Christopher M. Sanderson

Lysine‐63‐linked ubiquitination is required for endolysosomal degradation of class I molecules

MHC class I molecules display peptides from endogenous and viral proteins for immunosurveillance by cytotoxic T lymphocytes (CTL). The importance of the class I pathway is emphasised by the remarkable strategies employed by different viruses to downregulate surface class I and avoid CTL recognition. The K3 gene product from Kaposis sarcoma‐associated herpesvirus (KSHV) is a viral ubiquitin E3 ligase which ubiquitinates and degrades cell surface MHC class I molecules. We now show that modification of K3‐associated class I by lysine‐63‐linked polyubiquitin chains is necessary for their efficient endocytosis and endolysosomal degradation and present three lines of evidence that monoubiquitination of class I molecules provides an inefficient internalisation signal. This lysine‐63‐linked polyubiquitination requires both UbcH5b/c and Ubc13‐conjugating enzymes for initiating mono‐ and subsequent polyubiquitination of class I, and the clathrin‐dependent internalisation is mediated by the epsin endocytic adaptor. Our results explain how lysine‐63‐linked polyubiquitination leads to degradation by an endolysosomal pathway and demonstrate a novel mechanism for endocytosis and endolysosomal degradation of class I, which may be applicable to other receptors.

Antisense transcripts in the human genome

By a systematic search of vertebrate mRNA sequences, we have identified a surprisingly large number of human antisense transcripts. These data suggest that regulation of gene expression by antisense and double-stranded RNAs could be a common phenomenon in mammalian cells.

The Use of Edge-Betweenness Clustering to Investigate Biological Function in Protein Interaction Networks

BackgroundThis paper describes an automated method for finding clusters of interconnected proteins in protein interaction networks and retrieving protein annotations associated with these clusters.ResultsProtein interaction graphs were separated into subgraphs of interconnected proteins, using the JUNG implementation of Girvan and Newmans Edge-Betweenness algorithm. Functions were sought for these subgraphs by detecting significant correlations with the distribution of Gene Ontology terms which had been used to annotate the proteins within each cluster. The method was implemented using freely available software (JUNG and the R statistical package). Protein clusters with significant correlations to functional annotations could be identified and included groups of proteins know to cooperate in cell metabolism. The method appears to be resilient against the presence of false positive interactions.ConclusionThis method provides a useful tool for rapid screening of small to medium size protein interaction datasets.

Roles of vaccinia virus EEV-specific proteins in intracellular actin tail formation and low pH-induced cell-cell fusion.

During vaccinia virus (VV) morphogenesis intracellular mature virus (IMV) is wrapped by two additional membranes to form intracellular enveloped virus (IEV). IEV particles can nucleate the formation of actin tails which aid movement of IEVs to the cell surface where the outer IEV membrane fuses with the plasma membrane forming cell-associated enveloped virus (CEV) which remains attached to the cell, or extracellular enveloped virus (EEV) which is shed from the cell. In this report, we have used a collection of VV mutants lacking individual EEV-specific proteins to compare the roles of these proteins in the formation of IEV and IEV-associated actin tails and fusion of infected cells after a low pH shock. Data presented here show that p45-50 (A36R) is not required for IEV formation or for acid-induced cell-cell fusion, but is required for formation of IEV-associated actin tails. In contrast, gp86 (A56R), the virus haemagglutinin, is not required for formation of either IEV or IEV-associated actin tails. Data presented also confirm that p37 (gene F13L), gp42 (B5R) and gp22-24 (A34R) are needed for formation of IEV-associated actin tails and for cell-cell fusion after low pH shock. The phenotypes of these mutants were not affected by the host cell type as similar results were obtained in a range of different cells. Lastly, comparisons of the phenotypes of VV strains Western Reserve, deltaA34R and deltaA36R demonstrate that actin tails are not required for low pH-induced cell-cell fusion.

The MIT Domain of UBPY Constitutes a CHMP Binding and Endosomal Localization Signal Required for Efficient Epidermal Growth Factor Receptor Degradation

We have identified and characterized a Microtubule Interacting and Transport (MIT) domain at the N terminus of the deubiquitinating enzyme UBPY/USP8. In common with other MIT-containing proteins such as AMSH and VPS4, UBPY can interact with CHMP proteins, which are known to regulate endosomal sorting of ubiquitinated receptors. Comparison of binding preferences for the 11 members of the human CHMP family between the UBPY MIT domain and another ubiquitin isopeptidase, AMSH, reveals common interactions with CHMP1A and CHMP1B but a distinct selectivity of AMSH for CHMP3/VPS24, a core subunit of the ESCRT-III complex, and UBPY for CHMP7. We also show that in common with AMSH, UBPY deubiquitinating enzyme activity can be stimulated by STAM but is unresponsive to its cognate CHMPs. The UBPY MIT domain is dispensable for its catalytic activity but is essential for its localization to endosomes. This is functionally significant as an MIT-deleted UBPY mutant is unable to rescue its binding partner STAM from proteasomal degradation or reverse a block to epidermal growth factor receptor degradation imposed by small interfering RNA-mediated depletion of UBPY.

Analysis of the human E2 ubiquitin conjugating enzyme protein interaction network

In eukaryotic cells the stability and function of many proteins are regulated by the addition of ubiquitin or ubiquitin-like peptides. This process is dependent upon the sequential action of an E1-activating enzyme, an E2-conjugating enzyme, and an E3 ligase. Different combinations of these proteins confer substrate specificity and the form of protein modification. However, combinatorial preferences within ubiquitination networks remain unclear. In this study, yeast two-hybrid (Y2H) screens were combined with true homology modeling methods to generate a high-density map of human E2/E3-RING interactions. These data include 535 experimentally defined novel E2/E3-RING interactions and >1300 E2/E3-RING pairs with more favorable predicted free-energy values than the canonical UBE2L3-CBL complex. The significance of Y2H predictions was assessed by both mutagenesis and functional assays. Significantly, 74/80 (>92%) of Y2H predicted complexes were disrupted by point mutations that inhibit verified E2/E3-RING interactions, and a approximately 93% correlation was observed between Y2H data and the functional activity of E2/E3-RING complexes in vitro. Analysis of the high-density human E2/E3-RING network reveals complex combinatorial interactions and a strong potential for functional redundancy, especially within E2 families that have undergone evolutionary expansion. Finally, a one-step extended human E2/E3-RING network, containing 2644 proteins and 5087 edges, was assembled to provide a resource for future functional investigations.

Molecular Basis for the Sorting of the SNARE VAMP7 into Endocytic Clathrin- Coated Vesicles by the ArfGAP Hrb

Summary SNAREs provide the specificity and energy for the fusion of vesicles with their target membrane, but how they are sorted into the appropriate vesicles on post-Golgi trafficking pathways is largely unknown. We demonstrate that the clathrin-mediated endocytosis of the SNARE VAMP7 is directly mediated by Hrb, a clathrin adaptor and ArfGAP. Hrb wraps 20 residues of its unstructured C-terminal tail around the folded VAMP7 longin domain, demonstrating that unstructured regions of clathrin adaptors can select cargo. Disrupting this interaction by mutation of the VAMP7 longin domain or depletion of Hrb causes VAMP7 to accumulate on the cells surface. However, the SNARE helix of VAMP7 binds back onto its longin domain, outcompeting Hrb for binding to the same groove and suggesting that Hrb-mediated endocytosis of VAMP7 occurs only when VAMP7 is incorporated into a cis-SNARE complex. These results elucidate the mechanism of retrieval of a postfusion SNARE complex in clathrin-coated vesicles.

Dissecting molecular cross-talk between Nrf2 and NF-κB response pathways.

In most tissues, cells are exposed to frequent changes in levels of oxidative stress and inflammation. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and nuclear factor-κB (NF-κB) are the two key transcription factors that regulate cellular responses to oxidative stress and inflammation respectively. Pharmacological and genetic studies suggest that there is functional cross-talk between these two important pathways. The absence of Nrf2 can exacerbate NF-κB activity leading to increased cytokine production, whereas NF-κB can modulate Nrf2 transcription and activity, having both positive and negative effects on the target gene expression. This review focuses on the potentially complex molecular mechanisms that link the Nrf2 and NF-κB pathways and the importance of designing more effective therapeutic strategies to prevent or treat a broad range of neurological disorders.

Proteomic analysis of Nrf2 deficient transgenic mice reveals cellular defence and lipid metabolism as primary Nrf2-dependent pathways in the liver

The transcription factor Nrf2 regulates expression of multiple cellular defence proteins through the antioxidant response element (ARE). Nrf2-deficient mice (Nrf2−/−) are highly susceptible to xenobiotic-mediated toxicity, but the precise molecular basis of enhanced toxicity is unknown. Oligonucleotide array studies suggest that a wide range of gene products is altered constitutively, however no equivalent proteomics analyses have been conducted. To define the range of Nrf2-regulated proteins at the constitutive level, protein expression profiling of livers from Nrf2−/− and wild type mice was conducted using both stable isotope labelling (iTRAQ) and gel electrophoresis methods. To establish a robust reproducible list of Nrf2-dependent proteins, three independent groups of mice were analysed. Correlative network analysis (MetaCore) identified two predominant groups of Nrf2-regulated proteins. As expected, one group comprised proteins involved in phase II drug metabolism, which were down-regulated in the absence of Nrf2. Surprisingly, the most profound changes were observed amongst proteins involved in the synthesis and metabolism of fatty acids and other lipids. Importantly, we show here for the first time, that the enzyme ATP-citrate lyase, responsible for acetyl-CoA production, is negatively regulated by Nrf2. This latter finding suggests that Nrf2 is a major regulator of cellular lipid disposition in the liver.

The vaccinia virus A27L protein is needed for the microtubule-dependent transport of intracellular mature virus particles.

The vaccinia virus (VV) A27L gene encodes a 14 kDa protein that is required for the formation of intracellular enveloped virus (IEV) and, consequently, normal sized plaques. Data presented here show that A27L plays an additional role in VV assembly. When cells were infected with the VV WR32-7/Ind 14K, under conditions that repress A27L expression, transport of intracellular mature virus (IMV) from virus factories was inhibited and some IMV was found in aberrant association with virus crescents. In contrast, other VV mutants (vDeltaB5R and vDeltaF13L) that are defective in IEV formation produce IMV particles that are transported out of virus factories. This indicated a specific role for A27L in IMV transport. Induction of A27L expression at 10 h post-infection promoted the dispersal of clustered IMV particles, but only when microtubules were intact. Formation of IEV particles was also impaired when cells were infected with WR32-7/14K, a VV strain expressing a mutated form of the A27L protein; however, this mutation did not inhibit intracellular transport of IMV particles. Collectively, these data define two novel aspects of VV morphogenesis. Firstly, A27L is required for both IMV transport and the process of envelopment that leads to IEV formation. Secondly, movement of IMV particles between the virus factory and the site of IEV formation is microtubule-dependent.