Targets of ubiquitin like system in mycobacteria and related actinobacterial species.

Protein turnover and recycling is a prerequisite in all living organisms to maintain normal cellular physiology. Many bacteria are proteasome deficient but they possess typical protease enzymes for carrying out protein turnover. However, several groups of actinobacteria such as mycobacteria harbor both proteasome and proteases. In these bacteria, for cellular protein turnover the target proteins undergo post-translational modification referred as pupylation in which a small protein Pup (prokaryotic ubiquitin-like protein) is tagged to the specific lysine residues of the target proteins and after that those target proteins undergo proteasomal degradation. Thus, Pup serves as a degradation signal, helps in directing proteins toward the bacterial proteasome for a turnover. Although the Pup-proteasome system has a multifaceted role in environmental stresses, pathogenicity and regulation of cellular signaling, but the fate of all types of pupylation such as mono and polypupylation on the proteins is still not completely understood. In this review, we present the mechanisms involved in the activation and conjugation of Pup to the target proteins, describing the structural sketch of pupylation and fundamental differences between the eukaryotic ubiquitin-proteasome and bacterial Pup-proteasome systems. We are also presenting a concise classification and cataloging of the complete battery of experimentally identified Pup-substrates from various species of actinobacteria.

A deubiquitinating enzyme encoded by HSV-1 belongs to a family of cysteine proteases that is conserved across the family Herpesviridae.

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 ubiquitin-related protein PLIC-1 regulates heterotrimeric G protein function through association with Gbetagamma.

PLIC-1, a newly described ubiquitin-related protein, inhibited both Jurkat migration toward SDF-1alpha and A431 wound healing, but the closely related PLIC-2 did not. PLIC-1 prevented the SDF-1alpha-induced activation of phospholipase C, decreased ligand-induced internalization of SDF-1alpha receptor CXCR4 and inhibited chemotaxis signaled by a transfected Gi-coupled receptor. However, PLIC-1 had no effect on Gs-mediated adenylyl cyclase activation, and inhibited only the Gbetagamma-dependent component of Gq-initiated increase in [Ca2+]i, which is consistent with selective inhibition of Gbetagamma function. PLIC-1 colocalized with G proteins in lamellae and pseudopods, and precipitated Gbetagamma in pull downs. Interaction with Gbetagamma did not require PLIC-1's ubiquitin-like or ubiquitin-associated domains, and proteasome inhibition had no effect on SDF-1alpha activation of phospholipase C, indicating that PLIC-1's inhibition of Gbetagamma did not result from effects on proteasome function. Thus, PLIC-1 inhibits Gi signaling by direct association with Gbetagamma; because it also interacts with CD47, a modulator of integrin function, it likely has a role integrating adhesion and signaling components of cell migration.

The HIV-TSG101 interface: recent advances in a budding field.

Efficient budding of HIV from the plasma membrane requires a small peptide motif, Pro-Thr/Ser-Ala-Pro (PTAP), located near the amino terminus of the p6 Gag protein. Studies from several laboratories have demonstrated that the ability of p6 to stimulate HIV budding requires a direct interaction between the PTAP motif and the host endosomal sorting protein TSG101. The structure of the PTAP-TSG101 binding site has recently been solved, providing valuable insights into this crucial protein-protein interaction.

Activation of the ATP-ubiquitin-proteasome pathway in skeletal muscle of cachectic rats bearing a hepatoma.

Rats implanted with Yoshida ascites hepatoma (YAH) show a rapid and selective loss of muscle protein due mainly to a marked increase (63-95%) in the rate of protein degradation (compared with rates in muscles of pair-fed controls). To define which proteolytic pathways contribute to this increase, epitrochlearis muscles from YAH-bearing and control rats were incubated under conditions that modify different proteolytic systems. Overall proteolysis in either group of rats was not affected by removal of Ca2+ or by blocking the Ca(2+)-dependent proteolytic system. Inhibition of lysosomal function with methylamine reduced proteolysis (-12%) in muscles from YAH-bearing rats, but not in muscles of pair-fed rats. When ATP production was also inhibited, the remaining accelerated proteolysis in muscles of tumor-bearing rats fell to control levels. Muscles of YAH-bearing rats showed increased levels of ubiquitin-conjugated proteins and a 27-kDa proteasome subunit in Western blot analysis. Levels of mRNA encoding components of proteolytic systems were quantitated using Northern hybridization analysis. Although their total RNA content decreased 20-38%, pale muscles of YAH-bearing rats showed increased levels of ubiquitin mRNA (590-880%) and mRNA for multiple subunits of the proteasome (100-215%). Liver, kidney, heart, and brain showed no weight loss and no change in these mRNA species. Muscles of YAH-bearing rats also showed small increases (30-40%) in mRNA for cathepsins B and D, but not for calpain I or heat shock protein 70. Our findings suggest that accelerated muscle proteolysis and muscle wasting in tumor-bearing rats result primarily from activation of the ATP-dependent pathway involving ubiquitin and the proteasome.