Role for Unique Protein in Diabetes

hypusine structure
The eukaryotic translation initiation factor eIF5A, which exists in two isoforms, was originally thought to be involved in formation of the first peptide bond during mRNA translation, but more recent work has implicated it as a translation elongation factor. In mammalian cells it has variously been associated with mediation of proliferation, apoptosis and inflammatory responses, although its mechanisms of action have remained vague. It has also been identified as a cofactor of the Rev trans-activator protein of HIV-1. eIF5A is unique in that it is the only known protein to contain the amino acid hypusine, formed posttranslationally via the sequential action of deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) acting at a specific lysine residue.

GC7 structure
Based on the role of eIF5A in inflammation, a multi-institutional research team led by scientists at Indiana University School of Medicine has explored involvement of the protein in pancreatic islet dysfunction during the development of diabetes. In a low-dose streptozotocin mouse model of diabetes the team found that depletion of eIF5A (using siRNA) protected the mice from pancreatic β-cell loss and hyperglycemia. The depletion of eIF5A resulted in impaired translation of inducible nitric oxide synthase (iNOS)-encoding mRNA within islet cells. Further studies using an inhibitor of DHS, N1-guanyl-1,7-diaminoheptane (GC7), demonstrated a requirement for hypusination in the action of eIF5A.

The study, published in the Journal of Clinical Investigation, demonstrates a role for eIF5A in inflammation-induced damage to islet cells. Since the negative effects of eIF5A depend on hypusination, DHS may represent a viable therapeutic target for diabetes. Further work will be necessary to establish the role of this pathway in development and progression of the human disease.

Study Identifies Receptor for Potentially Fatal Infection

mucor sp. sporangium
Photomicrograph showing a mature sporangium of a Mucor species mould; Image: Wikimedia commons; source – CDC
Mucormycosis is a potentially fatal infection of the sinuses, brain, or lungs, which is most commonly caused by the fungus Rhizopus oryzae. Even if the disease is successfully treated with antifungal agents together with surgery to remove necrotic tissue, survivors are typically left with considerable disfigurement. The condition is seen most often in people with diabetic ketoacidosis (DKA) who have elevated serum glucose and iron levels and a team of researchers at the University of California Los Angeles has now discovered why these individuals are more susceptible to infection. In mucormycosis, fungal invasion of blood vessels results in the formation of blood clots and destruction of local tissue and the team set out to identify the endothelial cell receptor that the fungus uses, and also whether iron and glucose play a role in regulating the expression of the receptor.

In human endothelial cells, glucose-regulated protein 78 (GRP78) was found to play a key role in endocytosis of R. oryzae and subsequent damage; enhanced expression of the protein in the presence of high concentrations of glucose, especially when iron levels are also elevated, offers an explanation of the increased susceptibility of individuals with DKA to mucormycosis. Mice with DKA, which have elevated levels of glucose and available iron, and which are also susceptible to mucormycosis, showed increased expression of GRP78 in sinus, lungs, and brain compared with normal mice. In further studies, treatment of DKA mice with GRP78-specific immune serum was shown to protect them from mucormycosis.

The study, which is published in the Journal of Clinical investigation, provides a new understanding of the pathogenesis of R. oryzae and may lead to new treatments for potentially lethal mucormycosis.

‘Resilience Factor’ Identified

bouncing ball
Image: Wikimedia Commons - Richard Bartz
Emotionally resilient people adapt to adversity and bounce back after stressful events; less resilient individuals find it difficult to cope with setbacks and may decline into depression or post-traumatic stress disorder (PTSD). Mice, like humans, vary widely in their reaction to stressful situations and a study led by researchers at Mount Sinai School of Medicine which was funded by the National Institute of Health’s National Institute of Mental Health (NIMH) has now uncovered a mechanism that helps to explain differences in resilience.

Mice experience stress when confronted by an aggressive, larger mouse and about two thirds of animals that repeatedly undergo this ‘social defeat’ have altered behaviours including long-lasting social avoidance and anxiety-like symptoms. The other third of the ‘defeated’ mice showed relatively few behavioural effects and these resilient animals were found to have higher levels of the transcription factor ΔFosB in the nucleus accumbens, an important brain reward-associated region or “pleasure centre”. Social behaviour in ‘defeated’ mice can be normalised by chronic antidepressant treatment and the action of fluoxetine was found to require induction of ΔFosB in the nucleus accumbens. Post-mortem examination shows that ΔFosB is also depleted in the brains of people who suffered from depression, suggesting that induction of this protein is a positive adaptation that provides resilience to stress.

ΔFosB is also known to be involved in regulating responses to both drugs of abuse and natural rewards such as food, sex and exercise, although the cell populations involved in these responses differ somewhat from those involved in protection from stress. The team suggest that concentrations of ΔFosB in the nucleus accumbens are important in setting the level of an individual’s reward-seeking motivation and that reduced concentrations of the protein are linked to the impaired motivation and ability to experience pleasure seen in many people with depression. The team now hope to discover small molecules that will augment the actions of ΔFosB and lead to resilience-boosting treatments for depression.

The study is published in the journal Nature Neuroscience.

Locking out or Locking in – It’s Not the Same Key for Malaria

Image: Flickr – Svadilfari
Plasmodium parasites, responsible for malaria in humans, have a complex lifecycle that is dependent on mosquito and human hosts. In human blood, the merozoite stage of the parasite invades red blood cells (erythrocytes), growing and multiplying before rupturing the cell and escaping to infect other erythrocytes. It is this profound effect on erythrocytes that is responsible for the symptoms of malaria – fevers, chills and anaemia. Untreated, the disease can be fatal and drug resistance is an increasing problem. With up to half a billion people infected each year and nearly a million deaths, mostly in sub-Saharan Africa, there is an urgent need for new treatments.

Researchers at Harvard School of Public Health (HSPH) were attempting to identify the mechanism by which Plasmodium falciparum merozoites enter erthyrocytes, but instead found a parasite protein that is essential for escape from the cells. When the protein, P. falciparum calcium-dependent protein kinase (PfCDPK5), was suppressed the parasites were trapped in the host cell and unable to infect new cells. In further experiments the team showed that these merozoites were still able to invade erythrocytes if released from their host cell by other means, indicating separate mechanisms for invasion and egress from erythrocytes.

The findings reveal an essential step in the biology of P. falciparum and suggest a new, parasite-specific, drug target for fighting one of the world’s most common and dangerous infections. Whilst many scientists are looking for inhibitors of parasite egress and invasion of red blood cells, no anti-malarial drugs yet target these stages of the parasite lifecycle.

The study is published in Science.

Regulating Cholesterol Levels

Image: Flickr – David Masters
Cholesterol is an essential component of all cellular membranes and is also required for synthesis of vitamin D and steroid hormones. Since it is poorly soluble in water, it is mainly transported through the bloodstream within lipoproteins – complex spherical particles composed of amphiphilic proteins and lipids whose outward-facing surfaces are water-soluble and inward-facing surfaces are lipid-soluble. Triglycerides and cholesterol esters are carried internally whilst phospholipids and cholesterol are transported in the surface monolayer of the lipoprotein particle. Several types of lipoproteins are found in blood, comprised of different apolipoproteins (which target specific tissues via receptor recognition) and with different capacities for cholesterol. These are usually referred to by their densities – the higher the ratio of cholesterol to lipoprotein, the lower the density. This gives rise to the so-called “bad cholesterol” (low density lipoprotein, LDL-cholesterol) and “good cholesterol” (HDL-cholesterol).

cholesterol structure
Problems arise when levels of the various lipoproteins are out of balance. Increased circulating levels of LDL-cholesterol are associated with the formation of foam cells, which can become trapped in the walls of blood vessels and contribute to artherosclerotic plaque formation leading to heart attacks and strokes. Conversely, HDL transports lipids to the liver for disposal and removes cholesterol from peripheral tissues, including the foam cells that form atherosclerotic plaques.

A new study by researchers at Massachusetts General Hospital (MGH) has now identified micro RNAs (miRNAs) that appear to play an important role in regulation of cholesterol/lipid levels. The team found that two members of the miR-33 family (miR-33a and miR-33b) target the ATP-binding cassette transporter A1 (ABCA1), an important regulator of HDL synthesis and reverse cholesterol transport, for posttranscriptional repression. Using antisense inhibition of miR-33 in mouse and human cell lines the researchers demonstrated up-regulation of ABCA1 expression and increased cholesterol efflux. Further, treatment of mice on a western-type diet with the antisense inhibitor resulted in elevated plasma HDL without affecting levels of LDL. The findings suggest that miR-33 may represent a therapeutic target for cardiovascular diseases.

The study is published in Science.

Newly Discovered Vulnerability in Tuberculosis

mycobacterium tuberculosis in sputum sample
Photomicrograph of a sputum sample containing Mycobacterium tuberculosis (stained red); Image - Wikipedia (source: CDC)
Tuberculosis (TB) is an airborne infectious disease caused by Mycobacterium tuberculosis (Mtb). TB is difficult to treat and the most commonly used antibiotics, rifampicin and isoniazid, need to be used for many months to eliminate the infection. The recent resurgence of TB, together with the emergence of drug-resistant strains of the bacterium, underscores the need for new treatments and researchers at Weill Cornell Medical College and the Novartis Institute for Tropical Diseases have identified a metabolic vulnerability in Mtb that could lead to new targets for drug therapy.

In vitro, Mtb is able to grow on a variety of carbon sources but fatty acids are believed to be the major source of carbon and energy for the bacterium during infection of a host. When bacterial metabolism is primarily fuelled by fatty acids, biosynthesis of sugars from intermediates of the tricarboxylic acid cycle is known to be essential for growth but the role of gluconeogenesis in the pathogenesis of Mtb had not been explored. Using genetic analyses and 13C carbon tracing, the team were able to show that phosphoenolpyruvate carboxykinase (PEPCK) – the enzyme that catalyses the first committed step of gluconeogenesis – is essential for the growth of Mtb supported by fatty acids. PEPCK was shown to be needed for growth of Mtb both in isolated macrophages derived from the bone marrow of mice and in infected mice.

Mtb lacking PEPCK failed to replicate in mouse lungs and silencing PEPCK during the chronic phase resulted in clearance of the infection, showing that Mtb relies on gluconeogenesis throughout the course of the infection. The finding that PEPCK plays a pivotal role in the growth and persistence of Mtb during both acute and latent infections in mice – and that PEPCK depletion also attenuates Mtb in IFNγ-deficient mice – suggests that this enzyme is an attractive target for chemotherapy.

The study is published in PNAS.

Stopping Cancer Spread

Fascin crystal structure
Human fascin complexed with macroketone, PDB ID=3LNA
The deadliest feature of cancer is its ability to spread, or metastasise. Migrastatin, a compound isolated from Streptomyces, was found to weakly inhibit tumour cell migration and, in 2005, researchers from Weill Medical College of Cornell University and the Sloan-Kettering Institute for Cancer Research described simplified analogues of migrastatin, including a compound they called macroketone, that inhibit mammary tumour metastasis in mice. Although the compounds were effective in preventing the spread of cancer cells, it wasn’t known how they achieved this. In a new study, published in the journal Nature, the team have revealed that macroketone exerts its anti-metastatic effect by targeting the actin-bundling protein, fascin. Cancer cells use invasive finger-like protrusions called invadopodia to spread into and degrade extracellular matrix and recent studies have shown that fascin is important for their assembly and stability.

Macroketone structure
Mice implanted with cancer cells and treated with macroketone lived out a full lifespan without any spread of the cancer whilst untreated animals all died from metastases. When treatment was delayed for one week after introduction of the cancer cells, metastasis was still blocked by more than 80%. Macroketone did not prevent implanted cancer cells from forming tumours or growing, suggesting that such compounds would need to be used in combination with chemotherapy drugs acting on the primary tumour. Because fascin is overexpressed in metastatic tumour cells but is only expressed at very low levels in normal epithelial cells, treatments that target fascin should have comparatively little effect on normal cells and may have fewer side effects than other treatments.

X-ray studies showed that macroketone binds to one of the actin-binding sites on fascin which prevents the actin fibres from bundling together and could form the basis for further drug design.

High Altitude Metabolism

High Altitude
Image: Flickr – Blue Turban Photography
The cellular responses to low oxygen levels (hypoxia), as occurs at high altitude, are critical for survival. The transcription factor, hypoxia inducible factor 1 (HIF-1), is a key player in this setting, upregulating genes that preserve function. Included in these are glycolysis enzymes, which allow ATP synthesis in an O2-independent manner, and vascular endothelial growth factor (VEGF), which promotes angiogenesis. HIFs are also important in development and deletion of HIF-1 in mammals is perinatally lethal.

HIF-1 occurs as a heterodimer of HIF-1α and the constitutively expressed HIF-1β. Under normal oxygen conditions, HIF-1α is a substrate for HIF-1 prolyl hydroxylases and the asparagine hydroxylase, factor inhibiting HIF-1α (FIH). The action of the prolyl hydroxylases results in the targeting of HIF-1α by an E3 ubiquitin ligase and subsequent degradation by the proteasome, whilst hydroxylation by FIH represses activity of its carboxy terminal transactivation domain (CAD). Both hydroxylation processes therefore serve to down-regulate the activity of HIF-1. When oxygen levels are low, however, the prolyl hydroxylases and FIH become inactive since they are dependent on O2.

A team led by researchers at University of California at San Diego have now reported on a FIH-knockout mouse. Despite the importance of HIF-1 in development, the FIH-deleted mice were healthy, although smaller than wild-type littermates. Where they differed significantly was in their metabolic profile. The FIH-null mice exhibited elevated metabolic rate, enhanced insulin sensitivity, hyperventilation and improved lipid and glucose homeostasis. On a high-fat diet, the animals were resistant to weight gain and had reduced central adiposity.

The team went on to explore the effects of tissue-specific FIH deletion, demonstrating that most of the features of the metabolic phenotype of the FIH-null mice could be replicated when only neuronal FIH was deleted.

The study, published in Cell Metabolism, identifies FIH as an essential regulator of metabolism and opens up the possibility of FIH inhibitors for the treatment of metabolic disorders.

Novel Strategy to Fight TB

chess board
Image: Flickr – gcfairch
The bacterium responsible for tuberculosis (TB), mycobacterium tuberculosis (Mtb), is notoriously difficult to kill. The most commonly used antibiotics, rifampicin and isoniazid, need to be used for extended periods of time (typically 6-24 months) to effectively eliminate infection. In addition, emergence of antibiotic-resistant strains is an increasing problem.

Researchers at Albert Einstein College of Medicine of Yeshiva University have now identified a new biochemical pathway in Mtb and two novel ways to kill the bacterium. The pathway involves four enzymatic steps in the conversion of the disaccharide, trehalose, to α-glucan mediated by TreS, Pep2, GlgE (which has been identified as a maltosyltransferase that uses maltose 1-phosphate) and GlgB. Focusing on GlgE, the researchers found that blocking the enzyme induced toxic accumulation of maltose-1-phosphate, killing the bacteria in vitro and in a mouse model of infection. Inhibition of another enzyme in the pathway was non-lethal until combined with inactivation of Rv3032, a glucosyltransferase involved in a distinct α-glucan pathway. Inhibition of Rv3032 alone was also non-lethal to the bacteria.

The research validates inhibition of GlgE as therapy for TB but also highlights the potential for targeting two α-glucan pathways – a strategy that potentially leads to reduced incidence of resistance. Both approaches are also distinct from the mechanisms of currently used antibiotics.

The study is published in Nature Chemical Biology.

Controlling Fat Distribution

Image: Patrick Hoesly
Extreme accumulation of fat in muscle tissue is associated with cardiovascular disease and is a contributory factor in insulin resistance and type II diabetes. It is therefore important to understand the mechanisms by which fat is taken up from the bloodstream and metabolised by tissues. Surprisingly, the role of blood vessels themselves in the transport of lipids has not been clearly established. Researchers at the Karolinska Institutet have now identified a role for vascular endothelial growth factor-B (VEGF-B) in endothelial targeting of lipids to peripheral tissues.

The VEGFs and their receptors are major regulators of angiogenesis and pharmacological intervention, for example with bevacizumab (a monoclonal antibody specific for VEGF-A), has been successfully exploited in oncology. This latest study has shown that VEGF-B, in mice, controls endothelial uptake of fatty acids via transcriptional regulation of vascular fatty acid transport proteins. Mice that were deficient in VEGF-B (Vegfb-/-) showed reduced uptake and accumulation of lipids in muscle, heart and brown adipose tissue. Instead, the Vegfb-/- mice preferentially transported lipids to white adipose tissue, resulting in a small weight increase. This regulation was mediated by VEGF receptor 1 and neuropilin 1 expressed by the endothelium.

The authors of the study, published in Nature, propose that this new role for VEGF-B could potentially lead to novel strategies to modulate pathological lipid accumulation in diabetes, obesity and cardiovascular diseases.