Facility Director of Biopolymer Facility & Mass Spectrometry
Roswell Park Cancer Institute
Elm and Carlton Streets
Buffalo NY 14263
Telephone: (716) 845-3055
Fax: (716) 845-7621
E-mail: latif.kazim@roswellpark.org

Education
Boston University, Boston, MA, BA, 1971, Chemistry/Biology
Wayne State University, Detroit, MI, MA, 1974, Biochemistry
University of Minnesota/Mayo Medical School, Rochester, MN, PhD, 1979, Biochemistry/Immunology
Mayo Medical School, Rochester, MN, Postdoctoral, 1979-1981, Immunology

General Research Interest
Research interests in this laboratory focus mainly on structure/activity relationships in proteins and peptides. An ongoing project involves the development of synthetic peptide agonists and antagonists of TGF-b that bind to TGF-b receptors and may therefore have therapeutic applications. We have recently synthesized conformationally constrained peptides of TGF-b and demonstrated that they bind to the TGF-b type III receptor. Using these peptides as templates we are currently attempting to design peptides with higher binding affinities for the TR III which may act as effective competitors (antagonists) of TGF-b .
In collaboration with Dr. John Subjeck, we are isolating and purifying heat shock proteins, including hsp110 and the related protein grp170, from tumor cells in order to determine whether these proteins are capable of inducing tumor-specific immunity. We are also collaborating on the identification of a consensus motif for peptides that bind to hsp110 and grp170.

With Dr. Thomas Tomasi, whose laboratory is studying the regulation of MHC class II gene expression, we are developing fluorescence-based quantitative PCR methods for determining the levels of expression in various cell types of mRNA coding for the MHC class II genes, and for genes that are thought to regulate class II gene expression.

Description of Research

Heat Shock Proteins and Hyperthermia Stress proteins are known to play essential roles in normal cellular functions and are involved in numerous pathways involving protein processing and interactions. They correspondingly protect cells from a variety of protein and cellular damaging environments at the molecular, cellular and organismal levels. Several of the stress proteins have come under intense scrutiny. However, from the earliest studies, major stress proteins known as hsp110 (in the cytoplasm) and grp170 (in the endoplasmic reticulum) were observed, but studies of their structure and function were not undertaken; that is, until recently. Recent cloning of these proteins in this laboratory indicated that they are related to one another and are also "distant" relatives of the intensively studied hsp70 family. Moreover, they have now been observed in every eucaryotic cell examined, from yeast to man. It is our overall goal to determine the functions of these large stress proteins and how they compare to and interact with the hsp70s as well as other molecular chaperones. Secondly, we are studying hsp110 and grp170 as potential cancer vaccines. We have demonstrated that both hsp110 and grp170 are highly efficient peptide chain binding proteins and it has been shown that other stress proteins possess specific functions in the immune response. Based on their ability to bind antigenic peptide, we are developing various approaches for the use of hsp110 and grp170 in cancer therapy. This specifically utilizes synthetic vaccines composed of recombinant proteins. Moreover, we are investigating the molecular/cellular mechanisms by which hsp110, hsc70 and grp170 interact with antigen presenting cells in eliciting an immune response.

Progress

Characterization of the molecular-biophysical properties of hsp110. Heat shock proteins are often observed to bind to denatured protein and inhibit protein aggregation. The ability of recombinant, full-length hsp110 to perform this function has been previously assessed using two reporter proteins, luciferase and citrate synthase. It was found that hsp110 inhibits heat induced protein aggregation and holds denatured protein in a folding competent state in a manner similar to hsc70, but is far more efficient than hsc70 in carrying out this important chaperoning function.

We have subsequently identified the functional domains of hsp110 which are responsible for its chaperoning activity by targeted deletion mutagenesis, using the known structure of DnaK as the model. The chaperoning ability of mutants was again assessed based on their ability to solubilize heat denatured luciferase as well as to refold luciferase in the presence of rabbit reticulocyte lysate. It was shown that these functions require only an internal region of hsp110 which includes the predicted peptide binding domain and two immediately adjacent C-terminal domains.

It is also shown that hsp110 is approximately four fold more efficient in stabilizing demature protein than is hsp70. Similar studies of the structure and function of grp170 are underway to define and near completion. Grp170 exhibits many of the structural/functional properties of hsp110, while also differing in important ways. Grp170 is also a potent peptide chain binding protein.

Lastly, several studies have confirmed that certain stress proteins can function as potent vaccines against a specific cancer when purified from the same tumor. This is based on the peptide binding characteristics of some stress proteins and their apparent involvement in antigen processing pathways. We are presently examining the vaccine potential of hsp110 and grp170. We have shown that prior vaccination with hsp110 or grp170 purified from Meth A fibrosarcoma caused complete regression of this tumor while hsp110 or grp170 purified from Colon 26 tumors lead to a significant growth inhibition of this highly aggressive tumor. In each case, a tumor specific cytotolytic T lymphocyte response developed in the mice immunized with tumor derived hsp110 or grp170. The process could be replicated by treatment of purified dendritic cells (DCs) in vitro and use of these cells as a vaccine. The mechanism of binding and intracellular trafficking of hsp110 and grp170 by DCs is being examined. Furthermore, since clinical use of "heat shock vaccine therapy" can be limited by the quantity of hsp/grp which can be purified from a human tumor (surgical) specimen, we have begun the characterization of synthetic, recombinant protein based vaccines. These studies are founded in our understanding of the structural/molecular chaperoning properties of hsp110 and grp170 referred to above. Recent data indicate that recombinant, molecular-targeted vaccines elicit powerful and specific CTL responses, suggesting that this may represent a new and highly potent approach to cancer therapy.

Key Publications

 

• Kazim AL and Atassi MZ: Prediction and Confirmation by Synthesis of Two Antigenic Sites in Human Haemoglobin by Extrapolation from the Known Antigenic Structure of Sperm-Whale Myoglobin. Biochem. J., 167:272-278, 1977. Kazim AL and Atassi MZ: Antibodies Against Protein Antigenic Sites that are Identical in the Homologous Protein of the Immunized Animal. Autoreactivity in Rabbits of Antibodies to Sperm-Whale Myoglobin. Biochem. Biophys. Acta, 494:277-282, 1977.
• Kazim AL and Atassi MZ: Production of Autoantibodies by Immunization with Rabbit Myoglobin. Immunochem., 15:67-70,1978.
• Kazim AL, Habeeb AFSA and Atassi MZ: Immunochemistry of Serum Albumin 7. Effects of Chemical Modification of Tyrosine and Arginine Residues on the Conformation and Immunochemistry of a Fragment Corresponding to the Last Third of Bovine Serum Albumin. Molecular Immunology, 16:457-464, 1979.
• Atassi MZ, Sakata S and Kazim AL: Localization and Verification by Synthesis of Five Antigenic Sites of Bovine Serum Albumin. Biochem. J., 179:327-331, 1979.
• Kazim AL and Atassi MZ: Haemoglobin Binding with Haptoglobin. Unequivocal Demonstration that the b-Chains of Human Haemoglobin Bind to Haptoglobin. Biochem. J., 185:285-287, 1980.
• Kazim AL and Atassi MZ: A Novel and Comprehensive Synthetic Approach for the Elucidation of Protein Antigenic Structures. Biochem. J., 191:261-264, 1980.
• Kazim AL and Atassi MZ: Nearest Neighbor Analysis of Myoglobin Antigenic Sites. Nearest Neighbor Residues Whose Replacements Can Alter the Environment of Binding Site Residue(s) and thus Change their Characteristics and Binding Capability. Biochem. J., 191:673-680, 1980.
• Kazim AL and Atassi MZ: Hemoglobin Binding with Haptoglobin: Localization of the Haptoglobin Binding Site on the a-Chain of Human Hemoglobin. Biochem. J., 197:507-510, 1981.
• Krco CJ, Kazim AL, Atassi MZ and David, CS: Genetic Control of the Immune Response to Hemoglobin. I. Demonstration of Separate Genetic Control of the Responses to the a- and b-Subunits by In Vitro Lymphocyte Proliferation. J. Immunogenetics, 8:315-322, 1981.
• Atassi MZ, Kazim AL and Sakata S.: High Yield Coupling of Peptides to Protein Carriers. Biochim. Biophys. Acta, 670:300-2, 1981.
• Kazim AL and Atassi MZ: Structurally Inherent Antigenic Sites. Localization of the Antigenic Sites of Human Hemoglobin in Three Host Species by a Comprehensive Synthetic Approach. Biochem. J. 203:201-208, 1982.
• McKean DJ, Nilson A, Infante AJ and Kazim, AL: Biochemical Characterization of B Lymphoma Cell Antigen Processing and Presentation to Antigen Reactive T-Cells. J. Immunol., 131:2726-2734, 1983.
• Griffith, JK, Kogoma, T, Corvo, DL, Anderson, W and Kazim, AL: Pleiotropic Effects of Tetracycline Resistance: Characterization of a TET Protein Domain that Confers Increased Susceptibility to Aminoglycoside Antibiotics and Complements Potassium Uptake in E. Coli. J. Bacteriology, 170:589-604, 1988.
• Klemm DJ, Kazim AL and Elias L: Phosphatidylglycerol Modulated Protein Kinase Activity From Human Spleen. I. Enzyme Purification and Properties. Arch. Biochem. Biophys., 265:496-505, 1988.
• Wildgoose, P, Kazim, AL and Kisiel, W.: The importance of residues 195-206 of human blood clotting factor VII in the interaction of factor VII with tissue factor. Proc. Natl. Acad. Sci. USA 87:7290-7294, 1990.
• Nakagaki, T, Kazim, AL and Kisiel, W: Isolation and characterization of a protein C activator from tropical moccasin venom. Thrombosis Res., 58:593-602, 1990.
• Kazim, AL : Identification of putative internalization signals in prion proteins. FEBS Letters, 331:1-3, 1993.
• Woodfin, BM and Kazim, AL . Interaction of the amino-terminus of an influenza virus protein with mitochondria. Arch. Biochem. Biophys., 306:427-430, 1993.
• Sarma, RH, Sarma, MH, Rein, R, Shibata, M, Setlik, RF, Ornstein, RL, Kazim, AL, Cairo, A and Tomasi, TB. Secondary Structure in Solution of Two Anti-HIV-1 Ribozymes as Investigated by Two-Dimensional 1H 500 MHz NMR Spectroscopy in Water. FEBS Letters, 357:317-323, 1995.
• Setlik RF, Shibata, M, Sarma, RH, Sarma, MH, Kazim, AL, Ornstein, RL, Tomasi, TB and Rein, R. Modeling of a Possible Conformational Change Associated with the Catalytic Mechanisms in the Hammerhead Ribozyme. J. Biomolec. Struc. Dynam., 13:515-522, 1995.
• Chadha, KC, Kazim, AL, Parthasarathy, R, Sulkowski, E, and Tomasi, TB. Heparin Binding Sites on Prions. Intl. J. BioChromatog., 2:211-223, 1997.
• Ojha, RP, Dhingra, MM, Sarma, MH, Myer, YP, Setlik, RF, Shibata, M, Kazim, AL, Ornstein, RL, Rein, R, Turner, CJ and Sarma, RH. Structure of an Anti-HIV Hammerhead Ribozyme Complex with a 17-mer DNA Substrate Analog of HIV-1 gag RNA and a Mechanism for the Cleavage Reaction: 750 MHz NMR and Computer Experiments. J. Biomolec. Struc. Dynam., 15:185-215, 1997.
• Wang, X-Y, Chen, X, Oh, HJ, Repasky, E, Kazim, AL and Subjeck, J. Characterization of Native Interaction of Hsp110 with Hsp70 and Hsp25. F.E.B.S. Letters 465:98-102, 2000.
• Magner WJ, Kazim AL, Stewart C, Romano MA, Catalano G, Grande C, Keiser N, Santaniello F, Tomasi TB. Avtivation of MHC class I, II and CD40 gene expression by histone deacetylase inhibitors. J. Immunol. 165:7017-7024, 2000.
• Wang X, Kazim AL, Kaneko Y, Repasky E, Subjeck J. Antitumor immunity elicited by two high-molecular weight heat shock proteins. J. Immunol., in press.
• Zheng, X, Salzler, RR, Kunz, DP, Baer, MR, Kazim, AL, Baumann, H and Wetzler, M A Novel Serine-Dependent Proteolytic Activity is Responsible for Truncated Signal Transducer and Activator of Transcription Proteins in Acute Myeloid Leukemia Blasts. Cancer Res., in press.