Papers by Ilya Serebriiskii
BMC cancer, Jan 28, 2015
Overexpression or mutation of the epidermal growth factor receptor (EGFR) potently enhances the g... more Overexpression or mutation of the epidermal growth factor receptor (EGFR) potently enhances the growth of many solid tumors. Tumor cells frequently display resistance to mechanistically-distinct EGFR-directed therapeutic agents, making it valuable to develop therapeutics that work by additional mechanisms. Current EGFR-targeting therapeutics include antibodies targeting the extracellular domains, and small molecules inhibiting the intracellular kinase domain. Recent studies have identified a novel prone extracellular tetrameric EGFR configuration, which we identify as a potential target for drug discovery. Our focus is on the prone EGFR tetramer, which contains a novel protein-protein interface involving extracellular domain III. This EGFR tetramer is computationally targeted for stabilization by small molecule ligand binding. This study performed virtual screening of a Life Chemicals, Inc. small molecule library of 345,232 drug-like compounds against a molecular dynamics simulation...
Methods in molecular biology (Clifton, N.J.), 2004
To characterize a protein's function, it is often advantageous to identify other proteins wit... more To characterize a protein's function, it is often advantageous to identify other proteins with which it interacts. The yeast two-hybrid system is one of the most versatile methods available for detection and characterization of protein-protein interactions, and in the recent years it has become a mature and robust technology. A further improvement to this technique is the ability to examine and distinguish more than one interaction simultaneously. This is achieved in the Dual Bait, which has successfully been used to detect proteins and peptides that target specific motifs in larger proteins, to facilitate rapid identification of specific interactors from a pool of putative interacting proteins obtained in a library screen, and to score specific drug-mediated disruption of protein-protein interaction.
BMC biotechnology, Jan 29, 2004
Whether for cell culture studies of protein function, construction of mouse models to enable in v... more Whether for cell culture studies of protein function, construction of mouse models to enable in vivo analysis of disease epidemiology, or ultimately gene therapy of human diseases, a critical enabling step is the ability to achieve finely controlled regulation of gene expression. Previous efforts to achieve this goal have explored inducible drug regulation of gene expression, and construction of synthetic promoters based on two-hybrid paradigms, among others. In this report, we describe the combination of dimerizer-regulated two-hybrid and tetracycline regulatory elements in an ordered cascade, placing expression of endpoint reporters under the control of two distinct drugs. In this Dual Drug Control (DDC) system, a first plasmid expresses fusion proteins to DBD and AD, which interact only in the presence of a small molecule dimerizer; a second plasmid encodes a cassette transcriptionally responsive to the first DBD, directing expression of the Tet-OFF protein; and a third plasmid e...
Matrix biology : journal of the International Society for Matrix Biology, 2008
Recent studies have emphasized the importance of cellular microenvironment in modulating cell gro... more Recent studies have emphasized the importance of cellular microenvironment in modulating cell growth and signaling. In vitro, collagen matrices, Matrigel, and other synthetic support systems have been used to simulate in vivo microenvironments, and epithelial cells grown in these matrices manifest significant differences in proliferation, differentiation, response to drugs, and other parameters. However, these substrates do not closely resemble the mesenchymal microenvironment that is typically associated with advanced carcinomas in vivo, which is produced to a large extent by fibroblasts. In this study, we have evaluated the ability of a fibroblast-derived three-dimensional matrix to regulate the growth of a panel of 11 human tumor epithelial cell lines. Although proliferative and morphological responses to three-dimensional cues segregated independently, general responsiveness to the matrix correlated with the ability of matrix to influence drug responses. Fibroblast-derived three...
Methods in Enzymology, 2001
Methods in Molecular Biologyâ„¢, 2007
Yeast two-hybrid systems are artificial genetic systems that allow identification and characteriz... more Yeast two-hybrid systems are artificial genetic systems that allow identification and characterization of protein-protein interactions. One common limit to the use of these techniques is when the intrinsic property of "bait" proteins of interest transcriptionally autoactivates reporters, eliminating the basis for interaction detection. To circumvent this problem, autoactivating baits can be alternatively used in bacteria wherein such activation does not occur. A single-vector system has been developed, which can be used either in yeast or in bacteria, streamlining and expanding capacity for protein-protein interaction screens. A concise proposal is provided for use of this system in bacteria; a companion article, chapter 15, describes use of the system in yeast.
The Proteomics Protocols Handbook, 2005
Protein-protein interactions play an essential role in all living systems, and hence their analys... more Protein-protein interactions play an essential role in all living systems, and hence their analysis is of foremost importance in molecular biology. Although there are num-ber of methods to detect protein-protein interactions, the yeast two-hybrid system is probably the ...
Current Protocols in Neuroscience, 2001
This unit presents protocols designed to detect interacting proteins. Using yeast as a "... more This unit presents protocols designed to detect interacting proteins. Using yeast as a "test tube" and transcriptional activation of a reporter system, interacting proteins can be identified. The system can also be used to test complex formation for proteins for which there exists a reason to expect interaction.
Methods in Microbiology, 2007
Proceedings of the National Academy of Sciences of the United States of America, 2002
The interaction of activated Ras with Raf initiates signaling cascades that contribute to a signi... more The interaction of activated Ras with Raf initiates signaling cascades that contribute to a significant percentage of human tumors, suggesting that agents that specifically disrupt this interaction might have desirable chemotherapeutic properties. We used a subtractive forward two-hybrid approach to identify small molecule compounds that block the interaction of Ras with Raf. These compounds (MCP1 and its derivatives, 53 and 110) reduced seruminduced transcriptional activation of serum response element as well as Ras-induced transcription by way of the AP-1 site. They also inhibited Ras-induced Raf-1 activation in human embryonic kidney 293 cells, Raf-1 and mitogen-activated protein kinase kinase 1 activities in HT1080 fibrosarcoma cells, and epidermal growth factor-induced Raf-1 activation in A549 lung carcinoma cells. The MCP compounds caused reversion of ras-transformed phenotypes including morphology, in vitro invasiveness, and anchorage-independent growth of HT1080 cells. Decreased level of matrix metalloproteinases was also observed. Further characterization showed that MCP compounds restore actin stress fibers and cause flat reversion in NIH 3T3 cells transformed with H-Ras (V12) but not in NIH 3T3 cells transformed with constitutively active Raf-1 (Raf⌬N). Finally, we show that MCP compounds inhibit anchorage-independent growth of A549 and PANC-1 cells harboring K-ras mutation. Furthermore, MCP110 caused G1 enrichment of A549 cells with the decrease of cyclin D level. These results highlight potent and specific effects of MCP compounds on cancer cells with intrinsic Ras activation.
Journal of immunology (Baltimore, Md. : 1950), 2014
Stable surface expression of human inhibitory killer cell Ig-like receptors (KIRs) is critical fo... more Stable surface expression of human inhibitory killer cell Ig-like receptors (KIRs) is critical for controlling NK cell function and maintaining NK cell tolerance toward normal MHC class I(+) cells. Our recent experiments, however, have found that Ab-bound KIR3DL1 (3DL1) readily leaves the cell surface and undergoes endocytosis to early/recycling endosomes and subsequently to late endosomes. We found that 3DL1 internalization is at least partially mediated by an interaction between the μ2 subunit of the AP-2 clathrin adaptor complex and ITIM tyrosine residues in the cytoplasmic domain of 3DL1. Disruption of the 3DL1/μ2 interaction, either by mutation of the ITIM tyrosines in 3DL1 or mutation of μ2, significantly diminished endocytosis and increased surface expression of 3DL1 in human primary NK cells and cell lines. Furthermore, we found that the 3DL1/AP-2 interaction is diminished upon Ab engagement with the receptor, as compared with untreated cells. Thus, we have identified AP-2-m...
Proteins: Structure, Function, and Bioinformatics, 2007
Functional complementation screens can identify known or novel proteins with important intracellu... more Functional complementation screens can identify known or novel proteins with important intracellular activities. We have isolated human enhancer of filamentation 2 (HEF2) in a screen to find human genes that promote pseudohyphal growth in budding yeast. HEF2 is identical to enhancer of rudimentary homolog (ERH), a highly conserved protein of 104 amino acids. In silico protein-interaction mapping implies that HEF2/ERH interacts with transcription factors, cell-cycle regulators, and other proteins shown to enhance filamentous growth in S. cerevisiae, suggesting a context for studies of HEF2/ERH function. To provide a mechanistic basis to study of HEF2/ERH, we have determined the crystal structure of HEF2/ERH at 1.55 Å . The crystal asymmetric unit contains a HEF2/ERH monomer. The two monomers of the physiological dimer are related by the y, x, Àz crystal symmetric operation. The HEF2/ERH structure is characterized by a novel a + b fold, a four-strand antiparallel b-sheet with three a-helixes on one side of the sheet. The b-sheets from the two monomers together constitute a pseudo-b-barrel, and form the center of the functional HEF2/ERH dimer, with a cavity channel at the dimer interface. Docking of this structure to the HEF2/ERH partner protein DCOH/PCD suggests that HEF2/ERH may regulate the oligomeric state of this protein. These data suggest that HEF2/ERH may be an important transcription regulator that also functions in the control of cell-cycle progression. Proteins 2007;68:427-437. V V C 2007 Wiley-Liss, Inc.
Molecular Biology of the Cell, 2008
For over a decade, p130Cas/BCAR1, HEF1/NEDD9/Cas-L, and Efs/Sin have defined the Cas (Crk-associa... more For over a decade, p130Cas/BCAR1, HEF1/NEDD9/Cas-L, and Efs/Sin have defined the Cas (Crk-associated substrate) scaffolding protein family. Cas proteins mediate integrin-dependent signals at focal adhesions, regulating cell invasion and survival; at least one family member, HEF1, regulates mitosis. We here report a previously undescribed novel branch of the Cas protein family, designated HEPL (for HEF1-Efs-p130Cas-like). The HEPL branch is evolutionarily conserved through jawed vertebrates, and HEPL is found in some species lacking other members of the Cas family. The human HEPL mRNA and protein are selectively expressed in specific primary tissues and cancer cell lines, and HEPL maintains Cas family function in localization to focal adhesions, as well as regulation of FAK activity, focal adhesion integrity, and cell spreading. It has recently been demonstrated that upregulation of HEF1 expression marks and induces metastasis, whereas high endogenous levels of p130Cas are associated with poor prognosis in breast cancer, emphasizing the clinical relevance of Cas proteins. Better understanding of the complete protein family should help inform prediction of cancer incidence and prognosis.
Molecular & Cellular Proteomics, 2005
Two-hybrid screening is a standard method used to identify and characterize protein-protein inter... more Two-hybrid screening is a standard method used to identify and characterize protein-protein interactions and has become an integral component of many proteomic investigations. The two-hybrid system was initially developed using yeast as a host organism. However, bacterial two-hybrid systems have also become common laboratory tools and are preferred in some circumstances, although yeast and bacterial two-hybrid systems have never been directly compared. We describe here the development of a unified yeast and bacterial two-hybrid system in which a single bait expression plasmid is used in both organismal milieus. We use a series of leucine zipper fusion proteins of known affinities to compare interaction detection using both systems. Although both two-hybrid systems detected interactions within a comparable range of interaction affinities, each demonstrated unique advantages. The yeast system produced quantitative readout over a greater dynamic range than that observed with bacteria. However, the phenomenon of "autoactivation" by baits was less of a problem in the bacterial system than in the yeast. Both systems identified physiological interactors for a library screen with a cI-Ras test bait; however, non-identical interactors were obtained in yeast and bacterial screens. The ability to rapidly shift between yeast and bacterial systems provided by these new reagents should provide a marked advantage for two-hybrid investigations. In addition, the modified expression vectors we describe in this report should be useful for any application requiring facile expression of a protein of interest in both yeast and bacteria.
Methods, 2004
The two-step two-hybrid approach described here is an adaptation of the classic two-hybrid system... more The two-step two-hybrid approach described here is an adaptation of the classic two-hybrid system. Its purpose is to identify proteins that interact with a relatively small, defined, functionally significant domain of a protein of interest. In this method, a first round of screening is performed to identify proteins that interact with bait comprised of the wild type protein. Next, each of the prey identified in this first round is tested for its ability to interact with functionally impaired, mutant bait. Any proteins that interact with the wild type bait, but not the mutant bait, are candidate effectors or regulators of the protein of interest.
Journal of Biological Chemistry, 2001
p21-activated kinase 1 (Pak1) is an effector for the small GTPases Cdc42 and Rac. Because Pak1 bi... more p21-activated kinase 1 (Pak1) is an effector for the small GTPases Cdc42 and Rac. Because Pak1 binds to and is activated by both these GTPases, it has been difficult to precisely delineate the signaling pathways that link extracellular stimuli to Pak1 activation. To separate activation of Pak1 by Cdc42 versus activation by Rac, we devised a genetic screen in yeast that enabled us to create and identify Pak1 mutants that selectively couple to Cdc42 but not Rac1. We recovered several such Pak1 mutants and found that the residues most often affected lie within the p21 binding domain, a region previously known to mediate Pak1 binding to GTPases, but that several mutations also map outside the borders of the p21 binding domain. Pak1 mutants that associate with Cdc42 but not Rac1 were also activated by Cdc42 but not Rac1. In rat 3Y1 cells expressing oncogenic Ha-Ras, the Pak1 mutants defective in Rac1 binding are not activated, suggesting that Ras signals through a GTPase other than Cdc42 to activate Pakl. Similar results were obtained when epidermal growth factor was used to activate Pak1. However, Pak1 mutants that are unable to bind Rac are nonetheless well activated by calf serum, implying that this stimulus may induce Pak activation independent of Rac.
Journal of Biological Chemistry, 1999
Biological regulatory systems require the specific organization of proteins into multicomponent c... more Biological regulatory systems require the specific organization of proteins into multicomponent complexes. Two hybrid systems have been used to identify novel components of signaling networks based on interactions with defined partner proteins. An important issue in the use of two-hybrid systems has been the degree to which interacting proteins distinguish their biological partner from evolutionarily conserved related proteins and the degree to which observed interactions are specific. We adapted the basic two-hybrid strategy to create a novel dual bait system designed to allow single-step screening of libraries for proteins that interact with protein 1 of interest, fused to DNA binding domain A (LexA), but do not interact with protein 2, fused to DNA binding domain B ( cI). Using the selective interactions of Ras and Krev-1(Rap1A) with Raf, RalGDS, and Krit1 as a model, we systematically compared LexA-and cI-fused baits and reporters. The LexA and cI baitr reporter systems are well matched for level of bait expression and sensitivity range for interaction detection and allow effective isolation of specifically interacting protein pairs against a nonspecific background. These reagents should prove useful to refine the selectivity of library screens, to reduce the isolation of false positives in such screens, and to perform directed analyses of sequence elements governing the interaction of a single protein with multiple partners.
Genomics, 2001
A notable difficulty in annotating genomic sequence is identifying the correct start codon in a g... more A notable difficulty in annotating genomic sequence is identifying the correct start codon in a gene. An important such case has been found with KRIT1, the cerebral cavernous malformation type 1 (CCM1) gene. Analysis of human and mouse genomic sequence encompassing the region containing KRIT1/Krit1 using exon/gene-prediction and comparative alignment programs revealed putative exons upstream of the previously described first exon. These additional candidate exons show significant matches to mouse and human ESTs that are contiguous with and extend upstream from the previously designated 5 end of the KRIT1 cDNA sequence. RT-PCR and 5RACE experiments confirm the presence of four additional upstream coding exons that encode an additional 207 amino acids. Importantly, a novel frameshift mutation in one of these newly identified KRIT1 exons has been found in a CCM1 family. These data establish the authentic KRIT1 amino acid sequence and suggest that the additional KRIT1 exons may harbor mutations in other CCM1 families. In addition, these results provide another example of the utility of rigorous computational and comparative sequence analysis for refining gene structure.
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Papers by Ilya Serebriiskii