Nickolay Neznanov

Differential Association of Products of Alternative Transcripts of the Candidate Tumor Suppressor ING1 with the mSin3/HDAC1 Transcriptional Corepressor Complex

The candidate tumor suppressor ING1was identified in a genetic screen aimed at isolation of human genes whose expression is suppressed in cancer cells. It may function as a negative growth regulator in the p53 signal transduction pathway. However, its molecular mechanism is not clear. The ING1locus encodes alternative transcripts of p47 ING1a , p33 ING1b , and p24 ING1c . Here we report differential association of protein products of ING1 with the mSin3 transcriptional corepressor complex. p33 ING1b associates with Sin3, SAP30, HDAC1, RbAp48, and other proteins, to form large protein complexes, whereas p24 ING1c does not. The ING1 immune complexes are active in deacetylating core histones in vitro, and p33 ING1b is functionally associated with HDAC1-mediated transcriptional repression in transfected cells. Our data provide basis for a p33 ING1b -specific molecular mechanism for the function of the ING1 locus.

Poliovirus Protein 3A Inhibits Tumor Necrosis Factor (TNF)-Induced Apoptosis by Eliminating the TNF Receptor from the Cell Surface

ABSTRACT Viral infections often trigger host defensive reactions by activating intrinsic (intracellular) and extrinsic (receptor-mediated) apoptotic pathways. Poliovirus is known to encode an antiapoptotic function(s) suppressing the intrinsic pathway. Here, the effect of poliovirus nonstructural proteins on cell sensitivity to tumor necrosis factor (TNF)-induced (i.e., receptor-mediated) apoptosis was studied. This sensitivity is dramatically enhanced by the viral proteinase 2A, due, most likely, to inhibition of cellular translation. On the other hand, cells expressing poliovirus noncapsid proteins 3A and 2B exhibit strong TNF resistance. Expression of 3A neutralizes the proapoptotic activity of 2A and results in a specific suppression of TNF signaling, including the lack of activation of NF-κB, due to elimination of the TNF receptor from the cell surface. In agreement with this, poliovirus infection results in a dramatic decrease in TNF receptor abundance on the surfaces of infected cells as early as 4 h postinfection. Poliovirus proteins that confer resistance to TNF interfere with endoplasmic reticulum-Golgi protein trafficking, and their effect on TNF signaling can be imitated by brefeldin A, suggesting that the mechanism of poliovirus-mediated resistance to TNF is a result of aberrant TNF receptor trafficking.

Poliovirus Protein 3A Binds and Inactivates LIS1, Causing Block of Membrane Protein Trafficking and Deregulation of Cell Division

Poliovirus infection results in resistance of infected cells to apoptotic stimuli. Viral proteins involved in such functions usually target key cellular regulators. Here we demonstrate that viral protein 3A binds and inactivates LIS1, a component of the dynein/dynactin motor complex, encoded by the gene mutated in patients with type I lissencephaly („smooth brain‰), thus causing rapid disappearence of TNF and interferon receptors from the plasma membrane. Like 3A, truncated derivatives of LIS, acting in a dominant negative manner, deregulate endoplasmatic reticilum -to-Golgi vesicular transport and eliminate unstable receptors from the cell surface. Protein 3A locks Golgi-targeted YFP in endoplasmatic reticilum, while expression of LIS1 mutants results in a dispersed cytoplasmic localization of the reporter protein. LIS1 dysfunction caused by ectopic expressing 3A or LIS1 mutants, as well as by overexpression of wild type LIS1, leads to cell trap at a postmitotic stage associated with inability to undergo cytokinesis. Thus, using vural protein as a research tool, we revealed the role of cellular protein LIS1 in endoplasmatic reticilum -to-Golgi transport, maintenance of Golgi integrity and cell cycle progression. Supplemental Material 1 Supplemental Material 2 Supplemental Material 3

Dominant negative form of signal-regulatory protein-alpha (SIRPa/SHPS-1) inhibits TNF-mediated apoptosis by activation of NF-kB

Genetic suppressor element (GSE) methodology was applied to identify new genes controlling cell response to tumor necrosis factor (TNF). A retroviral library of randomly fragmented normalized cDNA from mouse fibroblasts was screened for GSEs capable of protecting NIH3T3 cells from TNF-induced apoptosis. The most abundant among isolated GSEs represented a fragment of cDNA encoding the C-terminal cytoplasmic region of the immunoglobulin family inhibitory receptor, SHPS-1 (mouse homologue of human SIRPα). Ectopic expression of this fragment (both from human and mouse versions) increased the NF-κB-dependent transcription in three cell lines tested; this effect could be reduced by the expression of full-length SIRPα, suggesting that the isolated GSE acts through a dominant negative mechanism. GSE-mediated activation of NF-κB depended on the presence of serum, was abrogated by wortmannin, and was associated with phosphorylation of PKB/Akt, suggesting that Akt mediates it. These data indicate that SIRPα/SHPS-1 is involved in negative regulation of NF-κB signaling.

The Ability of Protein Tyrosine Phosphatase SHP-1 to Suppress NFκB Can Be Inhibited by Dominant Negative Mutant of SIRPα

In contrast with hematopoietic cells and fibroblasts, which express mainly one form of protein tyrosine phosphatase (PTP) SHP-1 or SHP-2, epithelial cells like A431, HeLa, and 293 express both forms of PTP. These two PTP regulate NFκB activity differently; SHP-1 inhibits and SHP-2 stimulates NFκB activation. In epithelial cells the process of NFκB activation depends on the combination of two PTP activities. The activity of PTP SHP-1 dominates in this tandem according to our data. The signal regulatory protein (SIRPα) is the adapter and the substrate of PTP SHP-1 and SHP-2. We investigated the role of SIRPα and its dominant negative mutant in PTP activities in 293 cells. The overexpression of wild-type SIRPα suppresses the activities of both PTP, but has a stronger effect on PTP SHP-2, especially when this protein is overexpressed in 293 cells. In contrast with wild-type SIRPα, its dominant negative mutant acts predominantly against PTP SHP-1, and can be detected in the complex with PTP SHP-1. The expressi...