Orphan receptors

Orphan receptors are receptors whose natural ligands have not been discovered. Ligands of many receptors which were once considered orphan have now been discovered. Many of these "adopted receptors" have been shown to play critical roles in the cell cycle, metabolism etc., and are often important drug targets.

Traditionally, receptors were discovered when unknown proteins binding to known ligands were identified. However, molecular techniques have now made it possible to identify proteins with unknown function as potential receptors based on their "similarity" to known receptors. This "similarity" may be based on amino acid sequence or on common domains shared by these proteins with known receptors.

All steroid hormones, vitamin D, retinoids, thyroid hormones etc. produce their effects by binding to intracellular receptors. Some receptors are in the cytosol (eg. glucocorticoid receptors), while others are in the nucleus (eg. thyroxine receptor). The ligand (the hormone) diffuses through the cell membrane and binds to its receptor. Once bound, these receptors can bind to specific areas of the DNA, resulting in an increase or decrease the expression of specific genes to which they can bind. Therefore, the receptors of these hormones act as transcription factors. These transcription factors bind to DNA via special domains called DNA binding domains. There are many types of characteristic DNA binding domains which are called zinc finger, leucine zipper, helix-turn-helix, helix-loop-helix etc. Therefore the presence of one of these DNA binding domains is a unique feature of receptors that act as transcription factors.

Molecular biologists use this knowledge to find new intracellular receptors in the cell. Following the completion of the human genome project, they have at their disposal, the sequence of the entire DNA in the human genome. These scientists scan this information to look for proteins that have the characteristic DNA domains. All proteins that are thus identified are potential receptors that act as transcription factors. This approach has been very productive and many previously unknown receptors with critical functions have been identified. A few receptors identified using this technique are PPAR (paroxisome proliferator activated receptor), LXR (liver X receptor), FXR (farnasoid X receptor) and RXR (retinoid X receptor). Many of these receptors had no known function or ligand when they were first discovered. Further research has identified their natural ligands and they have been shown to play fundamental roles in cell functioning. For example, the PPARs have been shown to be important regulators of carbohydrate and lipid metabolism in the cell. Thioglitazones are a group of anti-diabetic drugs that act by binding to the PPAR receptors. In the paper chosen for "Research Article of the Week", the LXR has now been shown to play an important role in mediating glucocorticoid effects in the cell.

Many more orphan receptors are likely to be shown to have important roles in the cell in the near future. Research in this area is particularly interesting because a number of these receptors can be targets for drugs. The PPARs are classical examples for this.

Designing newer and safter glucocorticoids

Glucocorticoids are used therapeutically for their anti-inflammatory and immunosuppressive properties. However, long-term glucocorticoid treatment is complicated by a number of side effects, mainly metabolic. It is known to exacerbate insulin resistance and worsen glycemic control in diabetics, accelerate osteoporosis and muscle wasting and cause fatty liver. Therefore glucocorticoid analogues that do not cause metabolic abnormalities, but retain their anti-inflammatory and immunosuppressive properties have long been sought by clinicians.

The authors of the paper, led by Carolyn Cummins of the University of Toranto in Canada, appear to have taken the first step towards achieving this goal. They have shown that a transcription factor called Liver X receptor (LXR) is required for dexamethasone (a synthetic analogue of glucocorticoids)-induced gluconeogenesis and hepatosteatosis. However, in LXR - beta knock-out mice, dexamethasone retained its immunosuppressive and anti-inflammatory effects.This showed that if scientists design glucocorticoid analogue which do not interact with LXR, such analogues may be anti-inflammatory and immunosuppressive without inducing metabolic side-effects.

It is known that glucocorticoids act by binding to cytoplasmic receptors (glucorticoid receptors) which translocate to the nucleus to induce or repress selective genes. LXRs are also a group of such receptors which can bind to selective genes and turn it on (induce) or turn it off (repress). However, the nature of glucocorticoid receptor interaction with LXR is unknown and future studies will be aimed at elucidating this.

Probiotics: disease preventing bacteria!!

Probiotic Effects on Cold and Influenza-Like Symptom Incidence and Duration in Children
Gregory J. Leyer, Shuguang Li, Mohamed E. Mubasher, Cheryl Reifer, and Arthur C. Ouwehand
Pediatrics 2009; 124: e172-e179.

http://pediatrics.aappublications.org/cgi/content/abstract/124/2/e172

In a double-blind, placebo controlled study done on 326 Chinese children aged 3 to 5 years, researches have found that twice daily administration of probiotics resulted in a marked reduction in incidence of cold and influenza-like symptoms.

The children who participated in this study were divided into three groups. One group received milk that contained Lactobacillus acidophilus (Lactobacillus group), the other received milk containing a combination of Lactobacillus acidophilus and Bifidobacterium animalis (Lactobacillus/Bifidobacterium group) and a third group received plain milk (placebo group). Each group had approximately 100 children. Milk was administered twice daily by school authorities during weekdays and at home during weekends for a period of 6 months. The children were closely monitored during this period and some of the important observations were as follows:

1. Compared to the placebo group, the Lactobacillus group had 53 percent fewer episodes of fevers, 41 percent fewer episodes of cough, and 28 percent fewer episodes of rhinorrhoea (runny nose).

2. The Lactobacillus/Bifidobacterium group had even larger reductions in symptom rates: 72 percent fewer fevers, 62 percent fewer coughs, and 59 percent fewer runny noses.

3. Duration of illnesses when they did occur in children receiving probiotics was significantly lower than the control group. Compared to placebo, the length of illness was decreased by 32 percent with Lactobacillus and by 48 percent with Lactobacillus/Bifidobacterium.

4. Antibiotic use was 68 percent lower in the Lactobacillus group and 84 percent lower in the Lactobacillus/Bifidobacterium group, compared to the placebo group.

5. Children who received the probiotics were absent from day care 28 to 32 percent less often than children in the placebo group.

Earlier studies had shown that probiotics were able to boost the immune response to certain types of bacterial and viral illnesses. However, this is the first time that probiotic use has been convincingly shown to prevent illnesses.

Probiotics are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host. They are usually given as dietary supplements. Lactobacillus and Bifidobacterium are the most commonly used probiotics.

A large amount of research is being done to explore the potential of probiotics in the prevention and treatment of a wide variety of clinical conditions. Some indications for probiotics have sound scientific basis, while others are still in the realms of speculation. Some of the approved indications for probiotics are:

1. Managing lactose intolerance

2. Prevention of colon cancer

3. Lowering cholesterol

4. Lowering blood pressure

5. Improving immune function and preventing infections

6. Helicobacter pylori induced peptic ulcers

7. Antibiotic-associated diarrhea

8. Reducing inflammation

9. Improving mineral absorption

10. Prevents harmful bacterial growth under stress

11. Irritable bowel syndrome and colitis

It was the Russian scientist Eli Metchnikoff who initially introduced the idea of supplementing the diet with friendly bacteria. Metchnikoff, while working at the Pasteur Institute in Paris, noticed that rural people in Bulgaria and other places in Europe, whose diet was rich in “milk fermented with lactic acid bacillus” were exceptionally long lived. He proposed that the lactic acid bacteria replaced harmful proteolytic bacteria (like Clostredia) in the gut. The proteolyic bacteria could produce toxic substances by digesting protein and these toxic substances eventually lead to age related changes and senility. This theory was called the “auto intoxication” theory of Metchnikoff. Metchnikoff himself started supplementing his diet with lactic acid bacilli called the “Bulgerian bacillus” and his health apparently improved. The practice soon caught on and patients were routinely put on a sour milk diet by physicians in Paris.

Five years after Metchnikoff death in 1915, it was demonstrated by Rettger that Metchnikoff’s “Bulgarian bacillus” was not capable of colonizing the human gut. After this discovery, Metchnikoff theory of longevity through consumption of lactic acid bacteria was questioned and soon became unpopular. However, other bacteria with health benefits were being discovered in the meantime. Henry Tissier (also of the Pasteur Institute) discovered the Bifidobacterium and advocated the use of this bacterium in the treatment of childhood diarrhea. A German doctor, Alfred Nissle isolated strains of E. coli that were able to prevent salmonellosis and shigellosis.

Since these discoveries, many more bacteria have been added to the probiotic list. Each bacterium has its own unique mechanisms of producing beneficial effects and mechanisms elucidated in one strain cannot be extrapolated to other probiotics even if they are closely related bacteria.

Recently there have been certain controversies regarding the use of probiotics. In a clinical trial conducted at the University of Western Australia aimed at showing the effectiveness of probiotics in reducing childhood allergies, 178 children were given either a probiotic or a placebo for the first six months of their life. Those given the good bacteria were more, not less, likely to develop a sensitivity to allergens.

In another instance, a double-blind, placebo-controlled therapeutic study on the effects of a probiotic cocktail on pancreatitis at University Medical Center Utrecht (UMC) showed that 24 out of 296 patients died between 2004 and 2007, with more deaths among those receiving the probiotic cocktail.

However, there have also been well designed studies that have shown beneficial effects of probiotics. The study that is described in this post is one such example. Clearly this is not the last we will hear of probiotics and their therapeutic benefits.

Acknowledgements:
Wikipedia article on Probiotics.

Eat less, live long?

Caloric restriction delays disease onset and mortality in rhesus monkeys.

Colman RJ, Anderson RM, Johnson SC, Kastman EK, Kosmatka KJ, Beasley TM, Allison DB, Cruzen C, Simmons HA, Kemnitz JW, Weindruch R.

Science. 2009 Jul 10;325(5937):201-4.

Caloric restriction has been proposed to increase the lifespan of a number of animals. However, this is for the first time that the benefits of calorie restriction have been shown in a primate. Preliminary results of study started in 1989 (and extended in 1994)on 76 Rhesus monkeys have shown that a 30% calorie restricted diet had many beneficial effects, including an increased disease-free lifespan and delayed onset of senility-associated diseases like diabetes mellitus, cardiovascular diseases, cancer and brain atrophy.

The authors claim that calorie-restricted monkeys "look" younger than their counterparts who were given a free rein at feeding time. In addition they show that there was significantly less mortality due to senility-associated diseases in the calorie restricted animals. These animals were also less prone to developing diabetes and colon adenocarcinoma which is known to be common in aged Rhesus monkeys. Functional MRI images of the brain showed that areas of the brain related to movement and memory were protected from age-related atrophic changes.

This study is interesting because this is probably as near as we can get to doing a large-scale experiment on caloric restriction in a species that is a close relative of man. Although a few descriptive studies in humans do suggest the beneficial effects of a calorie restricted diet in the elderly, ethical considerations virtually preclude a randomized controlled trial of the nature described in this study. The authors say that the study is on-going and the results at the conclusion of the study will reveal more interesting facts.

There have been a number of theories that have tried to explain the apparent ability of calorie restriction to extend life span. A few of the current theories are given below.

1. The "hormesis" theory
The term "hormesis" refers to a wide variety of phenomena where intentional application of a mild stress on an organism elicits a response that enables it to withstand a much stronger stress to which it may be subjected to in future. This kind of response is analogous to the effects of vaccination with a live, attenuated pathogen on the immune system.

I has been shown that when Caenorhabditis elegans is subjected to restriction of glucose availability for metabolism, it induces a state of mild oxidative stress. This elicits a robust antioxidant response in the organism which ultimately resulted in an increased resistance to further oxidative stress. This was probably the first experimental evidence for hormesis being the reason for extended life span following CR.

Read article

2. Sir2/SIRT1 and resveratrol
Sir2 or "silent information regulator 2" is a longevity gene, discovered in baker's yeast cells, that extends lifespan by suppressing DNA instability. In mammals Sir2 is known as SIRT1. Recent discoveries have suggested that the gene Sir2 might underlie the effect of CR. David Sinclair at Harvard Medical School, Boston, showed that in mammals the SIRT1 gene is turned on by a CR diet, and this protects cells from dying under stress.

Read article

Sirtris Pharmaceuticals, Inc., a GlaxoSmithKline-owed biotechnology company based in Cambridge, MA co-founded by Sinclair, is developing resvertrol and other SIRT1 activators for human use. Because life-span extension is not an FDA-approvable indication, the company is developing SIRT1 activators for the treatment of diseases associated with aging including type 2 diabetes and cancer.

3. Free radicals and glycation

A calorie restricted diet provides lesser amounts of energy, thus decreasing the flux through the mitochondrial electron transport chain (ETC). It is known that the ETC is "leaky" and 1-2% of oxygen normally gets converted to superoxide, a free radical that can cause oxidative tissue damage (oxidative stress). Decreased calorie intake can therefore lead to reduced oxidative stress.

Reduced caloric intake will also result in reduced mean glucose levels in blood. It is known that glucose can form adducts with a variety of macromolecules by a non-enzymatic, spontaneous process called "glycation". Glycation of proteins is one of the major mechanisms of pathogenesis of long-term diabetes mellitus. A reduced mean glucose levels would result in decreased glycation and tissue abnormalities associated with protein glycation.

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4. Longevity and subclinical hypothyroidism

A recent study in Ashkenazi Jews has shown that a low thyroid activity as evidenced by moderately elevated TSH levels is associated with extreme longevity. The authors suggest that lowered metabolism associated with low thyroid hormone levels is probably responsible for this effect. They cite the example of the elephant with a very low basal metabolism having a long average lifespan as compared to a mouse with a very high basal metabolism having a very short lifespan.

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5. Neuropeptide Y

One popular hypothesis suggests that the neuroendocrine response to low energy availability mediated by neuropeptide Y released by the arcuate nucleus of the hypothalamus plays a primary role in calorie restriction mediated lifespan extension. A recent review published in Molecular and Cellular Endocrinology explains the scientific basis of this hypothesis.

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References:
Wikipedia article on "Caloric restriction"

Genetically engineered children :)

Find below a link to an article published in Clinical Chemistry by Eleftherios P. Diamandis entitled, “How to Win Wimbledon Championships: Creating Beklof and Vamos”. It’s a funny take on the probability of genetically engineered children in the near future.
http://www.clinchem.org/cgi/content/full/55/6/1253

Rhes and Huntingtin - The odd couple!!

Rhes, a striatal specific protein, mediates mutant-huntingtin cytotoxicity.
Subramaniam S, Sixt KM, Barrow R, Snyder SH.
Science. 2009 Jun 5;324(5932):1327-30.

Huntington’s disease (HD) is an inherited neurodegenerative disorder, characterized by the gradual, irreversible impairment of psychological, motor, and cognitive functions. Symptoms typically appear in middle age, but onset can occur at almost any age. The course of the disease can last 15 to 20 years. The genetic mutation underlying HD has been traced to a gene encoding a protein called Huntingtin (Mr 350,000). The normal function of Huntingtin is not yet known. In individuals who will not develop HD, a region of the gene that encodes the amino terminus of the protein has a sequence of CAG codons (for glutamine) that is repeated 6 to 39 times in succession. In individuals with adult-onset HD, this codon is typically repeated 40 to 55 times. In individuals with childhood-onset HD, this codon is repeated more than 70 times. The length of this simple trinucleotide repeat indicates whether an individual will develop HD, and at approximately what age the first symptoms will occur.

The abnormal Huntingtin protein is produced in almost all cells of the body, but the pathology is limited to the corpus striatum in the brain. In fact, the corpus striatum is almost paper-thin in patients with advanced HD. The possible reason for localization of pathology in what is essentially a global genetic defect is not known. However, the authors of this paper have come up with an explanation to this perplexing phenomenon.

Rhes is a monomeric G protein that is almost exclusively expressed in the corpus striatum. The normal function of Rhes has not been elucidated but it is known to modify proteins by a process called sumolyation. Sumoylation is a post-translational modification. It involves the addition of a small protein called SUMO. SUMO proteins are similar to ubiquitin, and sumoylation is directed by an enzymatic cascade analogous to that involved in ubiquitination. In contrast to ubiquitin, SUMO is not used to tag proteins for degradation. SUMO modification of proteins has many functions like protein stability, nuclear-cytosolic transport, and transcriptional regulation.

In this paper, the authors have shown that Rhes causes the sumoylation of the mutated Huntingtin protein. In the absence of sumoylation, abnormal Huntingtin tends to aggregate within the cell. Sumoylation seems to inhibit this aggregation. It has long been thought that cellular toxicity was caused by the protein aggregates. However, this paper suggests that the soluble form of abnormal Huntingtin is harmful to the cell. Rhes by sumoylating Huntingtin, seemed to inhibit aggregation, thus increasing the concentration of soluble Huntingtin in the cell. Since, Rhes, as pointed out earlier, is exclusively expressed in the corpus striatum, the pathology in HD is therefore localized to the corpus striatum, though all cells in the body have the abnormal gene.

The obvious next step in research in this field would be to find out the normal function of Rhes. This could be done in vivo by using transgenic Rhes knock-out mice and in vitro by siRNA based techiniques. If Rhes is not shown to have critical functions in the cell, a drug that decreases Rhes expression could be the basis of treatment of HD in the future.

Why are patients with Down’s syndrome protected from cancer?

Discovery of novel anti-tumor proteins in patients with Down’s syndrome.

In a very interesting study published in Nature this month, scientists from Harvard Medical School have zeroed in on a gene that might explain why patients with Down’s syndrome have a very low rate of cancer compared to the general population (Baek et al., 2009).
Down syndrome is one of the most common inherited causes of mental retardation. It occurs with an approximate frequency of 1:700 births. The association between Down's syndrome and decreased cancer incidence had earlier been explored by Harvard's Dr. Judah Folkman who died last year. Dr. Folkman had noticed that cancer is rare among Down's patients, except for leukemia. He studied nearly 18,000 Down's patients and showed that they had 10 percent the expected rate of cancer.
Down’s syndrome is characterized by the presence of an extra copy of chromosome 21 in the genome (trisomy 21). Chromosome 21 has 231 genes in it. Scientists have proposed that a protein coded for by one of the genes on the 21st chromosome called “Down's syndrome candidate region-1” (DSCR1, also known as RCAN1) may explain the low incidence of cancer in Down’s syndrome. This protein inhibits Vascular Endothelial Growth Factor (VEGF). VEGF is one of the many factors that help cancer cells to stimulate formation of new blood vessels (angiogenesis). Inhibition of this factor by DSCR 1 can therefore retard the growth of cancers. People with Down syndrome have three copies of this gene while normal individuals only have two. It was shown that the levels of this protein is higher in patients with Down’s syndrome. In this study, the scientists also developed a mouse model of trisomy 21. The mouse model, like the humans, showed higher levels of DSCR-1 protein, and as expected, was resistant to development of tumors.

It is possible that more genes on the 21st chromosome can have anti-tumor properties that will explain the rarity of cancer in Down’s syndrome. Further research will reveal some of these genes. As of now, researchers say that three new proteins have been identified as potential targets in treatment of cancer. One is DSCR-1 and the others are calcineurin (a protein regulated by DSCR-1) and DYRK1A (a DSCR-1 like protein).

References:
Baek KH, Zaslavsky A, Lynch RC, Britt C, Okada Y, Siarey RJ, Lensch MW, Park IH, Yoon SS, Minami T, Korenberg JR, Folkman J, Daley GQ, Aird WC, Galdzicki Z, Ryeom S (2009) Down's syndrome suppression of tumour growth and the role of the calcineurin inhibitor DSCR1. Nature. 2009 May 20.

http://www.ncbi.nlm.nih.gov/pubmed/19458618?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum