Ziwei Huang

Molecular Determinants of the Caspase-promoting Activity of Smac/DIABLO and Its Role in the Death Receptor Pathway

Smac/DIABLO is a mitochondrial protein that is released along with cytochrome c during apoptosis and promotes cytochrome c-dependent caspase activation by neutralizing inhibitor of apoptosis proteins (IAPs). We provide evidence that Smac/DIABLO functions at the levels of both the Apaf-1-caspase-9 apoptosome and effector caspases. The N terminus of Smac/DIABLO is absolutely required for its ability to interact with the baculovirus IAP repeat (BIR3) of XIAP and to promote cytochrome c-dependent caspase activation. However, it is less critical for its ability to interact with BIR1/BIR2 of XIAP and to promote the activity of the effector caspases. Consistent with the ability of Smac/DIABLO to function at the level of the effector caspases, expression of a cytosolic Smac/DIABLO in Type II cells allowed TRAIL to bypass Bcl-xL inhibition of death receptor-induced apoptosis. Combined, these data suggest that Smac/DIABLO plays a critical role in neutralizing IAP inhibition of the effector caspases in the death receptor pathway of Type II cells.

Generation of Constitutively Active Recombinant Caspases-3 and -6 by Rearrangement of Their Subunits

Caspases play a major role in the transduction of the apoptotic signal and execution of apoptosis in mammalian cells. Ectopic overexpression of the short prodomain caspases-3 and -6 precursors in mammalian cells does not induce apoptosis. This is due to their inability to undergo autocatalytic processing/activation and suggests that they depend on the long prodomain caspases for activation. To investigate directly the apoptotic activity of these two caspases in vivo, we engineered constitutively active recombinant caspases-3 and -6 precursors. This was achieved by making contiguous precursor caspases-3 and -6 molecules, which have their small subunits preceding their large subunits. Unlike their wild type counterparts, these recombinant molecules were capable of autocatalytic processing in an in vitro translation reaction, suggesting that they are catalytically active. They were also capable of autoprocessing and inducing apoptosis in vivo independent of the upstream caspases. Furthermore, their autocatalytic and apoptotic activities were inhibited by the pancaspase inhibitor z-VAD-fluoromethylketone, but not by CrmA or Bcl-2, thus directly demonstrating that the targets of inhibition of apoptosis by CrmA and Bcl-2 are upstream of caspases-3 and -6. Since caspases-3 and -6 are the most downstream executioners of apoptosis, the constitutively active versions of these caspases could be used at very low concentrations in gene therapy model systems to induce apoptosis in target tissues or tumors.

Reactions of salicylaldehydes with alkyl cyanoacetates on the surface of solid catalysts: syntheses of 4H-chromene derivatives

Substituted 4H-chromene derivatives are a new class of compounds that bind Bcl-2 protein and induce apoptosis in tumor cells. Here we report an efficient synthetic method for the preparation of these compounds from salicylaldehyde derivatives and alkyl cyanoacetates under solid-phase catalysis.

CD4 dimerization and oligomerization: implications for T-cell function and structure-based drug design

Recent studies of CD4 structure and function have revealed possible mechanisms for CD4 self-association, with implications for its role in T-cell activation. Here, the authors discuss the formulation of a hypothetical three-dimensional model of CD4 oligomerization and how it impacts on the understanding of T-cell function and rational drug design targeting specific CD4 surface functional sites.

SYNTHETIC PEPTIDE SEQUENCE FROM THE C-TERMINUS OF THE INSULIN-LIKE GROWTH FACTOR-I RECEPTOR THAT INDUCES APOPTOSIS AND INHIBITION OF TUMOR GROWTH

Although the type 1 insulin‐like growth factor receptor (IGF‐IR) is a potent inhibitor of apoptosis, its C‐terminus sequence sends contradictory signals, including a clearly proapoptotic signal. We have synthesized a peptide, peptide 2, having the sequence of the IGF‐IR from residue 1282 to residue 1298 (C‐terminus of the β subunit). To favor its uptake into cells, we linked it to a stearic acid moiety at its NH‐terminus. Peptide 2 is taken up by the cells, where it inhibits DNA synthesis and causes apoptosis, while a scrambled peptide (with stearic acid) and peptide 2 without stearic acid are completely ineffective. Peptide 2 is more effective when cells are in anchorage‐independent conditions than when they grow in monolayer cultures. Accordingly, we find that peptide 2 can inhibit the growth of a human prostatic cell line in nude mice. The proapoptotic effect of peptide 2 is inhibited by the expression of Bcl‐2 or by a dominant negative mutant of caspase 9. These and other data indicate that peptide 2 does not seem to be competing directly with the IGF‐IR for common substrates, but that its proapoptotic effect is related to its ability to activate the caspase cascade. J. Cell. Physiol. 181:124–135, 1999.

Identification of the CD8 DE Loop as a Surface Functional Epitope IMPLICATIONS FOR MAJOR HISTOCOMPATIBILITY COMPLEX CLASS I BINDING AND CD8 INHIBITOR DESIGN

We used an approach of protein surface epitope mapping by synthetic peptides to analyze the surface structure-function relationship of the CD8 protein. Small synthetic peptide mimics of the CD8 DE loop were shown to effectively block CD8 binding to major histocompatibility complex (MHC) class I molecules and possess significant inhibitory activity on in vitroCD8+ T cell function. These results suggested that the DE loop region of the CD8 protein is an important functional epitope mediating CD8-MHC class I interaction and the activation of CD8+ T cells, a finding that is consistent with the recently reported crystal structure of the CD8-MHC class I complex. The structural basis for the biological activity of the DE loop peptide was further analyzed in a series of analogs containing alanine substitutions. This study provides support for the concept of bioactive peptide design based on protein surface epitopes and suggests that such an approach may be applicable to other protein-protein complexes, particularly those of immunoglobulin superfamily molecules.

A natural motif approach to protein design: a synthetic leucine zipper peptide mimics the biological function of the platelet factor 4 protein

The design of smaller functional mimics of large proteins has long been an important challenge. In this study we use the natural leucine zipper as a structural template to design a 31‐residue peptide analog that mimics the function of the larger platelet factor 4 (PF4) protein. The heparin binding activity of PF4 has been introduced into an unrelated leucine zipper sequence only by virtue of incorporating four lysines of PF4. Circular dichroism and binding experiments have shown that the designed leucine zipper peptide adopts a stable helical conformation and shows significant PF4‐like heparin binding activity. These results strongly suggest that the lysine residues play an important role in the binding of PF4 to heparin. The de novo generation of the PF4 function in a designed leucine zipper peptide demonstrates that the leucine zipper motif is a useful scaffold for the design of functional peptides and proteins.

Control of apoptosis by using small molecule regulators of Bcl-2 family proteins

The potent biological activity of CPM-1285 suggests that it may represent a promising lead for the development of new anticancer agents. The cell permeable Bcl-2 inhibitor can also be used as a chemical probe to study the in vivo mechanism and signaling pathway of the Bcl-2 family. Unlike other peptides that are active only in vitro or in the cell-free system, the cell-permeable peptide approach described here provides a new tool to analyze the function of the Bcl-2 family in living cells and animals.

A natural motif approach to protein folding and design [1]

The design of smaller functional mimics of large proteins has long been an important challenge. In this study we use the natural leucine zipper as a structural template to design a 31-residue peptide analog that mimics the function of the larger platelet factor 4 (PF4) protein. The heparin binding activity of PF4 has been introduced into an unrelated leucine zipper sequence only by virtue of incorporating four lysines of PF4. Circular dichroism and binding experiments have shown that the designed leucine zipper peptide adopts a stable helical conformation and shows significant PF4-like heparin binding activity. These results strongly suggest that the lysine residues play an important role in the binding of PF4 to heparin. The de novo generation of the PF4 function in a designed leucine zipper peptide demonstrates that the leucine zipper motif is a useful scaffold for the design of functional peptides and proteins.

Identification of a novel human CD8 surface region involved in MHC class I binding

Human CD8 (hCD8) is a glycoprotein expressed on the surface of T cells that has specificity for antigens presented by major histocompatibility complex (MHC) class I proteins. hCD8 functions as a co-receptor with the T-cell antigen receptor (TCR) by binding to the MHC class I molecule on the antigen-presenting cell, thereby increasing the avidity of the TCR for its ligand. In this study, we have utilized an approach combining molecular modeling, synthetic peptide mapping, and hCD8-MHC class I binding assay to probe precise surface regions of hCD8 involved in MHC class I binding. These experiments have led to the identification of the DE loop of hCD8 protein that is implicated in the hCD8 interaction with MHC class I molecules. Synthetic conformationally restricted peptide analogs derived from the hCD8 DE loop have been shown to specifically inhibit hCD8-MHC class I binding. To further define the functional role of each residue of the DE loop, DE loop peptide analogs containing a series of alanine substitutions were synthesized and tested. These studies provide information about the structure-function relationship between the surface structural feature of the CD8 DE loop and CD8 mediated immunological function.