.41).
The 2′-5′-oligoadenylate synthetase is an enzyme also induced by Type I interferons which requires activation by ds-RNA like the protein kinase. The activated synthetase acts as an activator of an endonuclease, RNase L. The activated RNAse degrades viral ss-RNA (Fig. 6.42).
Another group of proteins, called Mx-proteins induced by α- and β-IFN are known to possess intrinsic antiviral activity, although the exact molecular mechanism by which they inhibit viral multiplication is not known. Mx-proteins have been reported to play a major controlling role in infections caused by influenza viruses in experimental animals as well as in humans.
Type II interferon includes g-IFN which is also known as immune IFN. Although g-IFN also possesses anti-viral activity, its major role is in the immunity through activation of cytotoxic T-lymphocytes which can destroy virus infected cells. Besides T-lymphocytes, other naturally occurring killer cells like macrophages and monocytes are also activated by g-IFN. Thus, in contrast to that of Type I interferons, the antiviral effect of g-IFN is expressed through activating the killer cells of the body which destroy the virus-infected cells.
Type II interferon induces the major histocompatibility antigens of human cells. Expression of these antigens is essential for immuno-potent cells to present foreign antigens to the T-lymphocytes during generation of specific immune responses.
IFN induced expression of these major histocompatibility antigens represents an important contribution of the antiviral activity of g-IFN through enhancement of the activity of cytotoxic T-lymphocytes. The activation of cytotoxic T-lymphocytes by y-IFN also implies its possible role in elimination of cancer cells which are recognized by the immune system of the body as foreign objects.
Applications of Interferons:
Interferons could be ideal agents for combating viral diseases. They inhibit viral multiplication at such low concentration which is non-toxic to uninfected cells. One interferon can inhibit many viruses. But there are certain draw-backs which stand in their use.
Firstly, for application in humans, interferon must be of human origin, though interferons produced in monkey kidney cell cultures are also effective in humans. Interferons are produced in very small quantities and it is difficult to get them in sufficient quantity in pure form for clinical application. Another factor is that interferons are effective only for short periods and as such can be used against only acute infections, like influenza.
The difficulty of obtaining sufficient quantity of pure interferon for clinical use has been overcome by cloning the α-IFN and β-IFN human genes in bacteria and yeast. By growing these transgenic organisms in mass culture, it has been possible to obtain clinically usable interferons in sufficiently large quantities. Alpha-interferon has been marketed in 1984 under the trade name Intron A.
In the following years, this biotechnologically produced interferon has been approved for clinical use against diseases like genital herpes caused by herpes-virus, hepatitis B and C. Beta-interferon has also been biotechnologically produced and marketed under the trade name Betaseron. It has been used in a disease called multiple sclerosis. A recombinant g-interferon has been found effective against an inherited chronic disease, called granulomatous disease.
The neutrophils of the affected individual are unable to kill the infectious bacteria. Application of y-IFN to such persons restores the ability of the neutrophils to kill bacteria. As the disease is chronic and inherited, the affected persons must take g-IFN throughout their life to remain normal.
Interferons are not only antiviral, but they have also anticancer activity. Clinical trials have shown that interferons have effect against only some types of tumours. Alpha-interferon has been approved for treating hairy-cell leukemia, and Kaposi’s sarcoma, a cancer that occurs in AIDS patients.
Gamma-interferon has been mainly used as an immuno-stimulant in cancer patients. Resistance against tumours in the body is controlled by the immune response against tumour antigens. The cytotoxic T-lymphocytes recognize these antigens as foreign and destroy them. Gamma-interferon can stimulate the cytotoxic function of T-lymphocytes and other natural killer cells of the body, thereby helping to control the tumour cells.
2- Beneficial viruses in agriculture
Genetic engineering and modification methods can be used to make modified genomes that can be transported to plants and animals by viruses that act as vectors or vehicles. This method can lead to more productive transgenic animals and plants. Scientists have successfully induced different genes like herbicide tolerance, high nitrogen fixation, high lysine (in corn), increased resistance to pests and diseases, in plants by using a virus as a gene carrier and vector.
In an experiment, it was found that all virus-infected plants were much more drought tolerant.
In Yellowstone National Park, soil temperatures can get quite high in geothermal areas, but some plants can grow very well in these locations with soil temperatures of 115 ° F.
After a few years, other researchers discovered that the plant was colonized by a fungus. Without the fungus, the plant could not tolerate heat. After that, it was discovered that there was a virus in the fungus. So, heat tolerance characteristic in plants was induced by viral induction in plants.
Drought-stressed rice plants after six days without water. The plant on the right is infected with the Brome mosaic virus the one on the left is “healthy” (that is, virus-free).
Viruses use in biological pest control
- can also be used to control harmful pests. Traditionally, this has been used in agriculture, but there are also applications in the control of agents important to human health.
- The types of agents used for this purpose may take advantage of the target species, maybe parasites of the target pests, be pathogenic or cause disease in the target species, or maybe competing species. used for pest control are common pathogens that cause diseases of the target species. Although they represent a small amount of total pesticide use, viruses are used for the control of multiple species of insects and also for rabbits.
- Biological agents can produce long-lasting effects and, in some cases, can spread among the target population. They have also been recognized as inherently less toxic than conventional pesticides by the US Environmental Protection Agency. USA
- Its disadvantages include a limited range of action, slow effects compared to chemical agents, high initial treatment costs, low environmental stability, particularly in sunlight.
Roles of viruses in the environment
San Diego State University, Department of Biology, San Diego, CA, USA.
Department of Microbiology, Institut Pasteur, 25, rue du Dr. Roux, 75724 Paris, France.
Faculty of Biology, Technion – Israel Institute of Technology, Technion City, Haifa 32000, Israel.
San Diego State University, Department of Biology, San Diego, CA, USA.
Department of Microbiology, Institut Pasteur, 25, rue du Dr. Roux, 75724 Paris, France.
Faculty of Biology, Technion – Israel Institute of Technology, Technion City, Haifa 32000, Israel.
Viruses are important microbial predators that influence global biogeochemical cycles and drive microbial evolution, although their impact is often under appreciated. Viruses reproduce after attaching and transferring their genetic material into a host cell. The host's cellular machinery is then redirected to the making of more viruses and results in the death of the host cell in the vast majority of cases. Viruses have developed intriguing mechanisms to utilize host proteins for their own defence and for shifting metabolism from host to virus, a topic that is reviewed for bacterial viruses in this special issue of Environmental Microbiology ( Roucourt and Lavigne, 2009 ).
Globally, there are an estimated 1e31 virus-like particles. Currently, it is thought that most of the viruses are phages that infect bacteria, but archaeal and eukaryotic viruses are certainly important components of most ecosystems. Since the average half-life of free viruses in most ecosystems is ∼48 h, an estimated 1e27 viruses are produced every minute. This means that roughly 1e25 microbes, or about 100 million metric tons, die every 60 s due to viruses. Viral predation, in combination with protist grazing, is able to maintain microbial numbers at values less then the carrying capacity of the system and as such plays an important role in controlling microbial communities. In this special issue Sandaa and colleagues (2009 ) report on the simultaneous control of microbial diversity by both viral predation (top-down control) and substrate availability (bottom-up control).
Much of our knowledge about the roles of viruses in natural environments comes from studies of marine microbial communities. In the world's oceans, about half of the organic matter produced by photosynthesis supports the production of new heterotrophic microbes (both Bacteria and Archaea). Viruses and protists then kill roughly equal proportions of these ( Fuhrman and Noble, 1995 ). The lysed cells become dissolved organic matter which can be used by other heterotrophic bacteria. This means that viral-mediated mortality increases net respiration, the release of CO2 and nutrient recycling in the world's oceans. Viruses and their microbial prey are also extremely diverse, abundant and active in marine sediments ( Danovaro et al., 2002 Breitbart et al., 2004a ). Moreover, viruses affect primary productivity by killing diatoms, dinoflagellates and cyanobacteria, as well as by releasing nutrients ( Suttle et al., 1990 ). Thus, viruses can account for a very significant part of the ocean's carbon cycling.
Viruses seem to be ubiquitous and have been reported from any environment where life is present, from freshwaters to the sands of the Sahara desert. However, very little is known of the ecological roles of viruses in most ecosystems. In soils, potentially the biggest biosphere on the planet, most viral studies have concentrated on estimating their abundance and taxonomy. Viruses are associated with the rhizosphere of plants ( Ackermann, 1997 ) and are also common in some of the harshest environments in the planet, ranging from hot springs ( Rice et al., 2001 Rachel et al., 2002 Breitbart et al., 2004b Redder et al., 2009 ) to hypersaline waters ( Nuttall and Dyall-Smith, 1993 ). In many of these extreme ecosystems, viruses are the only known microbial predators.
Metagenomic analyses of natural communities and man-created niches have shown that viruses are extremely diverse and novel. For example, 1 kg of marine sediment may contain over a million different viral types and 200 l of seawater may contain about 5000 viral types ( Breitbart et al., 2002 2004a ). And there may be at least 1000 different viruses living in the human gut ( Breitbart et al., 2003 ). The vast majority (> 70%) of genetic material carried by these viruses is completely uncharacterized and natural viral communities probably represent the largest unexplored area of genetic information space left on the planet. Metagenomics analyses of blood have also shown that healthy humans carry a number of novel, unknown viruses ( Breitbart and Rohwer, 2005 ), including phage to known human pathogens ( Gaidelytëet al., 2007 ). The impact of these viruses on human health is currently unknown, but we expect an explosion of research in this area over the next couple of years.
In addition to their influences on biogeochemical cycles, viruses drive microbial evolution by natural selection for microbes resistant to infection and via lateral gene transfer. Many viruses are strain-specific predators. Therefore as a particular microbial strain becomes dominate in a system, its viral predators will expand exponentially and kill it off. This will leave a niche for another microbial strain to grow into, which will be subsequently killed off by another viral type. This means that the dominant microbial species within a system will be constantly turned over. This ‘kill-the-winner’ hypothesis may explain much of the observed microbial diversity and changes in community structure ( Thingstad, 2000 ).
Viruses are also important exchangers of genetic information between hosts, because they inject their genomes into the host cells. For example, most of the completely sequenced microbial genomes contain proviral sequences. Proviruses are viruses that have integrated their genomes into the host's genome and are replicated with the host. Most proviruses can become active at a later date and subsequently end up killing their host. Many proviruses also express genes, which can dramatically alter the phenotype of the host cell. The majority of environmental strains of Vibrio cholerae, for example, are not human pathogens until they are infected with a provirus carrying the cholera toxin. Acquisition and loss of proviruses is one of the most common mechanisms of lateral gene transfer.
Viruses also move ecologically important genes from host to host. For example, viruses that infect the marine cyanobacteria Prochlorococcus and Synechoccus often carry a gene (psbA) that encodes a protein central to photosynthesis ( Mann et al., 2003 Lindell et al., 2004 ). These viruses express this and other photosynthesis genes during infection and are thought to use photosynthetic proteins to keep that host cell alive and produce energy during the infection cycle ( Lindell et al., 2005 Clokie et al., 2006 ). However, in some cases, when the virus moves this central gene from one microbial strain to another, a recombination event can incorporate parts of the viral gene into the host ( Zeidner et al., 2005 Sullivan et al., 2006 ). This event may simultaneously inactivate the viral genome, enabling the host to survive infection, and change the genotype of the host. Another intriguing example is of the horizontal transfer of the ceramide-producing sphingolipid biosynthesis genes from coccolithophores to their viruses ( Wilson et al., 2005 Monier et al., 2009 ). These genes, which may be involved in triggering programmed cell death, are expressed during infection ( Allen et al., 2006 ) as well as during a coccolithophore bloom in a natural mesocosm microbial community ( Pagarete et al., 2009 ). These examples, where viruses have maintained host genes and actively express them during infection, indicate that viruses are not merely vectors for horizontal gene transfer but that gene transfer impacts the evolution of both microbial hosts and viruses.
Viruses not only move genes from one organism to another, they are also able to move genetic material between ecosystems. Some viral sequences have been found to be ubiquitously spread through the biosphere ( Breitbart et al., 2004c Short and Suttle, 2005 ). There is also evidence that viruses from one environment can successfully infect and replicate on microbes from unrelated environments ( Wilhelm and Matteson, 2008 ). These results provide support that viruses can move throughout the world and move genes between ecosystems. Similarly a recent study of RNA viruses in human stool samples showed that plant viruses are efficiently passing though the human gut and are disseminated with the seeds ( Zhang et al., 2006 ). In this way, viruses can use animals to move from place to place.
Our knowledge of environmental viruses has increased greatly over the last decade, yet we still have much to learn about even the best-studied environments as the reports in this special issue show. New viruses are still being discovered and their genomes are continuing to reveal a multitude of novel genes and new potential functions ( Redder et al., 2009 Sullivan et al., 2009 ). Enormous viral diversity is being uncovered in every new habitat investigated ( Rosario et al., 2009 ) and we are just beginning to understand the temporal and spatial variability of the diversity of natural viral populations ( Chen et al., 2009 Short and Short, 2009 ). Seasonal and diel changes in viral production are also reported ( Winget and Wommack, 2009 ) and virus-mediated bacterial mortality is shown to significantly impact carbon cycling in the Southern Ocean ( Evans et al., 2009 ).
The extent of microbial mortality is dependent on their susceptibility to viral infection, yet many microbes are resistant to infection. Varying degrees of resistance and susceptibility are likely to be one of the factors facilitating long-term coexistence of both host and virus in nature. However little is known about the mechanisms and dynamics of resistance in environmental host–virus systems. In this special issue Tomaru and colleagues (2009 ) report on a phenomenon of reversible resistance of a dinoflagellate to an RNA virus and suggest that an intracellular suppression mechanism is responsible.
As the cost of sequencing declines, more comparative genomics studies are being carried out and are revealing striking similarities between viruses isolated on the same host at different times and places ( Angly et al., 2009 Ceyssens et al., 2009 Redder et al., 2009 Weynberg et al., 2009 ) indicating that, if the environment is adequately sampled, we will eventually gain an understanding of the viral diversity existing in nature. Clearly viral genomes are not static, and as mentioned above, their evolution is affected by the transfer of genes between hosts and viruses ( Sullivan et al., 2009 Weynberg et al., 2009 ), as well as by mutation and recombination between viruses ( Marston and Amrich, 2009 Redder et al., 2009 ).
Finally, in this special issue, Kropinski and colleagues (2009 ) put forward a rational scheme for the naming of newly isolated bacterial and archael viruses for us to consider and Brüssow (2009 ) presents an intriguing historical perspective on the animal origins of many human viruses.
Over the past decade, new and exciting findings in the field of environmental virology have spearheaded a recent revival into research of bacterial viruses. Furthermore the discovery of distinctly different archael viruses has opened the door to a whole new realm of viral research. Although the known archaeal viruses reveal an exceptional degree of diversity with regard to both morphotypes and genomes, this might still be an underestimation: they are presently the least studied component of the biosphere and have only been isolated from a limited number of habitats (nearly exclusively from geothermal and hypersaline environments).
Despite the recent increased interest in environmental viruses, our knowledge remains sparse. We are still only scratching the surface of discovery of global viral diversity, have little understanding of the functionality of the majority of genes in the global viral gene pool and the roles they play in the interaction with their hosts, and are still grappling with understanding viral impact on ecological and evolutionary processes. The decade to come promises much on these fronts.
Protein S-palmitoylation in immunity
S-palmitoylation is a reversible posttranslational lipid modification of proteins. It controls protein activity, stability, trafficking and protein–protein interactions. Recent global profiling of immune cells and targeted analysis have identified many S-palmitoylated immunity-associated proteins. Here, we review S-palmitoylated immune receptors and effectors, and their dynamic regulation at cellular membranes to generate specific and balanced immune responses. We also highlight how this understanding can drive therapeutic advances to pharmacologically modulate immune responses.
S-palmitoylation is a posttranslational modification of proteins with lipids. It typically involves the addition of a 16-carbon palmitic acid to cysteines of a protein via a thioester bond (figure 1), but other fatty acids like myristic acid and oleic acid can also be added . S-palmitoylation and other lipid modifications control protein-membrane association and trafficking, thereby playing critical roles in protein function and cell signalling . S-palmitoylation is unique among lipid modifications because the high energy thioester bond between the fatty acyl group and cysteine residue allows it to be a reversible modification and therefore can impart spatio-temporal control of protein function . For example, this dynamic fatty acid modification of H- and N-ras enables the protein cycling between the plasma membrane and Golgi apparatus, thus maintaining subcellular compartmentalization for diversification of signal transduction (figure 1) . In addition to protein targeting to different compartments and membrane microdomains, the role of S-palmitoylation is also implicated in protein stability, conformation, and homotypic and heterotypic interactions (figure 1). S-palmitoylation can exert multiple effects simultaneously to orchestrate the desired protein function. Moreover, it can act in concert with other co- and posttranslational modifications to regulate protein functions. Reported S-palmitoylated proteins include ion channels, receptors, transporters, enzymes, cell-adhesion proteins, innate immunity effectors and many others. Overall, S-palmitoylation is involved in a diverse array of physiological processes, including cellular signalling, differentiation, transcriptional regulation, metabolism and others [5–7].
Figure 1. Protein S-palmitoylation and regulation. Palmitoylation-depalmitoylation cycle regulates protein-membrane association, lipid raft targeting, protein stability and protein–protein interactions, among others. Dynamic S-palmitoylation is mediated by DHHC palmitoyl acyl transferases (DHHC-PATs) and depalmitoylases. Image created with Biorender.com.
S-palmitoylation of proteins is mediated by palmitoyl acyltransferases (PATs) in intracellular compartments including the endoplasmic reticulum, Golgi apparatus and plasma membrane . In humans, PATs are a family of 23 proteins with a characteristic Asp-His-His-Cys (DHHC) domain essential for catalysis. PATs were first discovered in yeast and are conserved across all eukaryotes, though their numbers and specificities can differ between species [9,10]. Regulation at transcriptional, translational and posttranslational levels, as well as PAT variable domains, determine localization, specific substrate profiles and functionality of individual enzymes . Recent structural studies of PAT family members have provided key understanding of reaction mechanism, substrate recognition, interaction, binding and fatty acyl chain selectivity [12,13]. Genetic, cell-based and animal-based studies have implicated PATs and PAT substrates in many patho-physiological conditions including cancer and schizophrenia [14,15].
Removal of palmitate from proteins is regulated by S-depalmitoylases, which catalyse hydrolysis of thioesters . Acyl-protein thioesterase 1 (APT1 also known as lysophospholipase 1, LYPLA1) and acyl-protein thioesterase 2 (APT2 also known as lysophospholipase 2, LYPLA2) were the first identified depalmitoylases for G proteins . Additionally, α/β-hydrolase domain-containing protein 17 (ABHD17) and other serine hydrolase superfamily proteins were identified as depalmitoylases for PSD-95 and oncogenic protein N-Ras [18,19]. Recently, ABHD10 was identified as a depalmitoylase for peroxiredoxin (PRDX5), a key antioxidant protein, and shown to regulate its antioxidant capacity . These findings provide evidence for roles of depalmitoylases in patho-physiological conditions including cancer and made them important targets for potent inhibitor development . Palmitoyl-protein thioesterase 1 (PPT1), an endo-lysosomal protein, is the first known depalmitoylating enzyme to be linked to a fatal genetic lysosomal storage disorder in neurons, neuronal ceroid lipofuscinoses (NCLs) . Development of inhibitors and chemical probes has advanced our understanding of individual depalmitoylase regulation and substrate specificity [23–25].
The earliest methods of detection of protein palmitoylation involved metabolic labelling of proteins with radioactive 3 H, 14 C or 125 I containing palmitic acids. These had many limitations including low sensitivity. Development of chemical approaches to study protein S-palmitoylation have revolutionized the understanding of the field (figure 2) [26–28]. Palmitic acid chemical reporters, including palmitic acid-like molecules containing azido or alkynyl groups (e.g. alk-16/ODYA), can label cysteines of target proteins using endogenous palmitoylation machinery (figure 2a). Using bioorthogonal ligation methods, they can be reacted with fluorophores for visualization or with biotin for enrichment and identification of palmitoylated proteins . Such reporters have further been incorporated in pulse-chase-like experiments to monitor palmitoylation turnover kinetics on proteins [19,30,31]. Moreover, incorporation of photocrosslinking chemical moieties in palmitic acid reporters enabled the identification of an S-palmitoylated protein interactome (figure 2b) . A complementary chemical approach for the study of S-palmitoylation involves modification of thioester linked cysteines in S-palmitoylated proteins. This strategy is exploited in methods like acyl-biotin exchange (ABE) and acyl-resin-assisted capture (acyl-Rac) (figure 2c) [33,34]. In these methods, free cysteines of palmitoylated proteins are capped and thioester linkages are subsequently cleaved to generate new thiols. These thiols are then selectively labelled by biotin for ABE or thiol-reactive resin for acyl-Rac, allowing further enrichment and detection of palmitoylated peptides or proteins. A variant of this method called acyl-PEG exchange (APE) exploits PEG labelling of newly generated thiols as a mass tag for mobility-shift based assays to identify levels of protein S-palmitoylation . Though the above chemical approaches each have individual limitations, they have been successfully applied for global profiling of palmitoylated proteins in yeast, protozoan and mammalian cells [36–39]. Furthermore, identification of S-palmitoylomes helped in the development of in silico predictive programmes for palmitoylation sites in proteins despite the lack of a consensus motif . Other methods developed for the analysis of specific palmitoylated proteins include in-cell imaging based on bioorthogonal fatty acid labelling and in situ proximity ligation or quantification of S-palmitoylation levels by gas/liquid chromatography and mass spectrometry [41,42]. Moreover, chemical synthesis or semi-synthesis of modified peptides/proteins has been important for mechanistic understanding of S-palmitoylated proteins, as has been exemplified by the classical study on palmitoylated Ras isoforms [4,27,28]. Recent reports of site-specific chemical modifications of Ras and transmembrane protein IFITM3 with palmitate mimics in live cells and evaluation of activity afford new opportunities to study S-palmitoylation and other lipid modifications in live cells [43,44].
Figure 2. Chemical methods for the study of S-palmitoylation. (a) Metabolic labelling of cells with palmitic acid reporter alk-16/ODYA and further copper catalysed azide-alkyne cycloaddition (CuAAC) reaction with azido-modified fluorescent or affinity tags for fluorescent visualization or affinity enrichment of S-palmitoylated proteins. (b) Metabolic labelling with a bifunctional fatty acid chemical reporter and in cell photocrosslinking allows detection and proteomic identification of S-palmitoylated protein interactome. (c) ABE and acyl-RAC methods involve capping of free cysteines with thiol-reactive reagent like N-ethylmaleimide (NEM) followed by removal of S-palmitoylation with NH2OH. Newly exposed cysteines are then reacted with a thiol-reactive biotin or resin, respectively. Further enrichment allows identification of S-palmitoylated proteins. Image created with Biorender.com.
We previously reviewed the application of these chemical tools for global profiling of palmitoylated proteins in immune cells including T cells, dendritic cells, macrophages and B cells . Since the publication of this review, there has been remarkable progress in identifying new palmitoylated immunity proteins, and also a renewed emphasis on functional characterization of individual S-palmitoylated immune proteins. In this review, we will highlight mechanistic studies on the roles of S-palmitoylation in the regulation of immune protein function in immune cells (table 1). This review is organized by proteins involved in adaptive and innate immune pathways (i.e. immune sensors, signal transducers, signal regulators and immune effectors). There are excellent reviews that describe the role of protein S-palmitoylation in other physiological processes .
Table 1. S-palmitoylated immunity-associated proteins.
2. S-palmitoylation of proteins in adaptive immunity
S-palmitoylation plays an important role in the regulation of host adaptive immune responses. Multiple studies investigated the S-palmitoylome of T cells using different methods and identified important factors involved in T cell activation and signal transduction (figure 3) [31,37,80–82]. The first step in T cell receptor (TCR) signal transduction involves the binding of TCR to peptide–major histocompatibility complexes (MHCs) on antigen-presenting cells (APCs). This binding step is followed by recruitment of Src family protein tyrosine kinase Lck to the cytoplasmic domains of CD4 and CD8 co-receptors. This triggers a signalling cascade leading to the formation of the LAT signalosome. Further signal propagation happens via Ca 2+ –calcineurin, mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signalling pathways.
Figure 3. S-palmitoylated proteins in T cell signalling. S-palmitoylated Src kinases (Lck) phosphorylates TCR complex leading to ZAP-70 recruitment and activation. Activated ZAP-70 phosphorylates palmitoylated LAT leading to downstream signalling pathways. Image adapted from ‘TCR Downstream Signalling’ by BioRender.com.
2.1. TCR coreceptors
S-palmitoylation of TCR subunits has not been reported, but radio labelling studies identified TCR coreceptor CD4 palmitoylation. CD4 is palmitoylated at Cys394 and Cys397, at the junction of the transmembrane and cytoplasmic domain . CD4 S-palmitoylation regulates clustering with TCR/protein kinase C (PKC) θ in lipid rafts . Coreceptor CD8 is a heterodimer of CD8α and CD8β. In humans, both CD8α and CD8β are S-palmitoylated, whereas in mice only CD8β is S-palmitoylated. Mouse CD8β S-palmitoylation is necessary for efficient CD8 coreceptor function as it increases CD8 association with Lck in lipid rafts .
2.2. Src family kinases
Src family kinase Lck is S-palmitoylated at Cys3 and Cys5 and this promotes its plasma membrane association [49,83]. Interestingly, Lck N-myristoylation at Gly2 is required for subsequent palmitoylation. Deletion of Lck S-palmitoylation reduces Lck-CD4 association and downstream signalling . Further studies show that site-specific S-palmitoylation at Cys3 is important for Lck lipid raft localization .
S-palmitoylation of Fyn, another Src family kinase involved in T cell signal transduction, has also been shown to play an important role in its membrane association . Fyn can be S-palmitoylated at Cys3 and Cys6 but Cys3 is the major site and is the most important for lipid raft association. Activation of Fyn by Lck in lipid rafts leads to TCR/CD3 activation and downstream signalling. DHHC2, 3, 7, 10, 15, 20, 21 have all been shown to mediate Fyn S-palmitoylation.
Activation of transmembrane adaptor proteins (TRAPs) is a crucial step in the T cell signal transduction pathway. TRAPs like LAT, phosphoprotein associated with GEMs (PAG) and Lck-interacting membrane protein (LIME) are S-palmitoylated and show lipid raft association . LAT activation leads to the recruitment of key molecules for downstream signal propagation. LAT is dually S-palmitoylated at a membrane juxtaposed Cys-X-X-Cys motif. S-palmitoylation of LAT is important for its stability and plasma membrane association [52,53]. Deletion of LAT S-palmitoylation hinders TCR interaction and recruitment of PLCγ and Grb2 for downstream signal propagation as evident from impaired calcium influx and Erk activation .
Beyond T cell activation, LAT S-palmitoylation is also shown to regulate T cell anergy, an inability of previously responsive T cell to respond to TCR stimulation. Anergic T cells show reduced LAT palmitoylation, lipid raft association and immunological synapse recruitment . This should be further explored for therapeutic interventions to inhibit or induce T cell anergy in cancer, transplantation and infectious disease.
2.4. Fas and FasL
Apoptosis of T cells that recognize self-antigens is necessary to prevent autoimmune diseases. Fas and Fas ligand (FasL) are critical regulators of T cell apoptosis. Fas binding to FasL leads to recruitment of death-inducing signalling complex (DISC) in Fas-bearing cells which activates caspase-3 mediated apoptosis. Fas is S-palmitoylated at single Cys199 in human and is required for its stability . DHHC7 is identified to palmitoylate Fas. Mutation of the Fas S-palmitoylation site reduces its lipid raft association and impairs apoptosis signalling.
An ABE-based assay identified FasL S-palmitoylation . FasL is palmitoylated at Cys82 located at the N-terminal end of the transmembrane domain. S-palmitoylation modulates FasL lipid raft partitioning and proteolytic cleavage by ADAM10 for efficient induction of Fas-mediated cell death.
2.5. PD-1 and PD-L1
Programmed death protein 1 (PD-1) is a T cell surface receptor that, upon activation, suppresses T cell proliferation and cytokine production. PD-1 ligands PD-L1 and PD-L2 are expressed on antigen-presenting cells and tumour cells. In silico motif-based prediction identified PD-1 S-palmitoylation at Cys192 between its transmembrane and cytosolic domain, which was confirmed by metabolic labelling studies . PD-1 S-palmitoylation is catalysed by DHHC9. PD-1 palmitoylation is necessary for its stability. Recent studies show that some cancer cells also express PD-1, thus enabling them to promote tumour growth independent of adaptive immunity. S-palmitoylation of PD-1 in tumour cells can modulate downstream mammalian target of rapamycin (mTOR) signalling and proliferation.
PD-L1 expressed on tumour cells is also S-palmitoylated and this modification inhibits PD-L1 ubiquitination and trafficking to lysosomes for degradation . DHHC3 catalyse PD-L1 palmitoylation. Inhibition of PD-L1 palmitoylation using 2-bromopalmitate or via DHHC3 silencing increases anti-tumour activity in cells and in mice. These discoveries of PD-1 and PD-L1 palmitoylation and targeting for modulation of T cell immune responses in cancer provide exciting opportunities for combinatorial approaches along with immune checkpoint therapy.
2.6. B cells
S-palmitoylation of critical proteins in B cells has also been identified. Indeed, an ABE-based profiling of B cells identified many candidate S-palmitoylated proteins . However, there have been limited studies on functional implications of B cell protein S-palmitoylation.
B cell receptor (BCR) coreceptor CD81 is S-palmitoylated at multiple membrane juxtaposed Cys residues . S-palmitoylation of CD81 is required for enhanced BCR-coreceptor complex lipid raft association . It is also important for CD81 association with other BCR coreceptors CD19 and CD21 . Inhibition of CD81 S-palmitoylation affects recruitment of downstream signalling molecules and activation of kinases PI3 K and PKC.
Human germinal centre-associated lymphoma (HGAL) is an adaptor protein involved in BCR signalling. Radio labelling studies identified HGAL S-palmitoylation . S-palmitoylation of HGAL regulates binding and activation of Syk kinase for downstream signalling. HGAL S-palmitoylation deletion mutant localizes in the cytoplasm and significantly impairs chemoattractant-induced cell motility.
3. Palmitoylation of proteins in innate immunity
3.1. Innate immune receptors and signalling adapter proteins
The innate immune system responds to microbial invasion by signalling through pattern recognition receptors (PRRs) that bind conserved microbial features or molecules associated with cellular damage. PRRs can be broadly classified into several families of related molecules. These families include the Toll-like receptors (TLRs), C-type lectin receptors, RIG-I-like receptors and NOD-like receptors. Additionally, a receptor known as cyclic GMP-AMP (cGAMP) synthase (cGAS) is involved in microbial DNA detection. Several of these molecules and/or their signalling adapter proteins have been demonstrated to be S-palmitoylated in recent years (figure 4). As we will describe below, S-palmitoylation, where it has been detected, is generally an activating modification for innate immune signalling through several of these microbial sensing pathways.
Figure 4. S-palmitoylated innate immune receptors and signalling adapter proteins. (a) Activation of S-palmitoylated TLR2 by pathogen associated molecular patterns (PAMPs) leads to MyD88 signalling. S-palmitoylated MyD88 forms a complex with IL-1 receptor-associated kinase (IRAKs) for downstream signalling and subsequent translocation of nuclear factor-κB (NF-κB) to the nucleus for cytokine induction. (b) STING binds to cyclic dinucleotides at endoplasmic reticulum and translocates to Golgi apparatus where it is palmitoylated. At the trans-Golgi network, S-palmitoylated STING is clustered and recruit TBK1 and IRF3 for downstream signalling. (c) DHHC5 mediated S-palmitoylation of NOD1/2 leads to its recruitment to bacteria-containing phagosome. Further exposure to bacteria derived molecules induces NF-κB signalling. Image created with Biorender.com.
Toll-like receptors (TLRs) were among the first of the microbe-sensing PRRs to be discovered. TLRs are transmembrane proteins that use leucine-rich repeat domains to detect diverse microbial products, such as proteins, nucleic acids and glycans. The human genome encodes 10 known TLRs, which localize either to the cell surface or within endosomes. Binding of a TLR to its ligand results in signalling through TLR-associated cytoplasmic adapter proteins, including MyD88. TLR signalling generally culminates in activation of the transcription factor NF-κB and production of inflammatory cytokines. S-palmitoylome profiling of murine dendritic cells using an alkynyl palmitate analogue with click chemistry-based biotin pulldowns identified TLR2 as a putative palmitoylated protein . S-palmitoylation was confirmed and mapped to Cys609 via mutagenesis of candidate cysteines. C609 is located directly adjacent to the cytoplasm-facing side of the TLR2 transmembrane domain. Cys609 mutant constructs showed less responsiveness than WT TLR2 to known TLR2 microbial ligands in NF-κB reporter assays. Further, 2-BP inhibition of TLR2 palmitoylation in murine dendritic cells partially reduced TLR2 levels at the cell surface, and significantly reduced cytokine production in response to TLR2 ligands. Together, these data indicate that TLR2 palmitoylation at Cys609 is required for its proper localization and full antimicrobial activity (figure 4a) .
S-palmitoylation levels on TLR2 were increased by overexpression of several DHHC proteins suggesting possible enzymatic redundancy for this activating modification . Cys609 is highly conserved among TLR2 proteins throughout evolution, further supporting a significant role for this modification in antimicrobial functionality. Several additional human TLRs possess cysteine residues near the cytoplasmic border of their transmembrane domains, including TLRs 1, 5, 6, 7, 8, 9 and 10. However, of the human TLRs, only TLRs 2, 5 and 10 showed robust labelling with alkynyl palmitate analogue, with TLR10 providing the strongest palmitoylation signal relative to total protein levels . Whether S-palmitoylation regulates the activity of TLRs 5 and 10, or whether other TLRs are palmitoylated under specific cellular conditions, remain to be determined.
MyD88 is a critical signalling adaptor protein for all TLRs, except for TLR3. The discovery of MyD88 S-palmitoylation came as a follow-up to the observation that inhibition of fatty acid synthase by the chemical inhibitor C75 reduced inflammatory responses upon TLR2, 4 or 7/8 stimulation of cells . This inhibition of TLR signalling by C75 was pinpointed to MyD88 through studies examining NF-κB activation upon overexpression of various signalling molecules downstream of TLRs. Given that the product of fatty acid synthase is palmitate, the S-palmitoylation of MyD88 was investigated and confirmed using chemical reporter labelling and ABE methods .
Candidate S-palmitoylated cysteines, Cys113 and Cys274, were identified on MyD88 by mass spectrometry, and were further confirmed to be sites of modification via cysteine to alanine mutagenesis of MyD88 constructs and ABE analysis . Interestingly, while single mutations of Cys113 or Cys274 both significantly decreased total MyD88 S-palmitoylation, a double mutant did not show a further decrease in palmitoylation. In terms of NF-κB and MAPK activation, mutation of Cys113 decreased responsiveness to the TLR4 ligand LPS, while mutation of Cys274 had no measurable effect. A Cys113 to Ala MyD88 mutant failed to recruit the IRAK4 signalling molecule upon TLR stimulation, indicating that S-palmitoylation is required for recruitment of IRAK4 to the Myddosome signalling complex downstream of TLR ligand binding (figure 4a). It will be interesting to determine whether MyD88 S-palmitoylation promotes localization with TLRs, such as TLR2, that are also palmitoylated . It is also of note that MyD88 S-palmitoylation was not detected in any of the published S-palmitoyl proteome studies , including those done in macrophages and dendritic cells, which may suggest that robust MyD88 S-palmitoylation must be induced or that palmitoylated MyD88 exists in poorly soluble protein aggregates.
DHHC6 was identified as a leading candidate palmitoyltransferase for MyD88 based on its ability to increase MyD88 S-palmitoylation when overexpressed and based on its high expression in myeloid cell types known to have potent TLR activities . As such, knockdown of DHHC6 decreased MyD88 S-palmitoylation and LPS responsiveness of macrophages. Overall, S-palmitoylation of MyD88 appears to be a required regulatory modification that can be targeted to decrease TLR-mediated inflammation either by directly inhibiting enzymatic palmitoylation of MyD88 or through limiting endogenous palmitate available for protein modification .
The stimulator of interferon genes (STING) is a signalling protein involved in the response to DNA in the cytosol. STING is a multipass transmembrane protein that is activated by the cyclic dinucleotide cGAMP, which is produced by cGAS upon sensing DNA in the cytosol. Ligand-activated STING moves from the ER to the Golgi apparatus and recruits signalling molecules to activate NF-κB and interferon regulator factor 3 (IRF3), resulting in the production of proinflammatory cytokines and type I interferons (IFNs). Mukai et al. , hypothesized that STING is post-translationally modified at the Golgi and discovered that STING could be modified with radiolabelled palmitic acid upon cellular activation with the chemical STING agonist DMXAA. Further, overexpression of Golgi-localized DHHCs 3, 7 and 15 increased STING S-palmitoylation. Conversely, 2-BP treatment of cells eliminated STING palmitoylation and prevented cytokine production upon DMXAA stimulation, suggesting that palmitoylation is necessary for inflammatory cytokine induction by STING. The primary sites of modification on STING were mapped to Cys88 and Cys91. A STING mutant construct with serine substitutions at these positions trafficked from the ER to the Golgi similarly to WT STING upon DMXAA stimulation, but could not induce activation of NF-κB and IRF3, and thus failed to induce proinflammatory downstream gene products, including type I interferons. These results demonstrate that S-palmitoylation of STING at specific cysteines is required for its inflammatory signalling function (figure 4b) .
Activating STING mutations in the human population have been associated with an inflammatory autoimmune interferonopathy known as STING associated vasculopathy with onset in infancy (SAVI). Known STING mutants associated with SAVI lost their ability to spontaneously activate the IRF3- and NF-κB-dependent gene promoters when combined with S-palmitoylation-impairing mutations at Cys88 and 91 or when palmitoylation was inhibited by 2-BP treatment of cells . These results suggested that palmitoylation of STING could potentially be targeted therapeutically in SAVI. Indeed, in an attempt to identify STING inhibitors by chemical screening, small molecule inhibitors that covalently react with Cys91 and block palmitoylation were discovered . These inhibitors prevented clustering of STING at the Golgi and prevented STING-dependent inflammation in mouse models. Concurrently, a second group identified endogenously formed nitro-fatty acids as inhibitors of STING palmitoylation via covalent reaction with Cys88 . Nitro-fatty acids were produced during DNA virus infection, suggesting that this may be a natural cellular feedback mechanism to prevent hyperactivation of the cGAS-STING pathway. Further, the addition of nitro-fatty acids to fibroblasts derived from SAVI patients prevented the constitutive type I IFN production by these cells . These exciting developments in small molecule inhibitors of STING provide compelling proof-of-concept studies toward therapeutic targeting of STING palmitoylation .
Nucleotide oligomerization domain (NOD) 1 and 2 proteins are cytosolic innate immune receptors that detect bacterial peptidoglycans. NOD1 and NOD2 are primarily soluble in the cytosol with a portion of these proteins associated with the cellular plasma membrane at steady state. However, they are rapidly redistributed to phagosomal membranes upon intracellular bacterial infection, where they activate NF-κB and MAPK signalling pathways. Since NOD1/2 lack transmembrane regions or traditional lipid-binding motifs, it was hypothesized that their rapid membrane association could be mediated by dynamic S-palmitoylation . Indeed, treatment of cells with palmitoylation inhibitor 2-BP altered the localization of both NOD1 and NOD2 as visualized by fluorescence microscopy, a phenomenon that was confirmed by biochemical fractionation techniques to be due to loss of membrane association. 2-BP treatment of macrophages abrogated the ability of the cells to respond to NOD1/2 ligands in terms of NF-κB and MAPK pathway activation. S-palmitoylation of NOD1 and NOD2 were both subsequently confirmed by ABE and chemical reporter labelling methods. S-palmitoylation of NOD1 was mapped to three cysteine residues, Cys558, 567 and 952. A triple cysteine mutant of NOD1 lost its membrane association and ability to activate NF-κB, an effect that could be rescued by fusion of a known S-palmitoylation amino acid motif to NOD1. Similar effects were seen for NOD2, though in this case S-palmitoylation was mapped to Cys395 and 1033 (figure 4c). A BioID screen for NOD1 and NOD2 interacting proteins identified DHHC5 as a probable candidate for modification of these immune effectors . Knockdown or knockout of DHHC5 resulted in decreased S-palmitoylation of NOD1 and NOD2, decreased NOD membrane association, and decreased activation of NF-κB and MAPK pathways in response to NOD ligands. Further, both DHHC5 and intact NOD S-palmitoylation sites were required for recruitment of NOD1/2 to Salmonella-containing phagosomes .
NOD2 variants with decreased peptidoglycan responsiveness have been associated with pathologies, such as Crohn's disease. Five of six disease variants that were tested showed major decreases in S-palmitoylation, suggesting that defective palmitoylation-dependent membrane association underlie their decreased functionality . Conversely, a gain-of-function NOD2 variant showed significantly increased baseline S-palmitoylation, and 2-BP treatment eliminated its hyperactivation of NF-κB. Overall, it has emerged that S-palmitoylation is required for NOD1/2 localization with bacteria-containing membranes and activation of downstream pathways. However, it remains to be determined precisely how bacterial sensing triggers a rapid increase in NOD1/2 S-palmitoylation and how this facilitates inflammatory signalling. Recent cross-linking studies with peptidoglycan fragment muramyl-dipeptide (MDP) photoaffinity reporter showed MDP-induced NOD2 interaction with the plasma membrane or endosome resident N-myristoylated GTP-ARF6 . It will therefore be interesting to understand the role of NOD2 palmitoylation in mediating interactions with membrane-bound host proteins.
3.1.5. Phagocytosis receptor
Phagocytosis for engulfing of pathogens or foreign particles by immune cells also involves initial surface receptor-mediated recognition. Phagocytosis receptor FCGR2A is S-palmitoylated and regulates its lipid raft association . Furthermore, S-palmitoylation of FCGR2A associated kinase ASAP2 can regulate FCGR2A mediated phagocytosis .
S-palmitoylation of scavenger receptor CD36 has also been well studied. CD36 is a transmembrane glycoprotein receptor expressed on immune cells including monocytes, macrophages and other cell types, such as adipocytes and cardiac myocytes. CD36 binds to and mediates uptake of oxidized phospholipids, oxidized lipoproteins and long-chain fatty acids, thus playing a major role in lipid homeostasis in cells. In addition, CD36 plays an important role as an innate immune sensor that can recognize and bind to bacterial cell wall components, erythrocytes infected with Plasmodium falciparum and apoptotic cells. S-palmitoylation of CD36 by DHHC4 and DHHC5 was shown to play a regulatory role in adipose tissue fatty acid uptake in mice . Binding of S-palmitoylated CD36 to fatty acids activates the kinase Lyn. Activated Lyn phosphorylates DHHC5 and inhibits its S-palmitoylation activity. Depalmitoylation of fatty acid bound CD36 by APT1 leads to downstream signalling to initiate endocytosis of fatty acids. Pharmacological or genetic perturbations of this dynamic palmitoylation–depalmitoylation cycle disrupts fatty acid uptake of cells . Further investigations of CD36 S-palmitoylation in downstream immune signalling will be helpful to understand connections between lipid metabolism and inflammation.
Moreover, many members of the G protein-coupled receptor (GPCR) family including β1-adrenergic receptor, S1P receptor subtype 1 (S1PR1), CCR5 are known to be S-palmitoylated [68–70]. CCR5 and other S-palmitoylated cell surface receptors like EGFR are also used by viruses like human immunodeficiency virus (HIV) and influenza A virus (IAV), respectively, to enter the host cell successfully. Moreover, It should be noted that S-palmitoylation not only plays a role in the host immune system but the host S-palmitoylation machinery is also exploited by many viral or bacterial proteins for host infection [93,94]. For example, S-palmitoylation of the currently pandemic SARS-CoV-2 spike protein by DHHC5 is important for virus entry in ACE2 expressing cells .
3.2. Palmitoylation of innate immune effectors
Activation of innate immune receptors initiates signalling pathways resulting in the production of proinflammatory cytokines including tumour necrosis factors (TNFs), IFNs, chemokines and immune effector proteins for the elimination of microbial pathogens.
3.2.1. IFN signalling
IFNs bind to cell surface IFN receptor leading to activation of Janus tyrosine kinase (Jak)/signal-transducing activators of transcription (STAT) pathways to induce expression of IFN stimulated genes (ISGs). Type I IFNs (IFNα/β) engage the IFNAR receptor complex which is a heterodimer of IFNAR1 and IFNAR2. Radio labelling with [ 3 H] palmitic acid identified S-palmitoylation of IFNAR1 and IFNAR2 . IFNAR1 is S-palmitoylated at Cys463 present in the cytoplasmic domain proximal to the transmembrane domain. Mutagenesis of Cys463 to Ala does not affect receptor stability, endocytosis or intracellular distribution. However, loss of IFNAR1 S-palmitoylation results in decreased STAT phosphorylation and nuclear translocation, thereby affecting IFNα-stimulated gene transcription.
Palmitoylome profiling of adipocytes identified S-palmitoylation of four proteins in the JAK/STAT pathway JAK1, STAT1, STAT3 and STAT5A . Palmitoylation of JAK1 and JAK2 was confirmed in adipocytes using an acyl-RAC-like assay. JAK1 palmitoylation was mapped to highly conserved Cys541 and Cys542. Mutation of these two Cys to Ser led to loss of S-palmitoylation and impaired JAK1 plasma membrane association. S-palmitoylation of STAT family members STAT1α, STAT1β, STAT3, STAT5B and STAT6 has been demonstrated using metabolic labelling and acyl exchange methods . Canonical type I and type III IFN pathway involves both STAT1 and STAT2 activation and heterodimerization, whereas type II IFN pathway involves STAT2 activation and homodimerization. STAT2 and STAT4 S-palmitoylation has not been reported. Recently, STAT3 palmitoylation has been implicated in the regulation of TH17 cell differentiation (figure 5a) . TH17 cells are proinflammatory T cells which express interlukin-17 (IL-17) and retinoic acid receptor-related orphan receptor gamma t (RORγt). STAT3 undergoes a palmitoylation–depalmitoylation cycle on Cys108 mediated by DHHC7 and APT2, respectively. This cycle accelerates TH17 cell differentiation by promoting membrane association, phosphorylation and nuclear translocation of STAT3. Interestingly, phosphorylated STAT3 is selectively depalmitoylated by APT2. Accelerated differentiation of TH17 has been connected to autoimmune diseases. DHHC7 and APT2 have been shown to be upregulated in patients with inflammatory bowel disease (IBD). Inhibition of APT2 activity or knockout of DHHC7 in a mouse model relieves IBD symptoms. Inhibition of STAT3 S-palmitoylation–depalmitoylation cycle could thus be a potential therapeutic strategy for IBD .
Figure 5. S-palmitoylated innate immune effectors. (a) STAT3 dynamic S-palmitoylation in cells. STAT3 palmitoylation by DHHC7 leads to membrane association and phosphorylation. Depalmitoylation of p-STAT3 by APT2 leads to nuclear translocation of p-STAT3 and expression of STAT3 target genes. (b) S-palmitoylated IFITM3 restricts enveloped virus entry in cells through the endocytic pathway. IFITM3 colocalizes with virus-containing endosomes and prevents the release of the virus genetic material into the cytoplasm by physically restricting virus membrane pore formation and shuttling the virus for lysosomal degradation. Image created with Biorender.com.
3.2.2. IFN effectors
Palmitoylome analysis also identified several ISGs in a murine dendritic cell line and murine embryonic fibroblasts, including bone marrow stromal antigen 2 (BST2) or tetherin, immunity-related GTPase M1 (IRGM1) and interferon-induced transmembrane protein 3 (IFITM3) . We have studied S-palmitoylation of IFITM3 and its regulation in our laboratories over the past 10 years. Interferon-induced transmembrane proteins (IFITM1, IFITM2 and IFITM3), which share highly conserved palmitoylation sites, are involved in host immune response to viral infections. IFITM3 is the most active isoform and restricts many viruses including influenza A virus (IAV), dengue virus (DENV), Ebola virus (EBOV), human immunodeficiency virus (HIV), hepatitis C virus (HCV) and Zika virus (ZIKV) [96,97]. IFITM3 also inhibits SARS coronavirus (SARS-CoV) infections . Surprisingly, IFITM3 promotes endemic human coronavirus OC43 infection [99,100]. Recent pseudovirus infection and cell–cell fusion studies provide conflicting results regarding a role of IFITM3 in pandemic SARS-CoV-2 infection [101–104]. Infection studies with genuine SARS-CoV-2 shows IFITM3 acts primarily as an antiviral factor but an endocytosis mutant which localizes to the plasma membrane acts as a proviral factor .
IFITMs are present in basal levels in many different cell types. They are intrinsically expressed in embryonic stem cells for protection against virus infections . IFITMs reduce the susceptibility of placental trophoblasts to viral infection, but they also inhibit trophoblast cell fusion, an essential process for placental development mediated by syncytin, which is derived from retroviral envelope [107,108]. Furthermore, the resistance of CD8+ resident memory T cells expressing IFITM3 to IAV infection extends the antiviral role of IFITMs to adaptive immune cells [109,110]. Two recent studies highlight broader roles of IFITMs in host immune pathways beyond antiviral activity. IFITM3 has been shown to be critical in phosphoinositide 3-kinase (PI3 K) signal amplification for the rapid expansion of B cells with high affinity to antigen . IFITM3 has also been implicated in neuroinflammation as it modulates amyloid-β production by regulating γ-secretase activity .
Palmitoylome profiling of mouse dendritic cells identified IFITM3 S-palmitoylation . IFITM3 is S-palmitoylated at three Cys residues (Cys71, C72 and 105) and loss of S-palmitoylation leads to abrogation of IFITM3 antiviral activity against influenza virus (figure 5b), and similar loss of antiviral activity was reported for palmitoylation-deficient IFITM1 . Further APE analysis showed that Cys72 is the major site of modification of IFITM3 and mutation of this residue significantly lowers antiviral activity . Also, mass spectrometric analysis of purified IFITM3 from mammalian cells revealed Cys72 as the predominant site of modification . Cys72 is highly conserved across most mammals and is required for the antiviral activity of IFITM3 orthologues from mice, bats and humans [114,115]. Indeed, palmitoylation of evolutionarily ancient IFITM homologues present in mycobacteria were found to be palmitoylated when expressed in human cells . Site-directed mutagenesis and live-cell imaging studies showed that Cys72 is essential for IFITM3 trafficking and colocalization with influenza virus in the endocytic pathway [117,118]. Recent NMR studies with IFITM3 chemically modified at Cys72 with maleimide-palmitate show that lipid modification at Cys72 stabilizes IFITM3 amphipathic helix membrane interaction which is important for restriction of virus infection [44,119]. These studies demonstrate the importance of site-specific palmitoylation of IFITMs in their antiviral activity. Overexpression of multiple palmitoyltransferases (DHHC 3, 7, 15 and 20) leads to increased IFITM3 S-palmitoylation but DHHC20 might be the most important for IFITM3 antiviral activity . Roles for palmitoylation of IFITM3 in PI3 K signalling and regulation of γ-secretase activity remain to be investigated.
3.2.3. TNF signalling
Tumour necrosis factors (TNFs) form another important superfamily of cytokines. TNFα regulates a variety of cellular processes including inflammation, proliferation, differentiation, and can induce various forms of cell death. TNF is synthesized as a transmembrane protein (tmTNF) and presented at the plasma membrane where it is cleaved and released as soluble TNF (sTNF). Membrane-bound N-terminal fragment of TNF (NTF) is further cleaved by signal peptide peptidase-like 2b to generate intracellular domain of TNFα (ICD-TNFα). All the TNF forms show biological activities. Metabolic labelling with [ 3 H]palmitic acid identified S-palmitoylation of tmTNF . Recent studies indicate a role of palmitoylation in tmTNF lipid raft partitioning, NTF stability and efficient cleavage for ICD-TNFα formation . Interaction of S-palmitoylated tmTNF with TNFR1 in lipid rafts diminishes sensitivity to sTNF, thus regulating downstream NFkB and ERK1/2 signalling pathway.
Dynamic S-palmitoylation of TNFR1 also regulates TNF signalling . Plasma membrane TNFR1 activation leads to recruitment of complex 1 adapter proteins to TNFR leading to NF-κB signalling. On the other hand, K63-ubiquitylation and internalization of TNFR1 triggers apoptosis signalling. TNFR1 can be palmitoylated at multiple Cys but dynamic S-palmitoylation at transmembrane proximal Cys248 regulates its plasma membrane localization. Depalmitoylation of activated TNFR1 by APT2 is necessary for enhanced NF-κB signalling, whereas knockdown of APT2 enhances Caspase-8 mediated cell death and reduced NF-κB signalling. Other members of TNF-receptor family have also been reported to be palmitoylated. S-palmitoylation of DR4 is promotes lipid raft association and cell death signalling, whereas DR6 S-palmitoylation prevents lipid raft association [77,78,122].
4. Conclusion and perspective
Over the past decade, we have developed greater understanding of S-palmitoylation-dependent regulation of proteins in innate and adaptive immune signalling. This is a testament to the development of chemical tools to study S-palmitoylation. Use of these chemical tools and proteomics methods allowed S-palmitoylome profiling of different cell types and helped in development of in silico prediction of S-palmitoylation sites in proteins. Further use of chemical inhibitors and genetic methods helped to understand the role of S-palmitoylation in the regulation of protein function. Coupling of these tools has led to the discovery of novel protein S-palmitoylation and mechanisms of protein regulation. However, it should be noted that functional analysis remains to be performed for many putative palmitoylated proteins identified in large scale S-palmitoylome profiling studies, making this a rich area for future investigations.
Though we have learned significantly about S-palmitoylation, we still have very little understanding of the implications of a dynamic lipid modification of proteins in cell signalling and regulation in comparison to other dynamic modifications like phosphorylation or ubiquitination.
We have little understanding of the mechanism and substrate specificity of the writers and erasers of palmitoylation. Structural characterization of individual DHHCs and DHHC selective inhibitor development will be necessary future advances.
In recent years, we have recognized that the role of S-palmitoylation and modifying enzymes in immune cells extends beyond antimicrobial functions to cancer and autoimmune diseases, among others. Moreover, there has been an increasing understanding of their potential as an attractive target for therapeutic interventions, as has been discussed in this review.
3.2.3 Transport across cell membranes
Opportunities for skills development
The basic structure of all cell membranes, including cell-surface membranes and the membranes around the cell organelles of eukaryotes, is the same.
The arrangement and any movement of phospholipids, proteins, glycoproteins and glycolipids in the fluid-mosaic model of membrane structure. Cholesterol may also be present in cell membranes where it restricts the movement of other molecules making up the membrane.
Movement across membranes occurs by:
- simple diffusion (involving limitations imposed by the nature of the phospholipid bilayer)
- facilitated diffusion (involving the roles of carrier proteins and channel proteins)
- osmosis (explained in terms of water potential)
- active transport (involving the role of carrier proteins and the importance of the hydrolysis of ATP)
- co-transport (illustrated by the absorption of sodium ions and glucose by cells lining the mammalian ileum).
Cells may be adapted for rapid transport across their internal or external membranes by an increase in surface area of, or by an increase in the number of protein channels and carrier molecules in, their membranes.
Students should be able to:
- explain the adaptations of specialised cells in relation to the rate of transport across their internal and external membranes
- explain how surface area, number of channel or carrier proteins and differences in gradients of concentration or water potential affect the rate of movement across cell membranes.
Required practical 3: Production of a dilution series of a solute to produce a calibration curve with which to identify the water potential of plant tissue.
Required practical 4: Investigation into the effect of a named variable on the permeability of cell-surface membranes.
Students could plot the data from their investigations in an appropriate format.
Students could determine the water potential of plant tissues using the intercept of a graph of, eg, water potential of solution against gain/loss of mass.
6. Comparative biology of telomeres
There is a general belief that something as fundamental to human tumor biology as telomere shortening and the reactivation of telomerase should be highly conserved and generally shared among mammals. Long-lived animals must protect the steady state (homeostasis) of their tissues by continuous replacement of the cells that regularly differentiate and die. An adult human contains approximately 10 12 rapidly multiplying cells. During an approximate 80 year lifespan (
30,000 days), each person makes and discards an enormous number of cells from the bone marrow, skin, and gastrointestinal tract each day . In the absence of a mechanism to deal with spontaneous rates of sporadic mutations, cancer in humans would be a lot more prominent and appear significantly earlier in life than already occurs. Mechanisms to minimize genomic damage are essential for large long-lived species. While much remains to be discovered, organ systems in large long-lived species must have evolved mechanisms that dramatically slow the rate of accumulation of replication errors (such as increased DNA repair). Alternatively, there could be DNA damage sensing mechanisms that increase cell turnover (e.g. apoptosis) rather to propagate an increased mutational load.
Based on a recent survey of telomere biology covering 㹠 mammalian species we now have a better conceptual framework for understanding the different uses of telomeres in different species [30,31]. Our results provide evidence that the ancestral mammalian phenotype had short telomeres and repressed telomerase, consistent with the hypothesis that the initial adaptation to homeothermy involved the adoption of replicative aging to compensate for the increased mutational load of elevated body temperatures. In addition, we observed that telomere length inversely correlates with lifespan while telomerase expression correlates with mass. The role of replicative aging as a tumor suppression mechanism is well accepted, however its contribution to lifespan remains controversial. The demonstration that telomere length inversely correlates with lifespan provides support for the interpretation that replicative aging is one of many factors contributing to lifespan in a large number of species. Also, the evidence that oxidative protection mechanisms are lower in species with long telomeres  suggests one evolutionary advantage of abandoning replicative aging in favor of long telomeres and not repressing telomerase in smaller mammals. These evolutionary studies now allow the role of telomeres in human cancer and aging to be put in the larger context of mammalian telomere biology.
The telomere shortening based tumor suppressor program is apparently not conserved in laboratory mice , which have long telomeres and constitutive telomerase in many tissues. While mice are much smaller and live only a few years compared to humans, they still get cancer at about the same incidence as humans. Thus, mice must have less well regulated telomerase and poorer oxidative damage protective mechanisms to explain these observations. However, more primary data are needed so a conceptual framework for understanding why most short lived animals have exceptionally long telomeres is obtained. For example, could exceptionally long telomeres provide some other advantage such as being G-rich in sequence to deal with increased oxidative damage?
To begin to address this central problem, we used phylogeny based statistical analyses to reconstruct mammalian ancestral states. The ancestral mammalian phenotype of short telomeres and repressed telomerase suggests the initial adaptation to the increased mutational load of homeothermy was the repression of telomerase and the consequent adoption of replicative aging. These studies demonstrated that telomere length inversely correlated with lifespan, while telomerase expression co-evolved with body size . Multiple independent times smaller, shorter lived species changed to having long telomeres and expressing telomerase. Trade-offs involving reducing the energetic/cellular costs of specific oxidative protection mechanisms (needed to protect short telomeres in the absence of telomerase) is one explanation for abandonment of replicative aging. These observations now provide a new framework for understanding different uses of telomeres in mammals, support a role for short telomeres in allowing longer lifespans to evolve, demonstrate the need to include telomere length in the analysis of comparative studies of oxidative protection in the biology of aging, and identify which mammals might be valuable to use as appropriate model organisms for the study of the role of telomeres in human cancer and aging.
Genomic Instability and Aging
Genomic instability has long been considered to be a central driving force in aging, and extensive evidence has accumulated over the years supporting DNA-damage accumulation with age and its role in genomic instability. The findings that compromised genome-maintenance results in shortened life spans of model organisms or in human premature-aging syndromes suggest that genome maintenance is indeed a central antiaging mechanism.
However, aging of an organism is a very complex process, during which different tissues gradually functionally deteriorate. Since each tissue has its own characteristics and challenges, it is likely that their functional deterioration also follows individual patterns. Vijg and Dolle have put forward a model, according to which aging is not a clonal phenomenon, but rather arises from increasing heterogeneity of the cells in a tissue  . At the level of cells, accumulation of random mutations can contribute to this heterogeneity. At the level of tissues, different challenges to genomic integrity may promote aging. For instance, neural cells, which have high respiratory requirements, may be more susceptible to the accumulation of oxidative damage and cell death, whereas T cells that rely on continued proliferation may be more susceptible to senescence caused by telomere shortening.
If genomic instability is considered as a mechanism driving this age-associated mosaicism by contributing accumulation of mutations and promoting downstream outcomes such as altered gene expression, cell-cycle arrest, or cell death ( Fig. 29.2 ), a better mechanistic understanding of what triggers age-related deterioration of genome-maintenance mechanisms or changes in nuclear architecture may provide valuable insights into the role of genomic instability in aging. In addition, a better understanding of interactions between cells and tissues in the aging organism will help in determining a hierarchy of age-related changes.
Figure 29.2 . Mechanisms contributing to genomic instability and their role during aging.
Gray bubbles represent DNA lesions, arrows indicate contributions of processes to the given outcomes, and two-directional arrows indicate interplay between two processes.
Chronic Myeloid Leukemia
Drugs Targeting the T315I Mutation
The fact that the T315I mutant is not responsive to either dasatinib or nilotinib and that this mutant has been detected in some patients with acquired resistance to dasatinib and nilotinib underscores the need for drugs with T315I inhibitory activity. 21 Ponatinib is a multitargeted kinase inhibitor active against all BCR-ABL mutants, including T315I. In a phase I study, more than 50% of patients in CP, mostly patients in whom two or more TKIs had failed, attained CCR. 21 The CCR rate was 92% in patients with the T315I mutation. Responses were less frequent and stable in patients with advanced disease. This was confirmed in a phase II study where 51% of patients resistant to or intolerant of second-generation TKIs and 70% of patients with the T315I BCR-ABL mutation experienced a major cytogenetic response. However, 1 year after its accelerated approval by the US FDA, a high number of vascular occlusive events, both arterial and venous thromboembolic events, began to be reported, leading to the US FDA placing a partial clinical hold on the drug. The hold was lifted following dose-reduction recommendations, but use is currently restricted to patients with the T315I mutation or for whom no alternative TKI is available.
Omacetaxine mepesuccinate is a semisynthetic formulation of homoharringtonine, an alkaloid extracted from various Cephalotaxus species. 22 It has a distinct mechanism of action related to inhibition of protein synthesis—interfering with initial protein elongation—leading to decreased levels of proteins important for leukemia cell survival, including BCR-ABL MYC and MCL1. Omacetaxine is approved for the treatment of adult patients with CP or AP CML with resistance or intolerance to two or more TKIs. Of 111 CP or AP CML patients who received two or more prior TKIs, major cytogenetic response was achieved in 18% of patients with CP, with median response duration of 12.5 months and major hematologic response in 14% of patients with AP, with a median response duration of 4.7 months. Omacetaxine is supplied as a single-use vial for parenteral administration. Common adverse reactions included thrombocytopenia, anemia, neutropenia, diarrhea, nausea, fatigue, asthenia, injection site reaction, pyrexia, and infection.