EBNA3C, one of the Epstein-Barr virus (EBV)-encoded latent antigens, is essential for primary B-cell transformation. Cyclin D1, a key regulator of G1 to S phase progression, is tightly associated and aberrantly expressed in numerous human cancers. Previously, EBNA3C was shown to bind to Cyclin D1 in vitro along with Cyclin A and Cyclin E. In the present study, we provide evidence which demonstrates that EBNA3C forms a complex with Cyclin D1 in human cells. Detailed mapping experiments show that a small N-terminal region which lies between amino acids 130–160 of EBNA3C binds to two different sites of Cyclin D1- the N-terminal pRb binding domain (residues 1–50), and C-terminal domain (residues 171–240), known to regulate Cyclin D1 stability. Cyclin D1 is short-lived and ubiquitin-mediated proteasomal degradation has been targeted as a means of therapeutic intervention. Here, we show that EBNA3C stabilizes Cyclin D1 through inhibition of its poly-ubiquitination, and also increases its nuclear localization by blocking GSK3β activity. We further show that EBNA3C enhances the kinase activity of Cyclin D1/CDK6 which enables subsequent ubiquitination and degradation of pRb. EBNA3C together with Cyclin D1-CDK6 complex also efficiently nullifies the inhibitory effect of pRb on cell growth. Moreover, an sh-RNA based strategy for knock-down of both cyclin D1 and EBNA3C genes in EBV transformed lymphoblastoid cell lines (LCLs) shows a significant reduction in cell-growth. Based on these results, we propose that EBNA3C can stabilize as well as enhance the functional activity of Cyclin D1 thereby facilitating the G1-S transition in EBV transformed lymphoblastoid cell lines.

Epstein Barr virus (EBV) is closely associated with the development of a vast number of human cancers. To develop a system for monitoring early cellular and viral events associated with EBV infection a self-recombining BAC containing 172-kb of the Epstein Barr virus genome BAC-EBV designated as MD1 BAC (Chen et al., 2005, J.Virology) was used to introduce an expression cassette of green fluorescent protein (GFP) by homologous recombination, and the resultant BAC clone, BAC-GFP-EBV was transfected into the HEK 293T epithelial cell line. The resulting recombinant GFP EBV was induced to produce progeny virus by chemical inducer from the stable HEK 293T BAC GFP EBV cell line and the virus was used to immortalize human primary B-cell as monitored by green fluorescence and outgrowth of the primary B cells. The infection, B-cell activation and cell proliferation due to GFP EBV was monitored by the expression of the B-cell surface antigens CD5, CD10, CD19, CD23, CD39, CD40 , CD44 and the intercellular proliferation marker Ki-67 using Flow cytometry. The results show a dramatic increase in Ki-67 which continues to increase by 6–7 days post-infection. Likewise, CD40 signals showed a gradual increase, whereas CD23 signals were increased by 6–12 hours, maximally by 3 days and then decreased. Monitoring the viral gene expression pattern showed an early burst of lytic gene expression. This up-regulation of lytic gene expression prior to latent genes during early infection strongly suggests that EBV infects primary B-cell with an initial burst of lytic gene expression and the resulting progeny virus is competent for infecting new primary B-cells. This process may be critical for establishment of latency prior to cellular transformation. The newly infected primary B-cells can be further analyzed for investigating B cell activation due to EBV infection.

Intramembrane proteases (IPs) cleave membrane-associated substrates in nearly all organisms and regulate diverse processes. A better understanding of how these enzymes interact with their substrates is necessary for rational design of IP modulators. We show that interaction of IP SpoIVFB with its substrate Pro-σ depends on particular residues in the interdomain linker of SpoIVFB. The linker plus either the N-terminal membrane domain or the C-terminal cystathione-β-synthase (CBS) domain of SpoIVFB was sufficient for the interaction but not for cleavage of Pro-σ Chemical cross-linking and mass spectrometry of purified, inactive SpoIVFB-Pro-σ complex indicated residues of the two proteins in proximity. A structural model of the complex was built via partial homology and by using constraints based on cross-linking data. In the model, the Proregion of Pro-σ loops into the membrane domain of SpoIVFB, and the rest of Pro-σ interacts extensively with the linker and the CBS domain of SpoIVFB. The extensive interaction is proposed to allow coordination between ATP binding by the CBS domain and Pro-σ cleavage by the membrane domain.

The CIII protein encoded by the temperate coliphage lambda acts as an inhibitor of the ubiquitous Escherichia coli metalloprotease HflB (FtsH). This inhibition results in the stabilization of transcription factor CII, thereby helping the phage to lysogenize the host bacterium. CIII, a small (54-residue) protein of unknown structure, also protects 32 , another specific substrate of HflB. In order to understand the details of the inhibitory mechanism of CIII, we cloned and expressed the protein with an N-terminal six-histidine tag. We also synthesized and studied a 28-amino-acid peptide, CIIIC, encompassing the central 14 to 41 residues of CIII that exhibited antiproteolytic activity. Our studies show that CIII exists as a dimer under native conditions, aided by an intersubunit disulfide bond, which is dispensable for dimerization. Unlike CIII, CIIIC resists digestion by HflB. While CIII binds to HflB, it does not bind to CII. On the basis of these results, we discuss various mechanisms for the antiproteolytic activity of CIII.An important event in the life cycle of bacteriophage lambda is the choice between its two alternate modes of development, viz., lytic and lysogenic (14,39). This choice involves the participation of proteins from the virus, as well as from its bacterial host, Escherichia coli. The small 54-residue phage protein CIII plays an important role in this process by stabilizing CII, the transcription factor essential for the establishment of lysogeny (24). In the absence of CIII, CII is rapidly degraded by the ATP-dependent host metalloprotease HflB (FtsH) (36, 37), driving toward the lytic cycle. On the other hand, overexpression of CIII promotes the lysogenic mode of phage development (1).The E. coli 32 protein, another substrate of HflB (22,38), is also protected by CIII, generating a heat shock response in the cell (6). Thus, the primary function of CIII appears to be inhibition of the protease activity of HflB, although another function, viz., acting as a molecular chaperone, has also been proposed (33). There has been just one recent report on the molecular mechanism of this antiproteolytic function of CIII (31), where it has been shown that CIII prevents the association between HflB and CII. Interestingly, CIII is itself an unstable protein and a substrate for HflB (23, 30), suggesting competitive inhibition as a possible mechanism for its action. An important development in the understanding of CIII action was the observation that the 24-residue central region of CIII (amino acids 14 to 37) is both necessary and sufficient for its activity (32). This domain is highly homologous among CIII proteins from , P22, and HK022 and is likely to form an amphipathic helix. Mutations in this region lead to loss of CIII activity, while those outside this region have little effect (32).Nevertheless, how this putative helical portion of CIII protects CII or 32 from HflB remains to be understood. To unravel the mechanism of CIII action, it is important to know the structure of CIII, as well as to understan...

Intramembrane metalloproteases (IMMPs) are conserved from bacteria to humans and control many important signaling pathways, but little is known about how IMMPs interact with their substrates. SpoIVFB is an IMMP that cleaves Pro-K during Bacillus subtilis endospore formation. When catalytically inactive SpoIVFB was coexpressed with C-terminally truncated Pro-K (1-126) (which can be cleaved by active SpoIVFB) in Escherichia coli, the substrate dramatically improved solubilization of the enzyme from membranes with mild detergents. Both the Pro(1-20) and K (21-126) parts contributed to improving SpoIVFB solubilization from membranes, but only the K part was needed to form a stable complex with SpoIVFB in a pulldown assay. The last 10 residues of SpoIVFB were required for improved solubilization from membranes by Pro-K (1-126) and for normal interaction with the substrate. The inactive SpoIVFB⅐Pro-K (1-126)-His 6 complex was stable during affinity purification and gel filtration chromatography. Disulfide cross-linking of the purified complex indicated that it resembled the complex formed in vivo. Ion mobility-mass spectrometry analysis resulted in an observed mass consistent with a 4:2 SpoIVFB⅐Pro-K (1-126)-His 6 complex. Stepwise photobleaching of SpoIVFB fused to a fluorescent protein supported the notion that the enzyme is tetrameric during B. subtilis sporulation. The results provide the first evidence that an IMMP acts as a tetramer, give new insights into how SpoIVFB interacts with its substrate, and lay the foundation for further biochemical analysis of the enzyme⅐substrate complex and future structural studies.Many critical cellular processes are regulated by intramembrane proteolysis (1). Intramembrane proteases (IPs) 3 cleave their substrates within a transmembrane segment (TMS) or near the membrane surface. There are three classes of IPs: rhomboids, aspartyl IPs, and IMMPs (often called site-2 proteases or S2Ps). Rhomboids are serine IPs that promote animal cellular signaling, coordinate bacterial quorum sensing, regulate mitochondrial homeostasis, and control protozoan infection (2-5). Presenilin, an aspartyl IP, is the catalytic component of ␥-secretase, which is involved in the processing of the amyloid precursor protein, Notch, and many other subtrates (6, 7). Dysfunction of ␥-secretase contributes to the pathogenesis of Alzheimer disease (8) and many other diseases (7). Aspartyl IPs also include preflagellin and prepilin peptidases involved in bacterial pathogenesis (9), and signal peptide peptidases, which facilitate the clearance of signal peptides from membranes, participate in viral infection, and generate small peptides as signal molecules for immune systems (10, 11). IMMPs also play critical roles in a wide variety of biological functions. In eukaryotes, cholesterol metabolism, the unfolded protein response, and the acute-phase response are regulated by IMMPs (1, 12, 13). In bacteria, IMMPs control sporulation, envelope stress responses, mating signal production, polar morphogenesis, virulence, ...