Ubiquitylation is a reversible protein modification that is implicated in many cellular functions. Recently, much progress has been made in the characterization of a superfamily of isopeptidases that remove ubiquitin: the deubiquitinases (DUBs; also known as deubiquitylating or deubiquitinating enzymes). Far from being uniform in structure and function, these enzymes display a myriad of distinct mechanistic features. The small number (<100) of DUBs might at first suggest a low degree of selectivity; however, DUBs are subject to multiple layers of regulation that modulate both their activity and their specificity. Due to their wide-ranging involvement in key regulatory processes, these enzymes might provide new therapeutic targets.

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Abstract. Small GTPases of the rab family are crucial elements of the machinery that controls membrane traffic. In the present study, we examined the distribution and function of rabll. Rabll was shown by confocal immunofluorescence microscopy and EM to colocalize with internalized transferrin in the pericentriolar recycling compartment of CHO and BHK cells. Expression of rabll mutants that are preferentially in the GTP-or GDP-bound state caused opposite effects on the distribution of transferrin-containing elements; rabll-GTP expression caused accumulation of labeled elements in the perinuclear area of the cell, whereas rabll-GDP caused a dispersion of the transferrin labeling. Functional studies showed that the early steps of uptake and recycling for transferrin were not affected by overexpression of rabll proteins. However, recycling from the later recycling endosome was inhibited in cells overexpressing the rabll-GDP mutant. Rab5, which regulates early endocytic trafficking, acted before rabll in the transferrin-recycling pathway as expression of rab5-GTP prevented transport to the rab11-positive recycling endosome. These results suggest a novel role for rabll in controlling traffic through the recycling endosome.

The past two years have seen an explosion in the structural understanding of the endosomal sorting complex required for transport (ESCRT) machinery that facilitates the trafficking of ubiquitylated proteins from endosomes to lysosomes via multivesicular bodies (MVBs). A common organization of all ESCRTs is a rigid core attached to flexibly connected modules that recognize other components of the MVB pathway. Several previously unsuspected key links between multiple ESCRT subunits, phospholipids and ubiquitin have now been elucidated, which, together with the detailed morphological analyses of ESCRT-depletion phenotypes, provide new insights into the mechanism of MVB biogenesis.

Autophagosome formation is a complex process that begins with the nucleation of a pre-autophagosomal structure (PAS) that expands into a phagophore or isolation membrane, the precursor of the autophagosome. A key event in the formation of the phagophore is the production of PtdIns3P by the phosphatidylinsitol kinase Vps34. In yeast the two closely related proteins, Atg18 and Atg21, are the only known effectors of PtdIns3P that act in the autophagy pathway. The recruitment of Atg18 or Atg21 to the PAS is an essential step in the formation of the phagophore. Our bioinformatic analysis of the Atg18 and Atg21 orthologues in all eukaryotes shows that WIPI1 and WIPI2 are both mammalian orthologues of Atg18. We show that WIPI2 is a mammalian effector of PtdIns3P and is ubiquitously expressed in a variety of cell lines. WIPI2 is recruited to early autophagosomal structures along with Atg16L and ULK1 and is required for the formation of LC3-positive autophagosomes. Furthermore, when WIPI2 is depleted, we observe a remarkable accumulation of omegasomes, ER-localized PtdIns3P-containing structures labeled by DFCP1 (double FYVE domain-containing protein 1), which are thought to act as platforms for autophagosome formation. In view of our data we propose a role for WIPI2 in the progression of omegasomes into autophagosomes.

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The JAMM (JAB1/MPN/Mov34 metalloenzyme) motif has been proposed to provide the active site for isopeptidase activity associated with the Rpn11/POH1 subunit of the 19S-proteasome and the Csn5-subunit of the signalosome. We have looked for similar activity in associated molecule with the SH3 domain of STAM (AMSH), a JAMM domain–containing protein that associates with the SH3-domain of STAM, a protein, which regulates receptor sorting at the endosome. We demonstrate isopeptidase activity against K48-linked tetraubiquitin and K63-linked polyubiquitin chains to generate di-ubiquitin and free ubiquitin, respectively. An inactivating mutation (D348A) in AMSH leads to accumulation of ubiquitin on endosomes and the concomitant stabilization of a ubiquitinated form of STAM, which requires an intact ubiquitin interaction motif (UIM) within STAM. Short interfering RNA knockdown of AMSH enhances the degradation rate of EGF receptor (EGFR) following acute stimulation and ubiquitinated EGFR provides a substrate for AMSH in vitro. We propose that AMSH is a deubiquitinating enzyme with functions at the endosome, which oppose the ubiquitin-dependent sorting of receptors to lysosomes.

Ubiquitylation is a major posttranslational modification that controls most complex aspects of cell physiology. It is reversed through the action of a large family of deubiquitylating enzymes (DUBs) that are emerging as attractive therapeutic targets for a number of disease conditions. Here, we provide a comprehensive analysis of the complement of human DUBs, indicating structural motifs, typical cellular copy numbers, and tissue expression profiles. We discuss the means by which specificity is achieved and how DUB activity may be regulated. Generically DUB catalytic activity may be used to 1) maintain free ubiquitin levels, 2) rescue proteins from ubiquitin-mediated degradation, and 3) control the dynamics of ubiquitin-mediated signaling events. Functional roles of individual DUBs from each of five subfamilies in specific cellular processes are highlighted with an emphasis on those linked to pathological conditions where the association is supported by whole organism models. We then specifically consider the role of DUBs associated with protein degradative machineries and the influence of specific DUBs upon expression of receptors and channels at the plasma membrane.