Group members
Prof T.S. Pillay	Chief Specialist Chemical Pathologist	Tel 021-406-6192
Wan Irani Ismail	PhD Student
Dr Ian Ross	PhD Student
Dr David Haarburger	Registrar/MMed Student
Dr Fierdoz Omar	Registrar/MMed Student
Ms Lauren Hartmann	MSc student

Proteomics has emerged as a relatively new field of protein science based on a “classical” electrophoretic technique and is now dominated by separation methods including traditional 2-D electrophoresis and liquid chromatography to separate proteins and methods to analyse proteins by mass spectrometry. Coupled with bioinformatics and rapidly evolving software, these techniques have become powerful methods for protein characterization and identification. In the diagnostic arena, these techniques have been used to characterize protein profiles from normal and diseased body fluids. Blood has direct contact with almost all of the tissues in the human body and therefore pathological changes are likely to be reflected by proteomic changes in serum. Biomarkers identified in serum may form the basis for simple, non-invasive diagnostic or monitoring tests. There have been a number of developments in proteomics which have enhanced its utility beyond the research  arena.

Proteomic analysis is being applied to the identification of biomarkers in a number of other disease states . Proteomic analysis of human serum for identification of disease-specific biomarkers promises to be a powerful diagnostic tool for defining the onset, progression and prognosis of human diseases. Serum provides an abundant sample for diagnostic analyses because of the expression and release of proteins (potential biomarkers) into the bloodstream in response to specific physiological states such as viral infections, bacterial infections, cancer and Alzheimer’s disease to name a few. Therefore, serum offers a medium to define differential expression characteristics specific to those physiological states.

The diagnosis of HIV/AIDS is dependent on the detection of viral protein or antiviral antibody in human serum .Recently, tests have also included the detection of viral nucleic acid in serum. CD4+ cells can also be counted as an index of immune status during infection and following antiretroviral therapy. The burden of HIV/AIDS on health services and economies worldwide and especially in Africa cannot be overstated. Treatment and prevention is critically dependent on the availability of these diagnostic tests. It will be useful to be able to develop tests to detect novel proteins that are induced during HIV infection. In order to develop such tests, biomarkers will have to be identified and then antibodies can be developed to these biomarkers. The development of such assays could potentially provide sensitive and highthroughput detection for screening, diagnosis and monitoring.
Assay techniques for the diagnosis of HIV, in common with serologic assays, have evolved over the past two decades, progressing from strictly research tools to routine methods that have proved to be valuable. Techniques such as Polymerase chain reaction (PCR), nucleic acid sequence based amplification (NASBA), and branched DNA (bDNA) methods offer increased sensitivity for early infection. These enhance the safety of blood supply, can be used to monitor viraemia and assess drug resistance by changes in copy number. However, even the best methods are not perfect and have limitations and false positives and negative do occur, depending on the “window” between infection and sero-conversion. This window can be often variable and is frequently responsible for false negative diagnoses.

We are interested in using proteomics for discovering biomarkers in biological fluids, particularly in the context of HIV/AIDS.

 

Metabolic diseases

The mechanism of metabolic dysregulation induced by anti-retroviral drugs.

 

The effects of anti-retroviral drugs on adipocytes and the insulin signalling pathway

 

The overall aims of the project are to understand the molecular basis for insulin resistance and lipodystrophy induced by antiretroviral drugs using a cell culture system

Background
Current antiretroviral treatment (highly active antiretroviral therapy [HAART]) has improved the prognosis of patients with HIV by dramatically suppressing HIV viral load, increasing CD4 counts, and reducing opportunistic infections associated with AIDS. However, as many as 83% of the patients receiving this treatment develop metabolic abnormalities, including dyslipidaemia (elevated triglycerides and cholesterol), central adiposity, and peripheral lipodystrophy.  Moreover, it is becoming increasingly acknowledged that these patients may be at high risk for developing premature cardiovascular morbidity as well as type 2 diabetes, emphasizing the medical significance of the metabolic  dysregulation associated with HAART.

It is not known whether the drugs or the virus itself causes the metabolic abnormality. AIDS is frequently associated with hypertriglyceridemia, but with reduced total, LDL, and HDL cholesterol. Nucleoside reverse transcriptase inhibitors may also be associated with hepatic steatosis and lactic acidemia, which are thought to result from mitochondrial damage caused by these agents. Yet, an increasing amount of clinical and epidemiological data attributes a central role for the HIV protease inhibitors (HPIs) in the induction of insulin resistance in HAART-treated patient. HPIs are a central component of the HAART regimen, as they are potent inhibitors of HIV aspartyl protease, an enzyme required for normal processing of HIV proteins  It appears that all HPI compounds induce insulin resistance and lipid abnormalities to various degrees but the mechanisms for these serious side effects are largely unknown. Only a few studies have been published recently regarding alterations in insulin signaling and insulin-regulated metabolism caused by HPIs at the cellular level. Using different agents and incubation periods, protease inhibitors have been shown to impair insulin signaling events in HepG2 cells to inhibit adipocyte differentiation and to decrease adipocyte insulin-stimulated glucose transport without affecting the insulin signaling cascade. Yet, despite the fact that impaired adipose tissue metabolism is at the center of the HPI-induced metabolic syndrome, a systematic evaluation of the effects of HPIs on fully differentiated adipocyte metabolism has not been reported.

In this study, we propose to analyse the effects of antiretroviral drugs on the insulin signalling pathway in adipocytes and in cells tranfected with the insulin receptor and to analyse the effects of these drugs on global protein expression in adipocytes using proteomics.

 

The molecular cell biology of insulin receptor signalling

Our group has previously worked on the signalling pathways downstream from the insulin receptor.  We previously elucidated the role of an adapter protein family, the APS/SH2B family in initiating the pathway leading to the phosphorylation of c-Cbl

 

SOME KEY PUBLICATIONS

Mehra A, Macdonald I, Pillay TS (2007) Variability in 3T3-L1 adipocyte differentiation depending on cell culture dish.
Anal Biochem. 2007 Mar 15;362(2):281-3.

Katsanakis KD, Pillay TS (2005) Crosstalk between the two divergent insulin signalling pathways is revealed by the protein kinase B (AKT)-mediated  phosphorylation of  adapter protein APS on serine 588.
Journal of  Biological Chemistry  Nov 11;280(45):37827-32.

Pillay TS (2004) A Fisherman’s Tale: phospho-dependent phage display as a discovery tool
(invited review) Discovery Medicine  Volume 4 (23) 315-318 October 2004

Wilcox A, Katsanakis  KD, Bheda F, Pillay TS (2004) Asb6, an adipocyte-specific ankyrin and SOCS box protein, interacts with APS to enable recruitment of  Elongins B and C to the insulin receptor signaling complex 
Journal of  Biological  Chemistry  Sep 10;279(37):38881-8.
(rated amongst the top 1 % of papers submitted to the Journal of Biological Chemistry- see JBC online September 3, 2004)

Ahn  M-Y, Katsanakis  KD, Bheda F, Pillay TS (2004) Primary and essential role of the adaptor protein APS for recruitment of both c-Cbl and its associated protein CAP in insulin signaling.
Journal of  Biological  Chemistry  May 14;279(20):21526-32

 Patel NA, Apostolatos HS, Mebert K, Chalfant CE, Watson JE, Pillay TS, Sparks J, Cooper DR. (2004)  Insulin Regulates PKC{beta}II Alternative Splicing in Multiple Target Tissues: Development of a Hormonally Responsive Heterologous Minigene.
Molecular  Endocrinology 2004 Apr;18(4):899-911

Khati M, Pillay TS (2004) Phosphotyrosine phosphoepitopes can be rapidly analyzed by co-expression of a tyrosine kinase in bacteria with a T7 bacteriophage display library.
Analytical Biochemistry Vol 325/1 pp 164-167

Pillay TS, Xiao S, Keranen L, Olefsky J (2004) Regulation of the insulin receptor by protein kinase C isoenzymes: preferential interaction with bII and interaction with the catalytic domain. Cellular Signalling    Jan;16(1):97-104

Ahmed Z, Pillay TS (2003)Adapter protein with a pleckstrin homology (PH) and an Src homology 2 (SH2) domain (APS) and SH2-B enhance insulin-receptor autophosphorylation, extracellular-signal-regulated kinase and phosphoinositide 3-kinase-dependent signalling.
Biochemical Journal. Apr 15;371(Pt 2):405-12.

Ahmed Z, Pillay  TS  (2001) Functional effects of APS and SH2-B on insulin receptor signalling.
Biochemical Society Transactions. Aug;29(4):529-34.  (Review)

Ahmed Z, Smith BJ, Pillay TS (2000) The APS adapter protein couples the insulin receptor to the phosphorylation of c-Cbl and facilitates ligand-stimulated ubiquitination of the insulin receptor.
FEBS Lett.  Jun 9;475(1):31-4

Ahmed Z, Smith BJ, Kotani K, Wilden P, Pillay TS (1999)  APS, an adapter protein with a PH and SH2 domain is a substrate for the insulin receptor kinase.
Biochemical Journal  Vol 341 (Part 3) 665-668

Kotani  K,  Wilden  P,  Pillay  TS (1998) SH2-Balpha is an adapter protein and substrate that interacts with the activation loop of the insulin receptor kinase.
Biochemical Journal Vol 335 (Part 1) 103-109

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