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Protein Malnutrition

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

A large part of  the world’s population is undernourished by the standards of Western Europe and North America. Scientists and nonscientists alike recognize as one of the major challenges of our time the problem of how to ensure that the production and distribution of food keep pace with the increasing number of mouths to be fed. In the world as a whole the most widespread and serious dietary deficiency is that of protein. This fact emerges clearly from the reports of the expert committees of WHO and FAO (World Health Organization, 1951, 1953). Nevertheless, many protein chemists, even those associated with medical research, may not realize the extent and severity of protein malnutrition, because it occurs chiefly in the technically underdeveloped countries far from where they work.

Dietary histories and response to treatment point to deficiency of total protein as the primary cause of the clinical syndrome kwashiorkor. The level of calorie intake has an important influence on the pattern of the disease. Deficiency of one or more specific amino acids, or amino acid imbalances in the diet, may perhaps be responsible for some of the symptoms and signs, particularly those whose incidence varies from one part of the world to another. All these variations on a theme are covered by the general term protein malnutrition. The onset is often precipitated by the added burden of diarrhea, infection, and parasitic infestation. The nutritional state influences the resistance to infection, and conversely the presence of an infection affects the state of nutrition. A further contributory factor may be the psychological upheaval in the child when the next baby in the family is born. At the root of all these causes lie poverty, ignorance, and disruption of the family life.

The planning of preventive measures cannot be effective unless it is based on some knowledge of the magnitude of the problem to be tackled. At a very rough estimate, in some countries perhaps 10% of the children suffer from severe protein malnutrition at some age between birth and 4 years. The marginal deficiency states must be much more common, Clinical signs and biochemical changes are of little value in diagnosing the early case; a deficit in body weight still seems to be the best criterion. Prevention ideally would be by greater production and consumption of animal protein, and by the increased use of skim milk and of surplus fish at present often wasted. However, animal protein is likely to remain scarce and expensive. Plant sources are being investigated with a view to encouraging not only domestic production, but also the production on an industrial scale of cheap foodstuffs rich in protein. A preventive program that is nutritionally sound may fail if account is not taken of local food habits, traditions, and customs. Protein requirements are affected by the quality of protein, the intake of calories, and by the state of the body (growth, the presence of disease, etc.). The maintenance requirement and the amount required for growth in children can be estimated, but the requirement for health is still unknown. For the time being, the allowances of protein recommended for people in the world as a whole are based empirically on the known physiological requirement with an arbitrarily added wide margin of safety.

The absorption of nitrogen is remarkably efficient even in severely malnourished infants. In general the nitrogen of plant proteins is less well absorbed than that of milk. When a baby receives a diet in which the protein is derived entirely from vegetabIe sources, incomplete absorption of nitrogen may play a significant part in the production of protein malnutrition. The malnourished baby who responds to treatment is able to retain and utilize nitrogen very efficiently; there is no evidence of any impairment in the mechanisms of protein synthesis. It is possible, however, that these mechanisms may be irreversibly damaged in babies who die, and that this may be the cause of death. The level of calorie intake has an important influence on the efficiency of utilization of nitrogen. An adequate calorie intake promotes conservation of nitrogen in the body as a whole when supplies of protein are short, but this protective effect may not be exerted equally in all organs. In this way the level of calorie intake may modify the pattern of protein depletion. A greater than normal calorie intake is needed for the restoration of depleted protein stores.

The discussion of protein metabolism in protein malnutrition has been purposely limited to a narrow field-to studies made on man, and to the few animal experiments that have a direct bearing on those studies. For technical reasons most of the work discussed relates to plasma proteins. There is a conflict of evidence between results obtained in man and animals about the effect of protein depletion or a low protein diet on the rate of catabolism of plasma albumin. It is of great importance to settle this point. A priori there seems no reason why the rate of protein catabolism should be affected by nutritional state. Preliminary studies with radioactive methionine in infants suggest, as working hypotheses, that in protein malnutrition there may be an increase in the reutilization of amino acids liberated by tissue catabolism, and an apparent concentration of protein synthesis in the more essential organs at the expense of the less essential. There is some experimental support for both these ideas, but further work is badly needed. The concept of protein stores or reserve protein is based entirely on dynamic and not on chemical considerations. It is suggested that the essential difference between a “labile” and a “fixed” protein is a difference in turnover rate. An attempt is made to show that the changes produced by protein depletion in the protein content of organs such as liver and muscle are a necessary consequence of the metabolic characteristics of proteins in those organs. There may be no need to invoke the help of homeostatic or compensatory regulations to explain the changes found in protein depletion.

Aging and growth are processes during which some metabolic adjustments must take place. It is believed that it may be better to regard the changes which are found in protein malnutrition in a similar light: as evidence of an alteration in functional pattern, rather than of damage or disease. Protein malnutrition in man has two aspects-a practical and a theoretical one. From the practical point of view it is an extremely common disease with a high mortality, and there is every reason to believe that it will become more common unless urgent preventive measures are taken. Theoretically it raises many questions that are of interest in relation to other branches of medicine and biochemistry. It is believed that the two aspects are linked, and that progress towards prevention is still impeded by our lack of basic knowledge as well as by our failure to apply what is already known. In protein malnutrition there is no sharp line between health and disease. The simple concept of specific deficiency diseases that grew from the discovery of vitamins is not applicable. We have to go back instead to the ideas of an earlier era, when nutrition was regarded as a branch of physiology, concerned with the functions, fate, and metabolic interrelationships of the major nutrients.

It is a characteristic of protein metabolism that nitrogen balance can be maintained at many different levels of protein intake. These different steady states are achieved by adjustments of the amount and distribution of proteins in the body as a whole, in organs, and in cells. It is believed that these changes in amount and distribution of proteins must result in alterations of metabolic pattern, with a gradation of change from an optimum, which cannot be defined, to a state of irreversible breakdown incompatible with life. In the intermediate stages function is modified and efficiency perhaps impaired. It seems possible that variations in diet, and particularly in the amount and quality of the protein, may underlie many of the differences in incidence and symptomatology of disease which are gradually being uncovered in different parts of the world.

Source References:

http://www.sciencedirect.com/science/article/pii/S0065323308603095#

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A Protease for ‘Middle-down’ Proteomics

Author and Reporter: Ritu Saxena, Ph.D.

Neil Kelleher and his research team at Northwestern University have developed a method for enzymatic proteolysis large peptides for mass spectrometry–based proteomics using a protease OmpT. The method was published in a recent issue of the journal Nature. http://www.ncbi.nlm.nih.gov/pubmed/22706673

Proteomics is defined as the study of the structure and function of proteins. Proteomic technologies will play an important role in drug discovery, diagnostics and molecular medicine because is the link between genes, proteins and disease. As researchers study defective proteins that cause particular diseases, their findings will help develop new drugs that either alter the shape of a defective protein or mimic a missing one. http://www.ama-assn.org/ama/pub/physician-resources/medical-science/genetics-molecular-medicine/current-topics/proteomics.page Proteomics, although refers to the study of the structure and function of proteins, it is often specifically used for protein purification and mass spectrometry.

‘Bottom-up’ and ‘Top-down’ are the two main strategies for proteomic studies using mass spectrometry. In Bottom-up proteomics referred to as the more common method, proteins are broken down into smaller pieces through enzymatic digestion followed by characterization into amino acid sequences and post translational modifications prior to analysis by mass spectrometry. By identifying and sequencing these smaller pieces, researchers can then determine the identity of the protein they make up. In Top-down proteomics, on the other hand, the process of proteolysis is skipped and it focuses on complete characterization of intact proteins and their post-translational modifications (PTMs).

“Although both the top-down and bottom-up approaches continue to mature, they each have limitations. The tryptic peptides used in the bottom-up approach are the primary unit of measurement, but their relatively small size (typically ~8–25 residues long) leads to problems such as sample complex­ity, difficulties in assigning peptides to specific gene products rather than protein groups, and loss of single and combinato­rial PTM information. The top-down approach handles these issues by characterizing intact proteins, but its success declines in the high-mass region. Therefore, a hybrid approach based on 2–20 kDa peptides could unite positive aspects of both bottom-up and top-down proteomics” says Kelleher et al in the research article.

The hybrid approach, referred to as ‘middle-down’ proteomics would enable the analysis of complex mixtures pre-sorted by protein size. Previously research efforts ‘middle-down’ proteomics included exploring the restricted proteolysis with enzyme alternatives to Trypsin and chemical methods (such as microwave-assisted acid hydrolysis), However, these methods generated peptides that were marginally longer than those produced by trypsin digestion. For the current study, Kelleher adds “We established an OmpT-based middle-down platform to analyze complex mixtures pre-sorted by protein size. After inte­grating the data from the middle-down workflow that was applied to ~20–100-kDa proteins fractionated from the HeLa cell proteome, we identified 3,697 unique peptides (average size: 6.3 kDa) from 1,038 unique proteins (26% average sequence coverage) at an esti­mated 1% false discovery rate”.

OmpT, a protease derived from Escherichia coli K12 outer membrane belongs to the novel omptin protease family10 and is known to cleave between two consecutive basic amino acid residues (Lys/Arg-Lys/Arg). The authors developed OmpT into an efficient rea­gent to generate >2-kDa peptides for middle-down proteomics, thus, utilizing OmpT to achieve robust, yet restricted, proteolysis of a complex genome. http://www.ncbi.nlm.nih.gov/pubmed/22706673

Researcher Kelleher and his team have been in news earlier for their work on ‘top-down’ proteomics when his team developed a new method that could separate and identify thousands of protein molecules quickly. In the first large-scale demonstration of the top-down method, the researchers were able to identify more than 3,000 protein forms created from 1,043 genes from human HeLa cells. The study was published in last year in the October issue of the journal Nature. http://www.ncbi.nlm.nih.gov/pubmed?term=22037311

Thus, Kelleher and his group was able to demonstrate that OmpT-based proteomic approach has a robust and restricted proteolysis capacity making it an attractive option for mass-spectrometry-based analysis of primary structure of protein.

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Reporter: Prabodh Kandala, PhD

Scientists at The Scripps Research Institute have found the first chemical compounds that act to block an enzyme that has been linked to inflammatory conditions such as asthma and arthritis, as well as some inflammation-promoted cancers.

The new study, published recently by the journal ACS Chemical Biology, describes new compounds that inhibit an important enzyme called PRMT1 (protein arginine methyltransferase 1). The new inhibitors will be useful to scientists who study PRMT1-related biological pathways in cells and who are developing drug treatments for PRMT1-related inflammatory conditions and cancers.

Standard screening techniques had been unable to distinguish between compounds that inhibit PRMT1 and those that inhibit other common PRMT family enzymes. In the new study, scientists from several Scripps Research laboratories collaborated to devise the first PRMT1-specific screening technique. “We were able to target a screening probe to a specific amino acid found on PRMT1, but not on most other PRMT enzymes,” said the study’s principal investigator Scripps Research Assistant Professor Kerri A. Mowen.

Mowen has been studying PRMT1 since her graduate school days, and, like others in the field, has been acutely aware of the need for selective PRMT1 inhibitors. The enzyme modifies the functionality of proteins by attaching a methyl group to their arginine amino acids; as such, it is involved in some of the most basic processes in cells. For example, Mowen and her colleagues showed in 2004 that PRMT1 helps drive the production of the key immune-stimulating proteins interferon-gamma and interleukin-4.

But although PRMT1 was known to be responsible for nearly all the arginine methylation that goes in mammalian cells, no one had been able to develop selective PRMT1 inhibitors, since the 10 other PRMT enzymes are nearly identical, structurally and biochemically. Even the removal of PRMT1’s gene from lab mice as an alternative way to study its functions was problematic, since mouse embryos can’t survive without the protein.

Inspiration Close By

The inspiration for the new PRMT1-selective screening technique came from research performed in the neighboring laboratory of co-author Benjamin F. Cravatt III, who chairs the Scripps Research Department of Chemical Physiology. As reported in 2010 in the journal Nature, Cravatt and his team screened tens of thousands of human and mouse proteins for the presence of hyper-reactive cysteine amino acids, which almost certainly mark functional sites on those proteins. PRMT1 was found to be one of the reactive-cysteine-containing proteins — and all but one other, comparatively rare PRMT enzyme was known to lack that cysteine.

“We took that discovery a step further, and we were able to find a probe that specifically would recognize that cysteine in PRMT1,” said Mowen, who was one of Cravatt’s collaborators on the 2010 study.

She and her colleagues first verified that the reactive cysteine in PRMT1 is in the active site of the enzyme. They then found a fluorescent probe that would bind to that cysteine. If a test compound acted as an inhibitor by fastening to PRMT1’s active site, it should interfere with the probe’s binding, and the probe’s fluorescence-based signal therefore should be lower. By contrast, if a test compound failed to bind to PRMT1’s active site, the probe should bind normally and its signal should remain elevated.

“We were able to verify, using available non-specific inhibitors of PRMT enzymes, that they did indeed bind to PRMT1 and prevent the probe from binding, and that was the proof-of-concept that enabled us to go ahead with a screen,” said Myles B. C. Dillon, a graduate student in Mowen’s lab who was lead author of the study.

Exploring Libraries of Potential

Dillon and Mowen turned to collaborator Scripps Research Professor Hugh Rosen, curator of a library of 16,000 chemical compounds known as the Maybridge Hitfinder Collection. By applying these compounds, one by one, along with the probe molecule, to solutions of PRMT1, the team was able to determine the compounds’ abilities to bind PRMT1’s active site and thus act as inhibitors. Importantly, the setup was simple enough to be adapted, with Rosen’s help, as an automated, “high-throughput” technique, capable of screening thousands of compounds.

In this way, the scientists were able to sift through the compound library to find two candidate PRMT1-selective inhibitors. “They have good efficacy and specificity, and we might be able to modify them to make them even better,” said Dillon.

Mowen, Dillon, and their colleagues now have a National Institutes of Health (NIH) grant to use their screening technique with a 300,000-compound NIH library, also curated at Scripps Research. “Once we get the results from this larger screen, we’ll consider our best inhibitor compounds and decide which ones to start optimizing,” said Dillon.

To Mowen, the success of the project owes much to the collaborative spirit at Scripps Research. “Many labs here are developing cutting-edge technologies that empower other labs’ work, and certainly we were able to benefit from that,” she said. “It’s a very supportive, synergistic environment.”

http://www.sciencedaily.com/releases/2012/05/120511104815.htm

Journal reference: Myles B. C. Dillon, Daniel A. Bachovchin, Steven J. Brown, M. G. Finn, Hugh Rosen, Benjamin F. Cravatt, Kerri A. Mowen. Novel Inhibitors for PRMT1 Discovered by High-Throughput Screening Using Activity-Based Fluorescence PolarizationACS Chemical Biology, 2012; : 120420132930009 DOI: 10.1021/cb300024c

 

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