Prof Gregory L Blatch
Director, Professor of Biochemistry
Professor Greg Blatch
Dr Adrienne Edkins - Lecturer, Biochemistry
Investigating the role of cell-surface expressed molecular chaperones
Adrienne's research interest is the function of molecular chaperones that are expressed on the plasma membrane of cells. Chaperones are proteins that control the stability and folding of other proteins in the cell and therefore play an important role in a range of cellular functions. Most chaperones are functional as intracellular proteins, although a subset of membrane-bound chaperones has been identified on the cell surface. We are using cancer cell and cancer stem cell models to analyse the mechanisms by which these extracellular chaperones control the growth, differentiation and migration of cancer cells.
Dr Adrienne Edkins
Adrienne completed a Master in Science at Rhodes University, before being awarded a Wellcome Trust 4 Year PhD Fellowship in Molecular Functions in Disease at the University of Glasgow. She spent four cold years in Glasgow studying the role of integrins in the production of cytokines by monocytic cells stimulated with CD23. She initially returned to Rhodes University in 2008 to take up a postdoctoral position with Professor Greg Blatch, attempting to identify novel anti-Hsp90 inhibitors from indigenous sources that could be developed into new chemotherapeutic drugs. She is currently employed as a Lecturer in Biochemistry and is pursuing research into the function of membrane-bound molecular chaperones.
Dr Petra Gentz - Research Officer
Dr Petra Gentz
Dr Eva-Rachele Pesce
Molecular characterization of the Hsp40 chaperone machinery of Plasmodium falciparum
Plasmodium falciparum, the protozoan parasite responsible for cerebral malaria, develops within mature human erythrocytes. The parasite genome contains several members of the Hsp40 protein family (Hsp40s), many of which are potentially exported into the parasitophorous vacuole and into the erythrocyte cytoplasm. Molecular chaperones are believed to provide cytoprotection to the parasite, especially during the febrile episodes associated with the disease, and to be involved in the remodelling of the infected erythrocyte caused by Plasmodium as well as in protein trafficking. This project encompasses an analysis of the Hsp40 chaperone machinery of P. falciparum with the main focus on heat inducible and potentially exported Hsp40 proteins and their possible interaction with parasite and human Hsp70 proteins (Hsp70s). The study involves the heterologous expression of P. falciparum Hsp40s and Hsp70s in Escherichia coli for in vitro assays as well as characterisation of the chaperones in parasites and infected erythrocytes.
Dr Eva Pesce
2008-present Claude Leon Foundation postdoctoral Fellow - Department of Biochemistry, Microbiology & Biotechnology, Rhodes University (South Africa)
2007 - 2008 Postdoctoral Fellow - Department of Biochemistry, Microbiology & Biotechnology, Rhodes University (South Africa)
2005-2007 Rhodes University Postdoctoral Fellow - Department of Biochemistry, Microbiology & Biotechnology, Rhodes University (South Africa)
2004 PhD (Plant Molecular Biology) - Institute for Plant Sciences, University of Bern (Switzerland)
2001 MSc (Molecular Biology) - Biophysics Institute, National Research Council & University of Genoa (Italy)
Dr Linda Stephens
Overall project: Development of heterologous expression systems for the production of malarial drug target proteins.
Specific project: Heterologous coexpression of the malarial chaperone, PfHsp70, increases the expression and solubility of the malarial drug target GTP cyclohydrolase I (PfGCHI).
Malaria, caused by Plasmodium strains, is the world's most important parasitic disease causing at least 1 million deaths annually. Drug resistant parasite strains have developed and research on putative malarial drug targets is frequently hampered by low yields of soluble protein obtained by heterologous expression in Escherichia coli host cells. This problem can be overcome by co-expression of Plasmodial molecular chaperones with the drug target in E. coli. GTP cyclohydrolase I (GCHI) from the malarial folate biosynthetic pathway is a validated antimalarial drug target. The molecular chaperone, PfHsp70 facilitates the correct folding and assembly of proteins in the parasite cell. We have developed a PfHsp70-drug target co-expression system for the production of soluble folded GCHI in E. coli. The co-expression of PfHsp70 resulted in enhanced heterologous expression of soluble GCHI. The GCHI was successfully purified in milligram quantities and its identity confirmed by mass spectroscopy. This is the first time malarial GCHI has been produced and purified in quantities suitable for structural and functional studies. This co-expression system should be applicable to other malarial proteins recalcitrant to soluble expression in heterologous systems.
Dr Linda Stephens
2005 PhD in Pharmacology, Rhodes University, Grahamstown, South Africa.
Thesis: An investigation into the antidepressant activity of Hypericum perforatum
2005 to present:
Postdoctoral research fellow working in Prof Greg Blatch's Chaperone
Research Laboratory, Department of Biochemistry, Microbiology and
Biotechnology, Rhodes University, Grahamstown, South Africa.
Dr Michael Ludewig
Characterisation of trypanosomal Hsp40 proteins essential to the survival and growth of the parasite
Dr Michael Ludewig
Dr Earl Prinsloo
The characterisation of the molecular determinants governing the interaction between STAT3 and Hsp90
STAT3 plays a significant role in the self-renewal of mouse embryonic stem cells (mESC) and is known to be involved in the establishment and maintenance of human cancer phenotypes. Using molecular and cell biological methods my work entails the elucidation of the determinants involved in the interaction of STAT3 and Hsp90 (and other chaperones and co-chaperones). This will offer new insights into the roles of molecular chaperones in mESC self-renewal and potentially the role of Hsp90 (and other chaperones) in the new paradigm of cancer stem cells.
Dr Earl Prinsloo
Claude Leon Postdoctoral Fellow 2008-2009 (Rhodes University)
NRF Innovation Fund Postdoctoral Fellow 2007 (Rhodes University)
Ph.D Microbiology 2003-2006 (Nelson Mandela Metropolitan University on behalf of University of Port Elizabeth)
M.Sc Microbiology 2000-2002 (University of Port Elizabeth)
B.Sc Hons Microbiology 1999 (University of Port Elizabeth)
B.Sc Biochemistry and Microbiology 1996-1998 (University of Port Elizabeth)
Characterization of Pfj1 and PfHsp40-2 partner proteins of Plasmodium falciparum Hsp70
Characterisation of novel trypanosomal Hsp70 chaperones
The malarial drug target protein PfDXR: Purification, Kinetic Characterisation and Inhibition Studies
1-deoxy-D-xylulose-5-phosphate reductoisomerase of Plasmodium falciparum (PfDXR) forms part of the mevalonate-independent pathway of isoprenoid biosynthesis. PfDXR catalyzes fatty acid biosynthesis using a distinct pathway that is dissimilar to that of the mevalonate pathway employed in the human host. This parasite enzyme therefore represents an effective target for anti-malarial drug design. A three-dimensional model of the malarial drug target protein PfDXR was generated, and validated using structure-checking programs and protein docking studies. Structural and functional features unique to PfDXR were identified using the model and comparative sequence analyses with apicomplexan and non-apicomplexan DXR proteins. The validated model was also used to develop an efficient in silico screening method for potential tool compounds for use in the rational design of novel DXR inhibitors. However, in drug development, the success of producing functional target proteins in vitro is subject to the heterologous over-production of soluble protein. The efficient over-production of malarial proteins in prokaryotic host systems often results in truncated protein forms or solubility-related problems that lead to protein aggregation, complicating purification. A number of co-expression strategies have been developed that may assist in protein folding, including co-expression with various bacterial and malarial chaperones, as well as co-expression with a lac repressor protein such that expression can be tightly regulated. Once a successful expression strategy has been developed to produce high enough yields of folded functional protein, PfDXR will be purified and used for in vitro inhibition studies with novel potential DXR inhibitors. Following in silico screening of potential DXR inhibitors, DXR inhibition will be assessed using an NADPH-dependant enzyme inhibition assay. Those ligands that demonstrate reasonable DXR inhibition will then be tested for antimalarial potential, using P.falciparum-infected erythrocyte tissue culturing. This may then assist in bridging the gap between R&D and the development of a novel, clinically effective antimalarial drugs.
Bachelor of Science (2004-2006)
B.Sc(Hons) Biochemistry (2007)
Structural and Functional characterization of the protein inhibitor of activated STAT3 (PIAS3)
The protein inhibitor of activated signal transducer and activator of transcription 3 (PIAS3) regulates the transcriptional activation activity of signal transducer of activated transcription 3 (STAT3). STAT3 is phosphorylated in the cytosol and forms a functional dimer and translocates into the nucleus where it activate transcription of genes involved in cell growth, proliferation and apoptosis. In the nucleus PIAS3 blocks the DNA binding domain of the STAT3 dimer and inhibits its transcription activation activity. PIAS3 has conserved four conserved domains: DNA binding scaffold attachment factor-A/B/ACINUS/ PIAS (SAP) domain, PINIT (proline, isoleucine, asparagine, isoleucine, threonine) domain, a putative really interesting new gene (RING) type zinc-finger binding domain, and the serine/ theroine-rich (S/T) acidic C-terminal domain. The PINIT domain may be the domain that determines the STAT3-PIAS3 interaction and this interaction has been suggested by in vivo analysis using the mutant version of PIAS3. My interest is to investigate the direct interaction of the PINIT domain (PIAS3115-316) with STAT3 in vitro and heterologous overexpression of functional PINIT protein capable of interacting with STAT3 in vitro and determine its solution structure using Nuclear Magnetic Resolution.
My academic qualifications are as follows: B.Sc Chemistry and Biochemistry (double major) obtained from the University of Zimbabwe (2002), B.Sc Honours in Structural Biology obtained from University of the Western Cape (2007). Other qualifications I hold include diplomas in Computer Studies and a Certificate in Quality Assurance (ISO). I have four years working experience gathered from engagements in Industries in Zimbabwe and the United Kingdom.
Jo-anne De La Mare
Elucidation of the mechanisms of action of novel anti-cancer compounds on the survival and proliferation of the MCF7 breast cancer cell line
In higher life forms there exists a need to be able to detect changes in the environment and to relay this information to cells and tissues, allowing the organism to respond to various external stimuli. This response is mediated, in part, by a diverse and complex array of signal transduction pathways and, in mammals, governs survival, proliferation, differentiation and apoptosis at the cellular level. In particular, two key pathways involved in survival and proliferation, namely the Akt/PKB and MAP kinase pathways, will be discussed in detail; together with an in-depth description regarding the mechanisms which regulate apoptosis. In human cancers, the tight control of these processes is lost through multiple alterations in the cellular signaling machinery which allow cancer cells to proliferate independently of exogenous growth signals, evade apoptosis, develop resistant to drugs and metastasize to other tissues. As a result, the focus in cancer treatment has moved away from the traditional DNA-damaging radiation therapy and chemotherapeutic drugs towards the targeting of specific signal transduction pathways and their central protein players. A wide range of specific inhibitors of pro-survival and anti-apoptosis signaling molecules have entered clinical trials. Examples include the Raf inhibitor, Sorafenib; the MEK inhibitor, PD98059; the anti-Hsp90 drug, Geldanamycin; the mTOR-specific inhibitor, Rapamycin and the NF-?B inhibitory agent, Sulindac.
Furthermore, a recent conceptual break-through in cancer research, termed the "stem-cell hypothesis", states that cancers originate within tissue stem cells (SCs) as a result of deregulation of their self-renewal pathways, and that these cells are resistant to chemotherapy and radiation. Thus, the targeting of specific signaling molecules involved in regulating self-renewal, namely members of the Notch, Hedgehog, and Wnt pathways, provides an opportunity for the complete eradication of tumours which would prevent recurrence of the disease.
This study will make use of a set of small organic molecules of indigenous marine plant origin provided by Dr Denzil Beukes (Department of Pharmacy, Rhodes University) and found to display cytotoxicity towards an oesophageal cancer cell line. A subset of these compounds also had a cytotoxic effect on MCF7 cells, with IC50 values in the low micromolar to nanomolar range. The cellular mechanisms by which these agents affect the MCF7 cells are unknown and will form the focus of this research
Jo-anne De La Mare
B.Sc (2005) Rhodes University
B.Sc Hons (2006) Rhodes University
M.Sc (2009) Rhodes University
Identifying the potential role of Hsp90 in differentiation and self-renewal pathways in the breast cancer (stem) cell model of MCF7 cells
Cell signaling and transcription pathways, such as the Hedgehog, Notch and (Wingless) Wnt pathways, which are involved in the self-renewal and differentiation of stem cells, are also related to cancer. Elucidation of these pathways that regulate differentiation of breast stem cells and breast cancer has increased the understanding of how dysregulation of these tightly controlled processes could play a role in carcinogenesis. Side population (SP) cells are a rare population of cells found in cancers which are found to contain a vast proportion of cancer stem-like cells. Breast cancer MCF7 SP cells are enriched with cancer stem cell-like cells and since there is difficulty in maintaining cancer stem cells from primary cell lines, the breast cancer MCF7 SP can be used as a model for research in breast cancer stem cells. There is an increased expression of the heat shock protein 90 (Hsp90) in tumors and cancers in comparison to normal tissues, suggesting its potential roles in assisting the tumor cells with maintaining homeostasis, as well as regulating growth and survival in a hostile environment. Very little is known about the direct association of Hsp90 and the various components in the signaling pathways, in particular the Wnt signaling pathway in breast cancer stem cells. It would be important to characterize the possible association between HSP90 and components of the Wnt signaling pathway in breast cancer stem cells in order to establish where therapeutic drugs involving Hsp90 inhibitors could be used as a potential for differentiation of the cancer stem cells.
2009 - M.Sc Rhodes University; NRF Grant Holders Bursary and Andrew Mellon half Scholarship; Duncan-Whitely Bursary2008 - B.Sc Hons. (Biochemistry) Rhodes University; Andrew Mellon Scholarship, SASOL Bursary
2005 - 2007 - BSc Rhodes University
The effect of DSG and DSG analogues on the in vitro chaperone activity of PfHsp70-I
My masters project involves investigating PfHsp70-I, a heat shock protein from the malaria parasite Plasmodium falciparum, as a potential drug target for malaria. PfHsp70-I has been found to be involved in promoting the malaria parasite's survival during its transition between the cold-blooded mosquito host, and the warm-blooded vertebrate (human) host. Finding a way of modulating this protein's activity could contribute to the vital search for effective malaria drugs. In this project, 15-deoxyspergualin (DSG), as well as a number of its analogues and related compounds, will be tested as potential modulators of PfHsp70-I. This will involve using ATPase assays and aggregation suppression assays to determine the effects of the test compounds on heat shock proteins, which will be recombinantly produced and purified. These proteins include PfHsp70, one of its partner proteins PfHsp40, as well as human heat shock proteins.
B.Sc in Chemistry and Biochemistry, Rhodes University (2005-2007)
B.Sc Hons in Biochemistry, Rhodes University (2008)
Currently M.Sc in Biochemistry at Rhodes _______________________________________________________________
Isolation of breast cancer stem cells: Characterisation and applicability to in vitro studies
One of the main problems in cancer research is in identifying the subpopulation within a tumour mass that is capable of initiating tumour growth. These cells are believed to be the cancer stem cells (CSCs), otherwise known as tumour-initiating cells. These cells have been associated with cancer reoccurrence. Recent research has provided evidence of CSCs in breast cancer, the most common malignancy affecting women. Breast CSCs need to be isolated in order to design therapies that will target these specific cells thereby eliminating renewal potential of tumours. Current isolation technologies utilize specific markers on the CSCs to isolate them from the tumour mass however there are currently no defining markers to identify these cells. The aims of the research are to develop a CSC isolation technique based on Percoll gradient centrifugation and to further characterize breast CSCs in terms of markers, morphology and possible resistance to cytotoxic compounds.
Undergrad: BSc (Microbiology and Biochemistry) (Rhodes)
Postgrad: BSc (Hons) (Biotechnology) (Rhodes)
Characterization of inhibitors of heat shock protein 90 (Hsp90) signalling pathways in breast cancer cells and breast cancer stem cells
Many tumours arise from a single cell that has been transformed into a cancer stem cell that has the capacity to proliferate and form tumours. Conventional anti-cancer therapies such as chemotherapy and radiation therapy may be limited in their effectiveness against most metastatic cancers due to the fact that they selectively target differentiated cells only. Therefore targeting of components of self-renewal pathways provides a more specific approach to the elimination of cancer stem cells. My work is focused on characterizing the effects of several molecules on the molecular chaperone, Hsp90, as is it involved in the maturation and stabilization of signal transducers and transcriptional regulators. A large number of this chaperone's client proteins have been found to play essential roles oncogenesis due to stimulation of signalling pathways. Therefore, the inhibition of Hsp90 represents a new target of anti-cancer therapy since it may influence many specific signalling and self-renewal pathways. Information regarding Hsp90-modulated signalling pathways and cancer stem cells is limited. This study will provide valuable insight into the Hsp-90 modulated pathways in cancer stem cells and will allow for the development of novel Hsp90 focused anti-cancer therapies.
2009 - M.Sc (Biochemistry) Rhodes University
2008 - B.Sc Hons. (Microbiology), Rhodes University
2005 - 2007 BSc (Microbiology and Biochemistry), Rhodes University
Descriptions for the following MSc students are under construction: Amy Kenyon, Lola Afolayan, Nana Maphumulo, Tarryn Willmer, & Ndumiso Mhlongo________________________________________________________