ScienceDaily (May 4, 2012) — Researchers from the University of Minnesota's Lillehei Heart Institute have effectively treated muscular dystrophy in mice using human stem cells derived from a new process that -- for the first time -- makes the production of human muscle cells from stem cells efficient and effective.
The research, published May 4 in Cell Stem Cell, outlines the strategy for the development of a rapidly dividing population of skeletal myogenic progenitor cells (muscle-forming cells) derived from induced pluripotent (iPS) cells. iPS cells have all of the potential of embryonic stem (ES) cells, but are derived by reprogramming skin cells. They can be patient-specific, which renders them unlikely to be rejected, and do not involve the destruction of embryos.
This is the first time that human stem cells have been shown to be effective in the treatment of muscular dystrophy.
According to U of M researchers -- who were also the first to use ES cells from mice to treat muscular dystrophy -- there has been a significant lag in translating studies using mouse stem cells into therapeutically relevant studies involving human stem cells. This lag has dramatically limited the development of cell therapies or clinical trials for human patients.
The latest research from the U of M provides the proof-of-principle for treating muscular dystrophy with human iPS cells, setting the stage for future human clinical trials.
"One of the biggest barriers to the development of cell-based therapies for neuromuscular disorders like muscular dystrophy has been obtaining sufficient muscle progenitor cells to produce a therapeutically effective response," said principal investigator Rita Perlingeiro, Ph.D., associate professor of medicine in the Medical School's Division of Cardiology. "Up until now, deriving engraftable skeletal muscle stem cells from human pluripotent stem cells hasn't been possible. Our results demonstrate that it is indeed possible and sets the stage for the development of a clinically meaningful treatment approach."
Upon transplantation into mice suffering from muscular dystrophy, human skeletal myogenic progenitor cells provided both extensive and long-term muscle regeneration which resulted in improved muscle function.
To achieve their results, U of M researchers genetically modified two well-characterized human iPS cell lines and an existing human ES cell line with the PAX7 gene. This allowed them to regulate levels of the Pax7 protein, which is essential for the regeneration of skeletal muscle tissue after damage. The researchers found this regulation could prompt naïve ES and iPS cells to differentiate into muscle-forming cells.
Up until this point, researchers had struggled to make muscle efficiently from ES and iPS cells. PAX7 -- induced at exactly the right time -- helped determine the fate of human ES and iPS cells, pushing them into becoming human muscle progenitor cells.
Once Dr. Perlingeiro's team was able to pinpoint the optimal timing of differentiation, the cells were well suited to the regrowth needed to treat conditions such as muscular dystrophy. In fact, Pax7-induced muscle progenitors were far more effective than human myoblasts at improving muscle function. Myoblasts, which are cell cultures derived from adult muscle biopsies, had previously been tested in clinical trials for muscular dystrophy, however the myoblasts did not persist after transplantation.
"Seeing long-term maintenance of these cells without major adverse side effects is exciting," said Perlingeiro. "Our research proves that these differentiated stem cells have real staying power in the fight against muscular dystrophy."
According to John Wagner, M.D., scientific director of clinical research at the University's Stem Cell Institute and blood and marrow transplant expert, "This research is a phenomenal breakthrough. Dr. Perlingeiro and her collaborators have overcome one of the most significant obstacles to moving stem cell therapies into the treatment of children with devastating and life threatening muscular dystrophies."
The U of M researchers say alternative methods of Pax7 induction will need to be investigated before this study can be turned into a human clinical trial. Their method of delivering the Pax7 protein involved genetic modification of cells with viruses and because viruses sometimes cause mutations, they add risk to a clinical trial. But the U of M researchers are committed to developing a safe and effective clinical protocol, and are actively testing alternate methods of delivering Pax7.
Radbod Darabi, Robert W. Arpke, Stefan Irion, John T. Dimos, Marica Grskovic, Michael Kyba, Rita C.R. Perlingeiro. Human ES- and iPS-Derived Myogenic Progenitors Restore DYSTROPHIN and Improve Contractility upon Transplantation in Dystrophic Mice. Cell Stem Cell, 2012; 10 (5): 610 DOI: 10.1016/j.stem.2012.02.015
http://www.sciencedaily.com/releases/2012/05/120504110554.htm?utm_source=feedburner&utm_medium=email&utm_campaign=Feed%3A+sciencedaily+%28ScienceDaily%3A+Latest+Science+News%29
Sunday, May 6, 2012
Wednesday, May 2, 2012
Telomeres in Disease
The ends of linear chromosomes have attracted serious scientific study—and Nobel Prizes—since the early 20th century. Called telomeres, these ends serve to protect the coding DNA of the genome. When a cell’s telomeres shorten to critical lengths, the cell senesces. Thus, telomeres dictate a cell’s life span—unless something goes wrong. Work over the past several decades has revealed an active, though limited, mechanism for the normal enzymatic repair of telomere loss in certain proliferative cells. Telomere lengthening in cancer cells, however, confers an abnormal proliferative ability.
In addition to cancer, telomeres have been found to be involved in numerous other diseases, including liver dysfunction and aplastic anemia, a condition in which the bone marrow does not produce a sufficient supply of new blood cells. Inadequate telomere repair and accelerated telomere attrition can be molecular causes of these diseases, and targeting these processes may lead to the development of novel therapies.
Chromosome tails
Telomeres consist of hexameric nucleotide sequences (TTAGGG in humans) that are repeated hundreds to thousands of times at each extremity of each chromosome. Telomeric DNA is coated by a group of proteins, collectively termed shelterin, which serves to protect telomere structure. Because DNA can only be synthesized in one direction, the RNA primers at the chromosome’s ends cannot be filled in, and thus a small amount of DNA is lost with every cell division—a loss that occurs in the telomeres. During normal aging of an animal or in cell culture, cells divide and telomeres shorten. As telomeric sequences do not contain genes, no important genetic information undergoes erosion during DNA replication. When telomeres become critically short, the cell becomes senescent—it ceases to divide—or undergoes apoptosis—it dies.
Telomere attrition explains the “Hayflick limit,” the number of divisions a cell is capable of undergoing in tissue culture before the cell stops dividing. Telomere length is therefore a type of “mitotic clock,” a measure of a cell’s proliferative history. Under circumstances in which cell proliferation continues despite critically short telomeres (usually about a few hundred hexanucleotide repeats), the telomere’s protective function is lost. Subtelomeric genetic information can be lost, and more importantly, recombination between chromosomes occurs, leading to chromosome instability, aneuploidy, and possible transformation to a cancer phenotype.
Some proliferative cells can elongate telomeres enzymatically through the telomerase complex. Telomerase (TERT) is a reverse transcriptase that employs a small RNA molecule (TERC) as a template to extend telomeres in cells. In this way, telomerase counterbalances the effects of cell division and cellular genetic “aging,” preventing senescence, apoptosis, and genetic instability. Telomerase-dependent telomere repair occurs naturally in some cells, such as embryonic and adult stem cells and some cells of the immune system—cell types that divide regularly to support development, maintain tissues, and combat infections, respectively.
Telomere maintenance is also possible by other mechanisms, such as the alternative pathway (ALT), which uses recombination between chromosomes to maintain telomere length. In ALT, telomeres are not newly elongated, but rather transferred from one chromosome to another, resulting in some daughter cells whose chromosomes have shorter telomeres and others with longer telomeres. The details of ALT’s components and regulation, however, are not well understood.
Telomere shortening and cancer
Most cells in which telomeres reach critically short lengths either die or enter senescence. In those few that survive, perhaps due to inadequate monitoring by p53 and related DNA damage-response safeguards, telomere repair would be subject to powerful selective pressure. Indeed, in most malignancies, telomerase gene upregulation or activation of the ALT pathway is thought to be necessary for the establishment of cellular immortality. Telomerase is so frequently increased in tumors and in cancer cell lines as to be considered an appropriate therapeutic target. Currently there are several clinical trials using telomerase inhibitors to treat a variety of cancers, but results have yet to be reported. Telomere shortening would also generate the equivalent of a “mutator phenotype” by increasing spontaneous chromosomal aberrations—from numerical changes to structural abnormalities—and would therefore increase the pool of aberrant cells upon which selection would act.
There are many sources of evidence suggesting that telomere attrition is associated with and likely etiologic of cancer. Patients with dyskeratosis congenita, a rare inherited bone marrow failure disease characterized by telomerase dysfunction, have a 1,000-fold increase in risk of tongue cancer and about a 100 fold increase in risk of acute myeloid leukemia. In aplastic anemia, patients with the shortest telomeres (absent mutations) are 4- to 5-fold more likely to have their disease undergo malignant transformation to myelodysplasia and leukemia. Telomere-free ends of chromosomes and aneuploidy may be apparent in cultured bone marrow years before progression to leukemia. Furthermore, some acute myeloid leukemia patients without prior bone marrow failure have inherited mutations in TERT and TERC.
Similarly, short leukocyte telomeres predict the development of cancer in patients with Barrett’s esophagitis, a condition in which the lining of the esophagus is damaged by stomach acid, or ulcerative colitis, a type of inflammatory bowel disease. In these diseases, the mechanism is less clear. Short telomeres in blood leukocytes may reflect the telomere length of the affected organ. Alternatively, they may be a biomarker of exposure to reactive oxygen species produced as a result of a chronic inflammatory process, which can both damage telomeres and cause cancer. Evidence for the latter hypothesis comes from the observation that cells cultured in room air have excessive telomere shortening in comparison to cells cultured at low oxygen tension.
More generally, genome-wide analyses have identified single nucleotide polymorphisms in TERT as risk factors in many cancers. In a recent report, short leukocyte telomeres were associated with increased risk of all cancers and of cancer fatalities in a large population followed over a decade. Circumstantially, telomere attrition is an accompaniment of aging, itself a major risk factor for cancer. Furthermore, secondary malignancies often occur after chemotherapy and radiation, which would be anticipated to cause marrow stress and telomere shortening. More direct data come from animal experiments. In a knockout mouse model, animals with reduced telomerase activity combined with diminished p53 surveillance of DNA damage developed a variety of epidermal cancers unusual in the rodent but typical of humans.
Other telomere diseases
In addition to cancer, other diseases have been linked to telomeres. Hematopoietic stem cells express telomerase in response to the enormous daily demand for red blood cells, white blood cells, and platelets. Thus while overexpression of telomerase in other tissues can cause malignant growth, faulty telomere repair in blood stem cells can also result in severe diseases caused by stem cell failure.
One such “telomere disease” is dyskeratosis congenita, an X-linked bone marrow disorder characterized by symptoms such as abnormal nails, a pigmented, net-like rash, a white patch or plaque in the mouth, and aplastic anemia. The disease usually presents in the first decade of life. The liver and lungs can also be affected, as is often observed after a hematopoietic stem-cell transplant is performed to correct the bone marrow disease. The reasons for liver and lung involvement are not clear, but the chemotherapy used for transplant conditioning and the new inflammatory cells in the transplanted bone marrow may accelerate the injury in these organs.
Patients suffering from dyskeratosis congenita inherit a mutation in a gene named DKC1, identified by Inderjeet Dokal at the Hammersmith Hospital in London, who performed linkage studies of large pedigrees. DKC1 encodes dyskerin, a protein that binds to the RNA component of the telomerase complex and stabilizes it. Later, heterozygous mutations in TERC were also found in some families with dyskeratosis congenita. The severe phenotype of X-linked dyskeratosis congenita is likely due to the loss of functional DKC1 and markedly reduced telomerase function, which results in defective telomere repair and leads to accelerated telomere attrition, causing cell senescence and organ failure.
Whereas dyskeratosis congenita caused by mutations in the DKC1 gene usually presents during infancy, mutations in TERC and in the enzymatic component encoded by TERT appear to have impacts later in life. The first TERT mutations found in humans were in adult patients with acquired aplastic anemia who lacked physical anomalies and did not have a family history of telomere-related disease. Penetrance of the phenotypes of TERT and TERC mutations is highly variable among and within families, as reflected by the severity, time of onset, and organs involved. Within pedigrees, members with the identical mutation may have minimal or no clinical manifestations, develop aplastic anemia late in life, or suffer pulmonary fibrosis (scarring of the lungs) or hepatic cirrhosis (scarring of liver tissue). Different organ systems may be affected in different family members at different times, and occasional patients have disease in marrow, lung, and liver. How faulty telomere repair leads to such diseases is not fully understood.
Even with the uncertainties that remain, the association of telomerase mutations with disease in such disparate organs systems has important practical consequences for patients and their physicians. In the family history, the presence of even mild blood count abnormalities, pulmonary fibrosis, and hepatic cirrhosis, as well as acute myeloid leukemia, are important clues for the diagnosis of a telomeropathy. Involvement of multiple medical subspecialties can be confusing; some patients have even made their own diagnoses after Internet searches. Telomere length in leukocytes can be measured commercially and is a reliable marker of these diseases when severely reduced. Sequencing TERT and TERC can also be diagnostic. The appropriate finding of a telomerase deficit has consequences for prognosis, treatment, and genetic counseling. But while the diagnosis of telomeropathies can be straightforward, there may be complications. Some typical families lack known mutations, and telomere length may be normal even in the presence of etiologic nucleotide substitutions. Furthermore, rare mutations in shelterin genes coding for the proteins that protect telomere structure can produce severe dyskeratosis but do not alter telomerase repair capacity. And regulatory regions of genes, not routinely screened, may be responsible in some cases.
Telomeres and aging
Telomeres shorten as we age. By analogy to the cellular mitotic clock, telomeres have been postulated as a marker of “genetic age,” and telomere length has been marketed as a simple predictor of longevity. Assays of telomere length have been bundled with recommendations for lifestyle modification and for drug therapy, neither based on appropriate clinical studies. Simple but appealing arguments relating telomeres and aging are currently controversial, likely simplistic, and potentially harmful. Telomere length does indeed reflect a cell’s past proliferative history and future propensity for apoptosis, senescence, and transformation. Cellular aging, however, is not equivalent to organ or organismal aging.
There are several considerations in relating telomere biology to aging. First, physiologically there is overlap between the shortest telomere length of young children and the longest telomeres of the elderly. Most telomere shortening occurs early in life, in association with growth, and when the rate of disease in general is low. The paradigmatic telomere syndrome of dyskeratosis congenita is not at all typical of progerias, inherited syndromes in which patients appear old and suffer diseases of aging such as premature atherosclerosis or dementia. Furthermore, the organ damage of dyskeratosis congenita is not very similar to normal aging of marrow, lungs, and liver. The marrow becomes mildly hypocellular in older individuals, but stem cell numbers may actually increase, and blood counts remain stable; and neither the liver nor lungs normally become fibrotic with advanced age, as they often do in dyskeratosis congenita patients. Although in adults, relatively short leukocyte telomeres have been associated with cardiovascular events—a common morbidity of the aging population—the clinical correlations have not been consistent, and may be related to overall reactive oxygen species exposure.
Studies in humans have attempted to relate telomere length to life span. In the provocative initial publication from the University of Utah in 2003, individuals around 60 years of age who had the longest telomeres lived longer than did subjects with the shortest telomeres, but the main cause of death in the latter group was, inexplicably, infectious disease; the persons with shorter telomeres did not have a higher rate of cancer deaths. Moreover, these findings have not been confirmed in other studies of older subjects. In another study evaluating a different population, telomere length failed to predict survival, but interestingly it correlated with years of healthy life. In a Danish study of people aged 73 to 101 years, telomeres correlated with life expectancy in a simple univariate analysis, but only before the researchers corrected for age, suggesting that the correlation was driven simply by the fact that younger subjects had longer telomeres. And a Dutch study of 78-year-old men found that while telomere lengths eroded with age, they failed to correlate with mortality. These discrepancies may have several causes. In some analyses, telomere lengths may have been studied as a surrogate marker of age. In addition, retrospective studies may identify “positive” associations that are random and cannot be reproduced in follow-up investigations.
The telomere hypothesis of aging also has been modeled in mice. For instance, in a murine model of telomerase deficiency and accelerated telomere attrition, researchers found that certain intracellular pathways involved in mitochondrial function and glucose metabolism were deregulated, a common occurrence in aging individuals, ultimately causing heart muscle disease. Interestingly, telomerase reactivation in these mice restored glucose production and heart function. However, these abnormalities observed in telomerase-deficient animals were not those typical of humans with very short telomeres; patients with telomeropathies usually do not suffer from heart disease. Indeed, the translation of mouse experiments on telomeres to human physiology and disease should be done with caution. Mice are not the ideal model for telomere attrition and its effects on aging as murine telomeres are 5 to 10 times longer than human telomeres, in spite of mice having a much shorter life span. When telomerase is knocked out in mice, they live a healthy life for several generations, and even late-generation animals with very short telomeres do not display the clinical phenotypes characteristic of human telomeropathies. Telomerase-deficient mice also do not have a higher incidence of cancer, which happens only if the p53 gene also is modulated, in contrast to humans with telomerase deficiency, who are at very high risk of developing cancer.
Implications for medicine
Telomeres and their repair are important in the growing field of regenerative medicine. Dolly the sheep had chromosomes with shorter telomeres probably because she was cloned from an adult mammary gland cell. This may have contributed to Dolly’s illnesses, especially her progressive lung disease. Embryonic stem cells, however, express telomerase and are able to maintain their telomere lengths despite numerous cell divisions. More recently, reprogramming mature adult skin cells to the pluripotent state has been achieved with introduction of just a few defined nuclear factors. During the process of reverting cells to a more immature and pluripotent state, the reprogrammed cells’ telomeres are highly elongated. In the first steps of reprogramming and likely in the early stages of embryogenesis, cells can elongate, and thus “rejuvenate,” their telomeres. Since telomere shortening limits cell proliferation, mechanisms that can elongate telomeres are highly desirable for effective regenerative medicine.
Telomerase expression is tightly regulated in the cell; just a few copies of the complex are present in the cell nucleus, and they exert their function during certain specific periods of the cell cycle. The mechanisms that modulate telomerase gene expression, resultant enzymatic activity, and telomere elongation are the focus of intensive research. MYC, a proto-oncogene that regulates the expression of many genes and cell pluripotency, activates telomerase expression. Sex hormones also activate telomerase expression in reproductive and nonreproductive organs, such as the bone marrow. The promoter region of the telomerase gene contains regulatory sequences that are modulated by estrogen; cells exposed to estrogens (or androgens converted into estrogens) upregulate telomerase expression. In retrospect, the clinical response of improved blood cell counts in patients with aplastic anemia, especially children with inherited marrow failure, to androgens may be attributable to this mechanism. However, whether higher blood levels of sex hormones or exposure to exogenous sex hormones causes telomere elongation is still unknown.
Conclusions
Telomeres and telomere repair are basic molecular processes in cells possessing linear DNA chromosomes. Accelerated telomere attrition due to genetic defects in telomerase and in the shelterin protein genes is etiologic in several human diseases not previously considered related in the clinic. These include aplastic anemia, pulmonary fibrosis, and hepatic cirrhosis. The telomeropathies, especially in their milder and more chronic forms, may not be rare and almost certainly are often unrecognized by physicians. The importance of telomere repair in tissues under regenerative stress is of special interest, particularly in the reactive responses of fibrogenesis in the liver and the lungs. The maintenance of adequate telomere lengths also may be important in embryonic and adult stem cells to enable proliferation while preventing chromosome instability, thus avoiding potential malignant transformation. Also of interest is the connection linking telomere attrition and chronic inflammation to cancer and other diseases. (See “An Aspirin for Your Cancer,” The Scientist, April 2011.)
There is still much to be learned about how telomerase gene mutations cause disease, why they only affect certain organs, and how telomeres can be targeted for therapies. Both the genetic regulation of telomerase expression and the effect of an organism’s environment on telomere attrition are poorly understood. Drugs or hormones that might modulate telomerase expression and maintain or elongate telomeres would be appealing in the treatment of the telomeropathies and in conditions in which telomere attrition has known medical consequences. Whether telomere shortening mediates human aging—and conversely, whether telomere elongation may reverse aging or prevent age-related diseases—are still controversial issues.
Rodrigo Calado is an Associate Professor of Medicine at the University of São Paulo. Neal Young is the Chief of the Hematology Branch at the US National Institutes of Health.
This article is adapted from a review in F1000 Medicine Reports, DOI:10.3410/M4-8 (open access).
http://the-scientist.com/2012/05/01/telomeres-in-disease/
In addition to cancer, telomeres have been found to be involved in numerous other diseases, including liver dysfunction and aplastic anemia, a condition in which the bone marrow does not produce a sufficient supply of new blood cells. Inadequate telomere repair and accelerated telomere attrition can be molecular causes of these diseases, and targeting these processes may lead to the development of novel therapies.
Chromosome tails
Telomeres consist of hexameric nucleotide sequences (TTAGGG in humans) that are repeated hundreds to thousands of times at each extremity of each chromosome. Telomeric DNA is coated by a group of proteins, collectively termed shelterin, which serves to protect telomere structure. Because DNA can only be synthesized in one direction, the RNA primers at the chromosome’s ends cannot be filled in, and thus a small amount of DNA is lost with every cell division—a loss that occurs in the telomeres. During normal aging of an animal or in cell culture, cells divide and telomeres shorten. As telomeric sequences do not contain genes, no important genetic information undergoes erosion during DNA replication. When telomeres become critically short, the cell becomes senescent—it ceases to divide—or undergoes apoptosis—it dies.
Telomere attrition explains the “Hayflick limit,” the number of divisions a cell is capable of undergoing in tissue culture before the cell stops dividing. Telomere length is therefore a type of “mitotic clock,” a measure of a cell’s proliferative history. Under circumstances in which cell proliferation continues despite critically short telomeres (usually about a few hundred hexanucleotide repeats), the telomere’s protective function is lost. Subtelomeric genetic information can be lost, and more importantly, recombination between chromosomes occurs, leading to chromosome instability, aneuploidy, and possible transformation to a cancer phenotype.
Some proliferative cells can elongate telomeres enzymatically through the telomerase complex. Telomerase (TERT) is a reverse transcriptase that employs a small RNA molecule (TERC) as a template to extend telomeres in cells. In this way, telomerase counterbalances the effects of cell division and cellular genetic “aging,” preventing senescence, apoptosis, and genetic instability. Telomerase-dependent telomere repair occurs naturally in some cells, such as embryonic and adult stem cells and some cells of the immune system—cell types that divide regularly to support development, maintain tissues, and combat infections, respectively.
Telomere maintenance is also possible by other mechanisms, such as the alternative pathway (ALT), which uses recombination between chromosomes to maintain telomere length. In ALT, telomeres are not newly elongated, but rather transferred from one chromosome to another, resulting in some daughter cells whose chromosomes have shorter telomeres and others with longer telomeres. The details of ALT’s components and regulation, however, are not well understood.
Telomere shortening and cancer
Most cells in which telomeres reach critically short lengths either die or enter senescence. In those few that survive, perhaps due to inadequate monitoring by p53 and related DNA damage-response safeguards, telomere repair would be subject to powerful selective pressure. Indeed, in most malignancies, telomerase gene upregulation or activation of the ALT pathway is thought to be necessary for the establishment of cellular immortality. Telomerase is so frequently increased in tumors and in cancer cell lines as to be considered an appropriate therapeutic target. Currently there are several clinical trials using telomerase inhibitors to treat a variety of cancers, but results have yet to be reported. Telomere shortening would also generate the equivalent of a “mutator phenotype” by increasing spontaneous chromosomal aberrations—from numerical changes to structural abnormalities—and would therefore increase the pool of aberrant cells upon which selection would act.
There are many sources of evidence suggesting that telomere attrition is associated with and likely etiologic of cancer. Patients with dyskeratosis congenita, a rare inherited bone marrow failure disease characterized by telomerase dysfunction, have a 1,000-fold increase in risk of tongue cancer and about a 100 fold increase in risk of acute myeloid leukemia. In aplastic anemia, patients with the shortest telomeres (absent mutations) are 4- to 5-fold more likely to have their disease undergo malignant transformation to myelodysplasia and leukemia. Telomere-free ends of chromosomes and aneuploidy may be apparent in cultured bone marrow years before progression to leukemia. Furthermore, some acute myeloid leukemia patients without prior bone marrow failure have inherited mutations in TERT and TERC.
Similarly, short leukocyte telomeres predict the development of cancer in patients with Barrett’s esophagitis, a condition in which the lining of the esophagus is damaged by stomach acid, or ulcerative colitis, a type of inflammatory bowel disease. In these diseases, the mechanism is less clear. Short telomeres in blood leukocytes may reflect the telomere length of the affected organ. Alternatively, they may be a biomarker of exposure to reactive oxygen species produced as a result of a chronic inflammatory process, which can both damage telomeres and cause cancer. Evidence for the latter hypothesis comes from the observation that cells cultured in room air have excessive telomere shortening in comparison to cells cultured at low oxygen tension.
More generally, genome-wide analyses have identified single nucleotide polymorphisms in TERT as risk factors in many cancers. In a recent report, short leukocyte telomeres were associated with increased risk of all cancers and of cancer fatalities in a large population followed over a decade. Circumstantially, telomere attrition is an accompaniment of aging, itself a major risk factor for cancer. Furthermore, secondary malignancies often occur after chemotherapy and radiation, which would be anticipated to cause marrow stress and telomere shortening. More direct data come from animal experiments. In a knockout mouse model, animals with reduced telomerase activity combined with diminished p53 surveillance of DNA damage developed a variety of epidermal cancers unusual in the rodent but typical of humans.
Other telomere diseases
In addition to cancer, other diseases have been linked to telomeres. Hematopoietic stem cells express telomerase in response to the enormous daily demand for red blood cells, white blood cells, and platelets. Thus while overexpression of telomerase in other tissues can cause malignant growth, faulty telomere repair in blood stem cells can also result in severe diseases caused by stem cell failure.
One such “telomere disease” is dyskeratosis congenita, an X-linked bone marrow disorder characterized by symptoms such as abnormal nails, a pigmented, net-like rash, a white patch or plaque in the mouth, and aplastic anemia. The disease usually presents in the first decade of life. The liver and lungs can also be affected, as is often observed after a hematopoietic stem-cell transplant is performed to correct the bone marrow disease. The reasons for liver and lung involvement are not clear, but the chemotherapy used for transplant conditioning and the new inflammatory cells in the transplanted bone marrow may accelerate the injury in these organs.
Patients suffering from dyskeratosis congenita inherit a mutation in a gene named DKC1, identified by Inderjeet Dokal at the Hammersmith Hospital in London, who performed linkage studies of large pedigrees. DKC1 encodes dyskerin, a protein that binds to the RNA component of the telomerase complex and stabilizes it. Later, heterozygous mutations in TERC were also found in some families with dyskeratosis congenita. The severe phenotype of X-linked dyskeratosis congenita is likely due to the loss of functional DKC1 and markedly reduced telomerase function, which results in defective telomere repair and leads to accelerated telomere attrition, causing cell senescence and organ failure.
Whereas dyskeratosis congenita caused by mutations in the DKC1 gene usually presents during infancy, mutations in TERC and in the enzymatic component encoded by TERT appear to have impacts later in life. The first TERT mutations found in humans were in adult patients with acquired aplastic anemia who lacked physical anomalies and did not have a family history of telomere-related disease. Penetrance of the phenotypes of TERT and TERC mutations is highly variable among and within families, as reflected by the severity, time of onset, and organs involved. Within pedigrees, members with the identical mutation may have minimal or no clinical manifestations, develop aplastic anemia late in life, or suffer pulmonary fibrosis (scarring of the lungs) or hepatic cirrhosis (scarring of liver tissue). Different organ systems may be affected in different family members at different times, and occasional patients have disease in marrow, lung, and liver. How faulty telomere repair leads to such diseases is not fully understood.
Even with the uncertainties that remain, the association of telomerase mutations with disease in such disparate organs systems has important practical consequences for patients and their physicians. In the family history, the presence of even mild blood count abnormalities, pulmonary fibrosis, and hepatic cirrhosis, as well as acute myeloid leukemia, are important clues for the diagnosis of a telomeropathy. Involvement of multiple medical subspecialties can be confusing; some patients have even made their own diagnoses after Internet searches. Telomere length in leukocytes can be measured commercially and is a reliable marker of these diseases when severely reduced. Sequencing TERT and TERC can also be diagnostic. The appropriate finding of a telomerase deficit has consequences for prognosis, treatment, and genetic counseling. But while the diagnosis of telomeropathies can be straightforward, there may be complications. Some typical families lack known mutations, and telomere length may be normal even in the presence of etiologic nucleotide substitutions. Furthermore, rare mutations in shelterin genes coding for the proteins that protect telomere structure can produce severe dyskeratosis but do not alter telomerase repair capacity. And regulatory regions of genes, not routinely screened, may be responsible in some cases.
Telomeres and aging
Telomeres shorten as we age. By analogy to the cellular mitotic clock, telomeres have been postulated as a marker of “genetic age,” and telomere length has been marketed as a simple predictor of longevity. Assays of telomere length have been bundled with recommendations for lifestyle modification and for drug therapy, neither based on appropriate clinical studies. Simple but appealing arguments relating telomeres and aging are currently controversial, likely simplistic, and potentially harmful. Telomere length does indeed reflect a cell’s past proliferative history and future propensity for apoptosis, senescence, and transformation. Cellular aging, however, is not equivalent to organ or organismal aging.
There are several considerations in relating telomere biology to aging. First, physiologically there is overlap between the shortest telomere length of young children and the longest telomeres of the elderly. Most telomere shortening occurs early in life, in association with growth, and when the rate of disease in general is low. The paradigmatic telomere syndrome of dyskeratosis congenita is not at all typical of progerias, inherited syndromes in which patients appear old and suffer diseases of aging such as premature atherosclerosis or dementia. Furthermore, the organ damage of dyskeratosis congenita is not very similar to normal aging of marrow, lungs, and liver. The marrow becomes mildly hypocellular in older individuals, but stem cell numbers may actually increase, and blood counts remain stable; and neither the liver nor lungs normally become fibrotic with advanced age, as they often do in dyskeratosis congenita patients. Although in adults, relatively short leukocyte telomeres have been associated with cardiovascular events—a common morbidity of the aging population—the clinical correlations have not been consistent, and may be related to overall reactive oxygen species exposure.
Studies in humans have attempted to relate telomere length to life span. In the provocative initial publication from the University of Utah in 2003, individuals around 60 years of age who had the longest telomeres lived longer than did subjects with the shortest telomeres, but the main cause of death in the latter group was, inexplicably, infectious disease; the persons with shorter telomeres did not have a higher rate of cancer deaths. Moreover, these findings have not been confirmed in other studies of older subjects. In another study evaluating a different population, telomere length failed to predict survival, but interestingly it correlated with years of healthy life. In a Danish study of people aged 73 to 101 years, telomeres correlated with life expectancy in a simple univariate analysis, but only before the researchers corrected for age, suggesting that the correlation was driven simply by the fact that younger subjects had longer telomeres. And a Dutch study of 78-year-old men found that while telomere lengths eroded with age, they failed to correlate with mortality. These discrepancies may have several causes. In some analyses, telomere lengths may have been studied as a surrogate marker of age. In addition, retrospective studies may identify “positive” associations that are random and cannot be reproduced in follow-up investigations.
The telomere hypothesis of aging also has been modeled in mice. For instance, in a murine model of telomerase deficiency and accelerated telomere attrition, researchers found that certain intracellular pathways involved in mitochondrial function and glucose metabolism were deregulated, a common occurrence in aging individuals, ultimately causing heart muscle disease. Interestingly, telomerase reactivation in these mice restored glucose production and heart function. However, these abnormalities observed in telomerase-deficient animals were not those typical of humans with very short telomeres; patients with telomeropathies usually do not suffer from heart disease. Indeed, the translation of mouse experiments on telomeres to human physiology and disease should be done with caution. Mice are not the ideal model for telomere attrition and its effects on aging as murine telomeres are 5 to 10 times longer than human telomeres, in spite of mice having a much shorter life span. When telomerase is knocked out in mice, they live a healthy life for several generations, and even late-generation animals with very short telomeres do not display the clinical phenotypes characteristic of human telomeropathies. Telomerase-deficient mice also do not have a higher incidence of cancer, which happens only if the p53 gene also is modulated, in contrast to humans with telomerase deficiency, who are at very high risk of developing cancer.
Implications for medicine
Telomeres and their repair are important in the growing field of regenerative medicine. Dolly the sheep had chromosomes with shorter telomeres probably because she was cloned from an adult mammary gland cell. This may have contributed to Dolly’s illnesses, especially her progressive lung disease. Embryonic stem cells, however, express telomerase and are able to maintain their telomere lengths despite numerous cell divisions. More recently, reprogramming mature adult skin cells to the pluripotent state has been achieved with introduction of just a few defined nuclear factors. During the process of reverting cells to a more immature and pluripotent state, the reprogrammed cells’ telomeres are highly elongated. In the first steps of reprogramming and likely in the early stages of embryogenesis, cells can elongate, and thus “rejuvenate,” their telomeres. Since telomere shortening limits cell proliferation, mechanisms that can elongate telomeres are highly desirable for effective regenerative medicine.
Telomerase expression is tightly regulated in the cell; just a few copies of the complex are present in the cell nucleus, and they exert their function during certain specific periods of the cell cycle. The mechanisms that modulate telomerase gene expression, resultant enzymatic activity, and telomere elongation are the focus of intensive research. MYC, a proto-oncogene that regulates the expression of many genes and cell pluripotency, activates telomerase expression. Sex hormones also activate telomerase expression in reproductive and nonreproductive organs, such as the bone marrow. The promoter region of the telomerase gene contains regulatory sequences that are modulated by estrogen; cells exposed to estrogens (or androgens converted into estrogens) upregulate telomerase expression. In retrospect, the clinical response of improved blood cell counts in patients with aplastic anemia, especially children with inherited marrow failure, to androgens may be attributable to this mechanism. However, whether higher blood levels of sex hormones or exposure to exogenous sex hormones causes telomere elongation is still unknown.
Conclusions
Telomeres and telomere repair are basic molecular processes in cells possessing linear DNA chromosomes. Accelerated telomere attrition due to genetic defects in telomerase and in the shelterin protein genes is etiologic in several human diseases not previously considered related in the clinic. These include aplastic anemia, pulmonary fibrosis, and hepatic cirrhosis. The telomeropathies, especially in their milder and more chronic forms, may not be rare and almost certainly are often unrecognized by physicians. The importance of telomere repair in tissues under regenerative stress is of special interest, particularly in the reactive responses of fibrogenesis in the liver and the lungs. The maintenance of adequate telomere lengths also may be important in embryonic and adult stem cells to enable proliferation while preventing chromosome instability, thus avoiding potential malignant transformation. Also of interest is the connection linking telomere attrition and chronic inflammation to cancer and other diseases. (See “An Aspirin for Your Cancer,” The Scientist, April 2011.)
There is still much to be learned about how telomerase gene mutations cause disease, why they only affect certain organs, and how telomeres can be targeted for therapies. Both the genetic regulation of telomerase expression and the effect of an organism’s environment on telomere attrition are poorly understood. Drugs or hormones that might modulate telomerase expression and maintain or elongate telomeres would be appealing in the treatment of the telomeropathies and in conditions in which telomere attrition has known medical consequences. Whether telomere shortening mediates human aging—and conversely, whether telomere elongation may reverse aging or prevent age-related diseases—are still controversial issues.
Rodrigo Calado is an Associate Professor of Medicine at the University of São Paulo. Neal Young is the Chief of the Hematology Branch at the US National Institutes of Health.
This article is adapted from a review in F1000 Medicine Reports, DOI:10.3410/M4-8 (open access).
http://the-scientist.com/2012/05/01/telomeres-in-disease/
Tuesday, May 1, 2012
Crystal Structure of Human Argonaute2
Scripps Research Institute Scientists Find the Structure of a Key Gene Silencer Protein
The Structure reveals potential therapeutic targets in area with untapped potential
LA JOLLA, CA, April 26, 2012:-
Scientists at The Scripps Research Institute have determined the three-dimensional atomic structure of a human protein that is centrally involved in regulating the activities of cells. Knowing the precise structure of this protein paves the way for scientists to understand a process known as RNA-Silencing and to harness it to treat diseases.
"Biologists have known about RNA-Silencing for only a decade or so, but it's already clear that there's an enormous untapped potential here for new therapies," said Ian MacRae, an assistant professor at Scripps Research and senior author of the new report.
The new report, which appeared on April 26,2012 in the Journal Science's advance online publication, Science Express, focuses on Argonaute2. This protein can effectively silence a gene by intercepting and slicing the gene's RNA transcripts before they are translated into working proteins.
Interception and Destruction of Messages
When a gene that code for a protein is active in a cell, its information is transcribed from DNA form into lengths of nucleic acid called messenger RNA. If all goes well, these coded mRNA signals make their way to the cell's protein factories, which use them as templates to synthesize new proteins. RNA-silencing, also called RNA-Interference is the interception and destruction of these messages and as such, is a powerful and specific regulator of cell activity, as well as a strong defender against viral genes.
The silencing process requires not only an Argonaute protein but also a small length of guide RNA, known as a short-interfering RNA or microRNA. The guide RNA fits into a slot on Argonaute and serves as a target recognition device. Like a coded strip of Velero(TM), it latches onto a specific mRNA target whose sequence is the chemical mirror image of its own- thus bringing Argonaute into contact with its doomed prey.
Argonaute2 is not the only type of human Argonaute protein, but it seems to be the only one capable fo destroying target RNA directly. "If the guide RNA is completely complementary to the target RNA, Argonaute2 will cleave the mRNA, and that will elicit the degradation of the fragments and the loss of the genetic message," said Nicole Schirle, the graduate student in MacRae's laboratory who was lead author of the paper.
Aimed at disease-causing genes or even a cell's own overactive guide RNAs, RNA-silencing could be a powerful therapeutic weapon. In principle, one needs only to inject target-specific guide RNAs, and these will link up with Argonaute protein in cells to find and destroy the target RNAs. Scientists have managed to do this successfully with relatively accessible target cells, such as in the eye. But they have found it difficult to develop guide RNAs that can get from the bloodstream into distant tissues and still function.
"You have to modify the guide RNA, in some way to get it through the blood and into cells, but as soon as you start modifying it, you disrupt its ability to interact with Argonaute,"said MacRae. Knowing the precise structure of Argonaute should enable researchers to clear this hurdle by designing better guide RNA.
More Points for Manipulation
Previous structural studies have focused mostly on Argonaute protein from bacteria and other lower organisms, which have key differences from their human counterparts. Schirle was able to produce the comparatively large and complex human Argonaute2 and to manipulate it into forming crystals for X-ray crystallography analysis a feat that structural biologists have wanted to achieve for much of the past decade. "It was just excellent and diligent crystallography on her part," said MacRae.
The team's analysis of Argonaute2's structure revealed that it has the same basic set of working parts as bacterial Argonaute proteins, except that they are arranged somewhat differently. Also, key parts of Argonaute2 have extra loops and other structures, not seen on bacterial versions, which may play roles in binding to guide RNA. Finally, Argonatue2 has what appear to be binding sites for additional co-factor proteins that are thought to perform other destructive operations on the target mRNA.
"Basically, this Argonaute protein is more sophisticated than its bacterial cousins; it has more bells and whistles, which give us more points for manipulation. With this structure solved, we no longer need to use the prokaryotic structures to guess at what human Argonaute protein look like," MacRae said.
He and Schirle and others in the lab now are analyzing the functions of Argonaute2's substructures, as well as looking for ways to design better therapeutic guide RNAs.
"Now with the structural data, we can see what synthetic guide RNAs will work with Argonaute and what won't, " MacRae said, "we might even be able to make guide RNAs that can outcompete natural ones."
The research that led to Schirle and MacRae's new paper, "The Crystal Structure of Human Argonaute2," was funded by the National Institute of General Medical Sciences, part of the National Institute of Health.
press@scripps.edu
The Structure reveals potential therapeutic targets in area with untapped potential
LA JOLLA, CA, April 26, 2012:-
Scientists at The Scripps Research Institute have determined the three-dimensional atomic structure of a human protein that is centrally involved in regulating the activities of cells. Knowing the precise structure of this protein paves the way for scientists to understand a process known as RNA-Silencing and to harness it to treat diseases.
"Biologists have known about RNA-Silencing for only a decade or so, but it's already clear that there's an enormous untapped potential here for new therapies," said Ian MacRae, an assistant professor at Scripps Research and senior author of the new report.
The new report, which appeared on April 26,2012 in the Journal Science's advance online publication, Science Express, focuses on Argonaute2. This protein can effectively silence a gene by intercepting and slicing the gene's RNA transcripts before they are translated into working proteins.
Interception and Destruction of Messages
When a gene that code for a protein is active in a cell, its information is transcribed from DNA form into lengths of nucleic acid called messenger RNA. If all goes well, these coded mRNA signals make their way to the cell's protein factories, which use them as templates to synthesize new proteins. RNA-silencing, also called RNA-Interference is the interception and destruction of these messages and as such, is a powerful and specific regulator of cell activity, as well as a strong defender against viral genes.
The silencing process requires not only an Argonaute protein but also a small length of guide RNA, known as a short-interfering RNA or microRNA. The guide RNA fits into a slot on Argonaute and serves as a target recognition device. Like a coded strip of Velero(TM), it latches onto a specific mRNA target whose sequence is the chemical mirror image of its own- thus bringing Argonaute into contact with its doomed prey.
Argonaute2 is not the only type of human Argonaute protein, but it seems to be the only one capable fo destroying target RNA directly. "If the guide RNA is completely complementary to the target RNA, Argonaute2 will cleave the mRNA, and that will elicit the degradation of the fragments and the loss of the genetic message," said Nicole Schirle, the graduate student in MacRae's laboratory who was lead author of the paper.
Aimed at disease-causing genes or even a cell's own overactive guide RNAs, RNA-silencing could be a powerful therapeutic weapon. In principle, one needs only to inject target-specific guide RNAs, and these will link up with Argonaute protein in cells to find and destroy the target RNAs. Scientists have managed to do this successfully with relatively accessible target cells, such as in the eye. But they have found it difficult to develop guide RNAs that can get from the bloodstream into distant tissues and still function.
"You have to modify the guide RNA, in some way to get it through the blood and into cells, but as soon as you start modifying it, you disrupt its ability to interact with Argonaute,"said MacRae. Knowing the precise structure of Argonaute should enable researchers to clear this hurdle by designing better guide RNA.
More Points for Manipulation
Previous structural studies have focused mostly on Argonaute protein from bacteria and other lower organisms, which have key differences from their human counterparts. Schirle was able to produce the comparatively large and complex human Argonaute2 and to manipulate it into forming crystals for X-ray crystallography analysis a feat that structural biologists have wanted to achieve for much of the past decade. "It was just excellent and diligent crystallography on her part," said MacRae.
The team's analysis of Argonaute2's structure revealed that it has the same basic set of working parts as bacterial Argonaute proteins, except that they are arranged somewhat differently. Also, key parts of Argonaute2 have extra loops and other structures, not seen on bacterial versions, which may play roles in binding to guide RNA. Finally, Argonatue2 has what appear to be binding sites for additional co-factor proteins that are thought to perform other destructive operations on the target mRNA.
"Basically, this Argonaute protein is more sophisticated than its bacterial cousins; it has more bells and whistles, which give us more points for manipulation. With this structure solved, we no longer need to use the prokaryotic structures to guess at what human Argonaute protein look like," MacRae said.
He and Schirle and others in the lab now are analyzing the functions of Argonaute2's substructures, as well as looking for ways to design better therapeutic guide RNAs.
"Now with the structural data, we can see what synthetic guide RNAs will work with Argonaute and what won't, " MacRae said, "we might even be able to make guide RNAs that can outcompete natural ones."
The research that led to Schirle and MacRae's new paper, "The Crystal Structure of Human Argonaute2," was funded by the National Institute of General Medical Sciences, part of the National Institute of Health.
press@scripps.edu
Nitric Oxide modulates bacterial biofilm formation
Scripps Research Institutes Scientists Solve a Mystery of Bacterial Growth and Resistance
Findings Shed Light on How Bacteria Form Protective Biofilms
LA JOLLA, CA, April 26,2012:-
Scientists at The Scripps Research Institute have unraveled a complex chemical pathway that enables bacteria to form clusters called biofilms. Such improved understanding might eventually aid the development of new treatments targeting biofilms, which are involved in a wide variety of human infections and help bacteria resist antibiotics.
The report, published online ahead of print on April 26,2012, by the journal Molecular Cell, explains how nitric oxide, a signaling molecule involved in the immue system, leads to biofilm formation.
"It is estimated that about 80 percent of human pathogens form biofilms during some part of their life cycle," said Scripps Research President and CEO Michael Marletta, PhD, who lead the work. "In this study, we have detailed for the first time the signaling pathway from nitric oxide to the sensor through cellular regulators and on to the biological output, biofilm formation."
"There's a lot of interest right now in finding ways to influence biofilm formation in bacteria", said lead author Lars Plate, a graduate student in Marletta's team, which recently moved to Scripps Research from the University of California, Berkeley. "Figuring out the signaling pathway is a prerequisite for that."
Dangerous Get Togethers
Biofilm formation is a critical phenomenon that occurs when bacterial cells adhere to each other and to surfaces, at times as part of their growth stage and at other time to gird against attack. In such aggregations, cells on the outside of a biofilm might still be susceptible to natural or pharmaceutical antibiotics, but the interior cells are relatively protected. This can make them difficult to kill using conventional treatments.
Biofilms can form on surgical instruments such as heart valves or catheters, leading to potentially deadly infections. Likewise, difficult-to-eliminate biofilms also play key roles in a host of conditions from gum disease to cholera, and from cystic fibrosis to Legionnaires disease.
For years, the Marletta lab and other groups have been studying how nitric oxide regulates everything from blood vessel dilation to nerve signals in humans and other vertebrates. Past research had also revealed that nitric oxide is involved in influencing bacterial biofilm formation.
Nitric oxide in sufficient quantity is toxic to bacteria, so it's logical that nitric oxide would trigger bacteria to enter the safety huddle of a biofilm. But nobody knew precisely how.
In the new study, the scientists set out to find what happens after the nitric oxide trigger is pulled. "The whole project was really a detective story in a way," said Plate.
The Detective Story
In vertebrates, nitric oxide can bind to something called the Heme-Nitric Oxide/Oxygen(H-NOX) binding domain on a specific enzyme, activating that enzyme and beginning the chemical cascades that lead to physiological functions such as blood vessel dilation.
Many bacteria also have H-NOX domains, including key pathogens, so this seemed the best starting point for the investigation. From there, the team turned to genomic data.
Genes for proteins that interact are often found adjacent to one another. Based on this fact, the researchers were able to infer a connection between the bacterial H-NOX domain and an enzyme called Histidine Kinase, which transfer phosphate chemical groups to other molecules in signaling pathways. The question was where the phosphate were going.
To learn more, the researchers used a technique called Phosphotrasfer profiling. This involved activating the histidine kinase and then allowing them to react separately with about 20 potential tragets. Those targets that the histidine kinase rapidly transferred phosphates to had to be part of the signaling pathway. "It's a neat method that we used to get an answer that was in fact very surprising," said Plate.
The experiments revealed that the histidine kinase phosphorylated three proteins called response regulators that work together to control biofilm formation for the project a primary study species, the bacterium Shewanells oneidensis, which is found in lake sediments.
Further work showed that each regulator plays a complementary role, making for an unusually complex system. One regulator activates gene expression, another controls the activity of an enzyme producing cyclic di guanosine monophosphate, an important bacterial messenger molecule that is critical in biofilm formation, and the third tunes the degree of activity of the second.
Divide and Conquer
Since other bacterial species use the same chemical pathway uncovered in this study, the finidings pave the way to further explore the potential for pharmaceutical application. As one example, researchers might be able to block biofilm formation with chemicals that interrupt the activity of one of the components of this nitric oxide cascade.
Marletta's group has already explored nitric oxide's role in controlling Legionnaires disease and, among other goals, will focus now on understanding biofilm formation in the bacterium that causes cholera.
The work for the paper, " Nitric Oxide modulates bacterial biofilm formation through a multi-component cyclic-di-GMP signaling network," was supported by the National Institutes of Health and a Chang-Lin Tien Graduate Fellowship in the Environmental Sciences.
press@scripps.edu
Findings Shed Light on How Bacteria Form Protective Biofilms
LA JOLLA, CA, April 26,2012:-
Scientists at The Scripps Research Institute have unraveled a complex chemical pathway that enables bacteria to form clusters called biofilms. Such improved understanding might eventually aid the development of new treatments targeting biofilms, which are involved in a wide variety of human infections and help bacteria resist antibiotics.
The report, published online ahead of print on April 26,2012, by the journal Molecular Cell, explains how nitric oxide, a signaling molecule involved in the immue system, leads to biofilm formation.
"It is estimated that about 80 percent of human pathogens form biofilms during some part of their life cycle," said Scripps Research President and CEO Michael Marletta, PhD, who lead the work. "In this study, we have detailed for the first time the signaling pathway from nitric oxide to the sensor through cellular regulators and on to the biological output, biofilm formation."
"There's a lot of interest right now in finding ways to influence biofilm formation in bacteria", said lead author Lars Plate, a graduate student in Marletta's team, which recently moved to Scripps Research from the University of California, Berkeley. "Figuring out the signaling pathway is a prerequisite for that."
Dangerous Get Togethers
Biofilm formation is a critical phenomenon that occurs when bacterial cells adhere to each other and to surfaces, at times as part of their growth stage and at other time to gird against attack. In such aggregations, cells on the outside of a biofilm might still be susceptible to natural or pharmaceutical antibiotics, but the interior cells are relatively protected. This can make them difficult to kill using conventional treatments.
Biofilms can form on surgical instruments such as heart valves or catheters, leading to potentially deadly infections. Likewise, difficult-to-eliminate biofilms also play key roles in a host of conditions from gum disease to cholera, and from cystic fibrosis to Legionnaires disease.
For years, the Marletta lab and other groups have been studying how nitric oxide regulates everything from blood vessel dilation to nerve signals in humans and other vertebrates. Past research had also revealed that nitric oxide is involved in influencing bacterial biofilm formation.
Nitric oxide in sufficient quantity is toxic to bacteria, so it's logical that nitric oxide would trigger bacteria to enter the safety huddle of a biofilm. But nobody knew precisely how.
In the new study, the scientists set out to find what happens after the nitric oxide trigger is pulled. "The whole project was really a detective story in a way," said Plate.
The Detective Story
In vertebrates, nitric oxide can bind to something called the Heme-Nitric Oxide/Oxygen(H-NOX) binding domain on a specific enzyme, activating that enzyme and beginning the chemical cascades that lead to physiological functions such as blood vessel dilation.
Many bacteria also have H-NOX domains, including key pathogens, so this seemed the best starting point for the investigation. From there, the team turned to genomic data.
Genes for proteins that interact are often found adjacent to one another. Based on this fact, the researchers were able to infer a connection between the bacterial H-NOX domain and an enzyme called Histidine Kinase, which transfer phosphate chemical groups to other molecules in signaling pathways. The question was where the phosphate were going.
To learn more, the researchers used a technique called Phosphotrasfer profiling. This involved activating the histidine kinase and then allowing them to react separately with about 20 potential tragets. Those targets that the histidine kinase rapidly transferred phosphates to had to be part of the signaling pathway. "It's a neat method that we used to get an answer that was in fact very surprising," said Plate.
The experiments revealed that the histidine kinase phosphorylated three proteins called response regulators that work together to control biofilm formation for the project a primary study species, the bacterium Shewanells oneidensis, which is found in lake sediments.
Further work showed that each regulator plays a complementary role, making for an unusually complex system. One regulator activates gene expression, another controls the activity of an enzyme producing cyclic di guanosine monophosphate, an important bacterial messenger molecule that is critical in biofilm formation, and the third tunes the degree of activity of the second.
Divide and Conquer
Since other bacterial species use the same chemical pathway uncovered in this study, the finidings pave the way to further explore the potential for pharmaceutical application. As one example, researchers might be able to block biofilm formation with chemicals that interrupt the activity of one of the components of this nitric oxide cascade.
Marletta's group has already explored nitric oxide's role in controlling Legionnaires disease and, among other goals, will focus now on understanding biofilm formation in the bacterium that causes cholera.
The work for the paper, " Nitric Oxide modulates bacterial biofilm formation through a multi-component cyclic-di-GMP signaling network," was supported by the National Institutes of Health and a Chang-Lin Tien Graduate Fellowship in the Environmental Sciences.
press@scripps.edu
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