Loss of Usp14 results in reduced levels of ubiquitin in ataxia mice

The ataxia (ax(J)) mutation is a spontaneous recessive mutation that results in reduced expression of ubiquitin-specific protease 14, Usp14. Mice homozygous for the ax(J) mutation are retarded for growth and exhibit several behavioral disorders, including a resting tremor and hindlimb paralysis. Although pathological defects appear to be limited to the central nervous system, reduction of Usp14 expression was widespread in the ax(J) mice. Usp14 co-fractionated with proteasomes isolated from livers and brains of wild-type mice. Proteasomes isolated from the ax(J) brains still possessed deubiquitinating activity and were functionally competent to hydrolyze 20S proteasomal substrates in vitro. However, the levels of monomeric ubiquitin were reduced approximately 35% in most of the ax(J) tissues examined. These results indicate that Usp14 functions to maintain the cellular levels of monomeric ubiquitin in mammalian cells, and that alterations in the levels of ubiquitin may contribute to neurological disease.     

Ubiquitin-dependent control of class II MHC localization is dispensable for antigen presentation and antibody production

Controlled localization of class II MHC molecules is essential for proper class II MHC-restricted antigen presentation and the subsequent initiation of an adaptive immune response. Ubiquitination of class II MHC molecules on cytosolic lysine (K225) of the β-chain has been shown to affect localization of the complex. We generated mice in which the endogenous β-chain locus is replaced with a GFP tagged mutant version that lacks the cytosolic lysine residue (I-A-β-K225R-EGFP). These mice have elevated levels of class II MHC as compared to I-A-β-EGFP mice, and immature bone marrow-derived dendritic cells show redistribution of class II MHC to the cell surface. Nonetheless, in these same cells efficiency of antigen presentation is unaffected in I-A-β-K225R-EGFP mice, as assayed for presentation of ovalbumin to appropriately specific T cells. The I-A-β-K225R-EGFP animals have normal CD4 T cell populations and are capable of generating antigen-specific antibody in response to model antigens and viral infection. We therefore conclude that in our experimental system modulation of trafficking by ubiquitination of residue K225 of the β-chain is not essential for the function of class II MHC products in antigen presentation or antibody production.     

High-molecular-weight protein (pUL48) of human cytomegalovirus is a competent deubiquitinating protease: mutant viruses altered in its active-site cysteine or histidine are viable

We show here that the high-molecular-weight protein (HMWP or pUL48; 253 kDa) of human cytomegalovirus (HCMV) is a functionally competent deubiquitinating protease (DUB). By using a suicide substrate probe specific for ubiquitin-binding cysteine proteases (DUB probe) to screen lysates of HCMV-infected cells, we found just one infected-cell-specific DUB. Characteristics of this protein, including its large size, expression at late times of infection, presence in extracellular virus particles, and reactivity with an antiserum to the HMWP, identified it as the HMWP. This was confirmed by constructing mutant viruses with substitutions in two of the putative active-site residues, Cys24Ile and His162Ala. HMWP with these mutations either failed to bind the DUB probe (C24I) or had significantly reduced reactivity with it (H162A). More compellingly, the deubiquitinating activity detected in wild-type virus particles was completely abolished in both the C24I and H162A mutants, thereby directly linking HMWP with deubiquitinating enzyme activity. Mutations in these active-site residues were not lethal to virus replication but slowed production of infectious virus relative to wild type and mutations of other conserved residues. Initial studies, by electron microscopy, of cells infected with the mutants revealed no obvious differences at late times of replication in the general appearance of the cells or in the distribution, relative numbers, or appearance of virus particles in the cytoplasm or nucleus.     

A functional ubiquitin-specific protease embedded in the large tegument protein (ORF64) of murine gammaherpesvirus 68 is active during the course of infection

All herpesviruses contain a ubiquitin (Ub)-specific cysteine protease domain embedded within their large tegument protein, based on homology with the corresponding sequences of UL36 from herpes simplex virus type 1 and M48 from murine cytomegalovirus. This type of activity has yet to be demonstrated for cells infected with a gammaherpesvirus. By activity-based profiling, we show that the large tegument protein of murine gammaherpesvirus (MHV-68) ORF64 (273 kDa) is a functional deubiquitinating protease, as assessed by tandem mass spectrometry of adducts in extracts from MHV-68-infected cells that had been labeled with ubiquitin vinylmethylester, a ubiquitin-based active site-directed probe. The recombinantly expressed amino-terminal segment of ORF64 displays deubiquitinating activity toward Ub C-terminal 7-amido-4-methylcoumarin in vitro. The findings reported here for MHV-68 ORF64 extend those made for the alpha- and betaherpesvirus families and are consistent with an important, conserved enzymatic function of the tegument protein.     

Site-Specific Chemoenzymatic Labeling of Aerolysin Enables the Identification of New Aerolysin Receptors

Aerolysin is a secreted bacterial toxin that perforates the plasma membrane of a target cell with lethal consequences. Previously explored native and epitope-tagged forms of the toxin do not allow site-specific modification of the mature toxin with a probe of choice. We explore sortase-mediated transpeptidation reactions (sortagging) to install fluorophores and biotin at three distinct sites in aerolysin, without impairing binding of the toxin to the cell membrane and with minimal impact on toxicity. Using a version of aerolysin labeled with different fluorophores at two distinct sites we followed the fate of the C-terminal peptide independently from the N-terminal part of the toxin, and show its loss in the course of intoxication. Making use of the biotinylated version of aerolysin, we identify mesothelin, urokinase plasminogen activator surface receptor (uPAR, CD87), glypican-1, and CD59 glycoprotein as aerolysin receptors, all predicted or known to be modified with a glycosylphosphatidylinositol anchor. The sortase-mediated reactions reported here can be readily extended to other pore forming proteins.     

A 19F NMR study of fluorobenzoate biodegradation by Sphingomonas sp. HB-1

While several microorganisms readily degrade 2- and 4-fluorobenzoates, only a very small number appear to catabolise the 3-fluoro isomer, owing to the accumulation of toxic intermediates. Here we describe the isolation of a bacterium capable of using 3-fluorobenzoate as a sole source of carbon and energy, and the experiments conducted to define the steps involved in the biodegradation of this compound. The organism was identified as a strain belonging to the genus Sphingomonas by sequence analysis of its 16S rRNA gene. To date no other organism from this genus is known to degrade this compound. Using fluorine nuclear magnetic resonance spectroscopy (19F NMR) to analyse the culture supernatant it was possible to observe the disappearance of 3-fluorobenzoate and the appearance of fluoride ion and four other fluorinated compounds. These were identified as 3-fluorocatechol, 2-fluoromuconic acid and 3- and 5-fluoro-1,2-dihydro-1,2-dihydroxybenzoates. Thus, the likely catabolic pathway involves dioxygenation of 3-fluorobenzoate yielding fluorocatechol and subsequent intra-diol cleavage to yield fluoromuconic acid. The organism can also use 2- and 4-fluorobenzoates as growth substrates.     

Protein ligation in living cells using sortase

Sortagging is a versatile method for site‐specific modification of proteins as applied to a variety of in vitro reactions. Here, we explore possibilities of adapting the sortase method for use in living cells. For intracellular sortagging, we employ the Ca²⁺‐independent sortase A transpeptidase (SrtA) from Streptococcus pyogenes. Substrate proteins were equipped with the C‐terminal sortase‐recognition motif (LPXTG); we used proteins with an N‐terminal (oligo)glycine as nucleophiles. We show that sortase‐dependent protein ligation can be achieved in Saccharomyces cerevisiae and in mammalian HEK293T cells, both in the cytosol and in the lumen of the endoplasmic reticulum (ER). ER luminal sortagging enables secretion of the reaction products, among which circular polypeptides. Protein ligation of substrate and nucleophile occurs within 30 min of translation. The versatility of the method is shown by protein ligation of multiple substrates with green fluorescent protein‐based nucleophiles in different intracellular compartments.     

A viral deubiquitylating enzyme restores dislocation of substrates from the endoplasmic reticulum (ER) in semi-intact cells

Terminally misfolded glycoproteins are ejected from the endoplasmic reticulum (ER) to the cytosol and are destroyed by the ubiquitin proteasome system. A dominant negative version of the deubiquitylating enzyme Yod1 (Yod1C160S) causes accumulation of dislocation substrates in the ER. Failure to remove ubiquitin from the dislocation substrate might therefore stall the reaction at the exit site from the ER. We hypothesized that addition of a promiscuous deubiquitylase should overcome this blockade and restore dislocation. We monitored ER-to-cytosol transport of misfolded proteins in cells permeabilized at high cell density by perfringolysin O, a pore-forming cytolysin. This method allows ready access of otherwise impermeant reagents to the intracellular milieu with minimal dilution of cytoplasmic components. We show that addition of the purified Epstein-Barr virus deubiquitylase to semi-intact cells indeed initiates dislocation of a stalled substrate intermediate, resulting in stabilization of substrates in the cytosol. Our data provide new mechanistic insight in the dislocation reaction and support a model where failure to deubiquitylate an ER-resident protein occludes the dislocon and causes upstream misfolded intermediates to accumulate.     

N-linked glycosylation does not impair proteasomal degradation but affects class I major histocompatibility complex presentation

The addition of N-linked glycans to nascent polypeptides occurs cotranslationally in the endoplasmic reticulum (ER). For many proteins the state of the glycans serves as an indicator, which allows the ER quality control system to monitor the conformation of polypeptides upon folding. Proteins that fail to fold in the ER are often dislocated to the cytoplasm, where they are subjected to proteasomal degradation. Although the addition of N-linked glycans occurs within the ER, non-lysosomal removal of the glycans occurs in the cytosol by the action of peptide N-glycanase (PNGase). In this study, we investigated the interplay between PNGase action and proteasomal degradation of ER misfolded proteins (i.e. whether PNGase acts prior to or following proteasomal degradation). Interestingly, we found that glycan removal from N-terminally extended peptides modulates the presentation of class I major histocompatibility complex-restricted epitopes. Our findings provide direct evidence that the proteasome is capable of degrading glycoproteins without prior removal of their glycans. This degradation is independent of either the identity of the glycosylated protein or the type and number of N-linked glycans it harbors. We also captured and characterized glycopeptides generated following proteasomal degradation of RNaseB. Although the carbohydrate moiety reduced the variability of the degradation products that include the glycosylated residue (local effect), the overall global digestion pattern of RNaseB was unaffected. Together with earlier findings by others, our data support a model in which PNGase may act both upstream and downstream to proteasomal degradation and demonstrates its important role in class I major histocompatibility complex antigen presentation.