We have discovered a ubiquitin (Ub)-specific cysteine protease encoded within the N-terminal approximately 500 residues of the UL36 gene product, the largest (3164 aa) tegument protein of herpes simplex virus 1 (HSV-1). Enzymatic activity of this fragment, UL36USP, is detectable only after cleavage of UL36USP from full-length UL36 and occurs late during viral replication. UL36USP bears no homology to known deubiquitinating enzymes (DUBs) or Ub binding proteins. Sequence alignment of the large tegument proteins across the family Herpesviridae indicates conservation of key catalytic residues amongst these viruses. Recombinant UL36USP exhibits hydrolytic activity toward Ub-AMC and ubiquitinated branched peptides in vitro. In addition, recombinant UL36USP can cleave polyUb chains and appears to be specific for Lys48 linkages. Mutation of the active site cysteine residue (Cys65) to alanine abolishes this enzymatic activity. The lack of homology between UL36USP and eukaryotic DUBs makes this new family of herpesvirus ubiquitin-specific proteases attractive targets for selective inhibition.

The largest tegument protein of herpes simplex virus 1 (HSV-1), UL36, contains a novel deubiquitinating activity embedded in it. All members of the Herpesviridae contain a homologue of HSV-1 UL36, the N-terminal segments of which show perfect conservation of those residues implicated in catalysis. For murine cytomegalovirus and Epstein-Barr virus, chosen as representatives of the beta-and gammaherpesvirus subfamilies, respectively, we here show that the homologous modules indeed display deubiquitinating activity in vitro. The conservation of this activity throughout all subfamilies is indicative of an important, if not essential, function.Modification of proteins by ubiquitin (Ub) plays a pivotal role in a multitude of cellular processes, including proteolysis, cell cycle control, receptor internalization, and sorting within the endo/lysosomal system (7,14,16). Ubiquitination is achieved by an enzymatic cascade comprising a Ub-activating enzyme (E1), several Ub-carrier proteins (E2s), and hundreds of Ub ligases (E3s). Ubiquitination can be reversed by several families of enzymes collectively designated deubiquitinating enzymes (DUBs) (1,15).A number of viruses have evolved strategies to manipulate the ubiquitination status of host cell proteins, both through conjugation and deconjugation (2,4,6,10,13). Recently, we reported the identification of a novel viral ubiquitin-specific protease (USP), UL36 USP , encoded by the herpes simplex virus 1 (HSV-1) genome (9). UL36USP is a polypeptide of approximately 420 amino acids (aa) carried within the N-terminal portion of UL36, the largest tegument protein (3,164 aa) of HSV-1. This activity was detected through the use of mechanism-based, activesite-directed probes and confirmed by expression in Escherichia coli of a corresponding fragment that cleaves ubiquitin-based substrates. UL36USP activity peaks at late stages of viral replication and appears to require proteolytic processing from fulllength UL36 (9). The N-terminal UL36 fragment is well conserved in alphaherpesviruses, and a low homology to corresponding genes of the betaherpesvirus and gammaherpesvirus subfamilies was apparent in sequence alignments, but with strict conservation of the proposed catalytic residues. DUB activity may therefore be well conserved across the herpesvirus family and, if this is proven to be correct, would suggest an important function for this type of activity.We therefore set out to investigate the possible DUB activity of two phylogenetically distant homologues of HSV-1 UL36 USP , each representing a different subfamily of the Herpesviridae. We chose UL36 homologues encoded by mouse cytomegalovirus (MCMV, M48) and Epstein-Barr virus (EBV, BPLF1) as representatives of the beta-and gammaherpesvirus subfamilies, respectively. In order to assess the degree of homology between UL36 from HSV-1 and its MCMV and EBV counterparts, a sequence alignment was generated, covering the first 336 aa (the numbering refers to HSV-1) of UL36 (Fig. 1). Overall, the homology to HSV-1 is rather low, with on...

We explored the role of antigen valency in B cell receptor (BCR) activation and rearrangement of intracellular MHC class II compartments as factors that contribute to the efficacy of antigen presentation. Using primary B cells that express a hen egg lysozyme (HEL)-specific BCR, we found that oligomeric HEL more efficiently promoted both BCR activation and internalization than did monovalent HEL, although monovalent HEL, unlike monovalent Fab fragments of anti-Ig, readily triggered the BCR. Nonetheless, oligovalent ligation positions the BCR in a membrane microdomain that is distinct from one engaged in the course of monovalent ligation, as judged by detergent extraction of the BCR. A ctivation of B cell receptors by specific antigen unleashes a cascade of downstream events which includes alteration of transcriptional programs that lead to cell proliferation and differentiation (1-3). B cells further use their B cell receptors (BCRs) to facilitate the capture of antigen, and in doing so, they improve antigen presentation via class II MHC molecules (4). The latter event is essential for B cells to receive help from antigen-specific CD4 ϩ T cells and to differentiate eventually into memory cells or plasma cells that secrete high-affinity antibody (5).Antigen-specific B cells present peptides derived from cognate antigens to CD4 ϩ T cells at concentrations far below those required for other B cells that lack an antigen-specific receptor (6). This highly efficient mode of antigen presentation is thought to be a consequence of the dual role of the BCR in the delivery of specific antigen to MHC class II-containing peptide-loading compartments and in signaling that leads to enhanced generation of immunogenic peptides (7,8).BCR-transduced signals promote efficient delivery of antigens through activation of Syk and B cell linker protein (BLNK) (9, 10). In addition, BCR-transduced signals induce physical and chemical remodeling of intracellular class II MHC-compartments (11). In the A20͞IIA1.6 mouse B lymphoma cell line, BCR activation by receptor cross-linking induced reorganization, fusion, and acidification of Lamp1-positive late endosomes where class II MHC molecules and invariant chain accumulate (12). In A20 B cells that express an anti-DNP IgM, BCR stimulation led to the transient intracellular accumulation of MHC molecules in newly formed multivesicular bodies, into which the peptide exchange catalyst H2-M was recruited (13).Although these studies were among the first to shed light on a role for BCR signaling in MHC class II-mediated antigen presentation, virtually all of the studies that describe behavior of MHC class II molecules in relationship to the activation of B cells have been performed in transformed cell lines. Moreover, stimulation of the BCR is usually accomplished either by cross-linking receptors with anti-Ig antibodies or by transfecting established cell lines to express BCRs of known specificity to enable their ligation in an antigen-specific manner, which then still involves extensive crosslinking...

Malondialdehyde (MDA), a known mutagen and suspected carcinogen, is a product of lipid peroxidation and byproduct of eicosanoid biosynthesis. MDA can react with DNA to generate potentially mutagenic adducts on adenine, cytosine, and particularly guanine. In addition, repair-dependent frame shift mutations in a GCGCGC region of Salmonella typhimurium hisD3052 have been attributed to formation of interstrand cross-links (Mukai, F. H. and Goldstein, B. D. Science 1976, 191, 868--869). The cross-linked species is unstable and has never been characterized but has been postulated to be a bis-imino linkage between N(2) positions of guanines. An analogous linkage has now been investigated as a stable surrogate using the self-complementary oligodeoxynucleotide sequence 5'-d(AGGCG*CCT)(2,) in which G* represents guanines linked via a trimethylene chain between N(2) positions. The solution structure, obtained by NMR spectroscopy and molecular dynamics using a simulated annealing protocol, revealed the cross-link only minimally distorts duplex structure in the region of the cross-link. The tether is accommodated by partially unwinding the duplex at the lesion site to produce a bulge and tipping the guanine residues; the two guanines and the tether attain a nearly planar conformation. This distortion did not result in significant bending of the DNA, a result which was confirmed by gel electrophoresis studies of multimers of a 21-mer duplex containing the cross-link.

Peptide:N-glycanase (PNGase) is ostensibly the sole enzyme responsible for deglycosylation of unfolded N-linked glycoproteins dislocated from the ER to the cytosol. Here we show the pan-caspase inhibitor, Z-VAD-fmk, to be an active site-directed irreversible inhibitor of yeast and mammalian PNGase at concentrations below those used to inhibit caspases in vivo. Through chemical synthesis we determined that the P1 residue, electrophile position, and leaving group are important structural parameters for PNGase inhibition. We show that Z-VAD-fmk inhibits PNGase in living cells and that degradation of class I MHC heavy chains and TCRalpha, in an identical cellular setting, is markedly different. Remarkably, proteasome-mediated turnover of class I MHC heavy chains proceeds even when PNGase is completely inhibited, suggesting that the function of PNGase may be to facilitate more efficient proteasomal proteolysis of N-linked glycoproteins through glycan removal.