A Degenerate Cohort of Yeast Membrane Trafficking DUBs Mediates Cell Polarity and Survival*

Deubiquitinating enzymes (DUBs), cysteine or metallo- proteases that cleave ubiquitin chains or protein conjugates, are present in nearly every cellular compartment, with overlapping protein domain structure, localization, and functions. We discovered a cohort of DUBs that are involved in membrane trafficking (ubp4, ubp5, ubp9, ubp15, and sst2) and found that loss of all five of these DUBs but not loss of any combination of four, significantly impacted cell viability in the fission yeast Schizosaccharomyces pombe (1). Here, we delineate the collective and individual functions and activities of these five conserved DUBs using comparative proteomics, biochemistry, and microscopy. We find these five DUBs are degenerate rather than redundant at the levels of cell morphology, substrate selectivity, ubiquitin chain specificity, and cell viability under stress. These studies reveal the complexity of interplay among these enzymes, providing a foundation for understanding DUB biology and providing another example of how cells utilize degeneracy to improve survival.Eukaryotic cells integrate signaling pathways to modulate their response to environmental changes, predominately through dynamic protein posttranslational modifications like ubiquitination (Ub''n) (2, 3). Cycles of Ub''n modulate protein stability, localization, and/or binding partners while maintaining cellular ubiquitin (Ub) homeostasis (3). Ub''n of substrate proteins is catalyzed by a linear sequence of enzymes (E1, E2, E3) and reversed by deubiquitinases (DUBs¹). Ub chains can be formed through any of Ub''s seven lysines (K6, K11, K27, K29, K33, K48, K63) or its N terminus (M1), generating a wide variety of Ub chain architectures that mediate specific cellular signals (4, 5). DUBs have been implicated in multiple essential cellular roles, including chromatin remodeling, DNA damage repair, kinase activation, endocytosis, ribosomal maturation, and immune responses (2, 3).Surprisingly, while multiple Ub''n enzymes (E1, E2, and E3) are essential in yeast (6–8), only a single DUB is essential for viability of both budding and fission yeasts (6–11), suggesting that considerable functional overlap may exist in yeast under standard laboratory conditions. In contrast, in metazoans, knockdown or loss of individual DUBs often results in developmental defects or disease states (3, 12, 13). Consistent with this possibility, we previously found that loss of five DUBs (5DUB delete: ubp4Δ1 ubp5Δ ubp9Δ ubp15Δ sst2Δ) but not any combination of four intracellular membrane trafficking DUBs significantly impacted cell polarity, Ub conjugate accumulation, and viability in S. pombe (1). To begin to make sense of this functional overlap, here we dissected the shared and specific functions of these five DUBs on multiple levels, defining their contributions to cell polarity, Ub chain specificities, shared and specific putative substrates, and individual and combined effects of DUB loss on cell survival under stress. We find that this cohort of five DUBs is degenerate (different elements that have overlapping but not fully redundant roles), forming a robust functional module for maintenance of cell polarity and viability.     

Endosomal Deubiquitinating Enzymes Control Ubiquitination and Down-regulation of Protease-activated Receptor 2

The E3 ubiquitin ligase c-Cbl ubiquitinates the G protein-coupled receptor protease-activated receptor 2 (PAR₂), which is required for postendocytic sorting of activated receptors to lysosomes, where degradation terminates signaling. The mechanisms of PAR₂ deubiquitination and its importance in trafficking and signaling of endocytosed PAR₂ are unknown. We report that receptor deubiquitination occurs between early endosomes and lysosomes and involves the endosomal deubiquitinating proteases AMSH and UBPY. Expression of the catalytically inactive mutants, AMSH(D348A) and UBPY(C786S), caused an increase in PAR₂ ubiquitination and trapped the receptor in early endosomes, thereby preventing lysosomal trafficking and degradation. Small interfering RNA knockdown of AMSH or UBPY also impaired deubiquitination, lysosomal trafficking, and degradation of PAR₂. Trapping PAR₂ in endosomes through expression of AMSH(D348A) or UBPY(C786S) did not prolong the association of PAR₂ with β-arrestin2 or the duration of PAR₂-induced ERK2 activation. Thus, AMSH and UBPY are essential for trafficking and down-regulation of PAR₂ but not for regulating PAR₂ dissociation from β-arrestin2 or PAR₂-mediated ERK2 activation.Ubiquitination of certain G protein-coupled receptors (GPCRs)³ is an essential signal for their postendocytic trafficking to lysosomes, which prevents uncontrolled signaling during chronic stimulation. Agonists stimulate ubiquitination of the β₂-adrenergic receptor (β₂AR), chemokine (CXC motif) receptor 4, and protease-activated receptor 2 (PAR₂), and the E3 ubiquitin ligases that mediate ubiquitination of these GPCRs and associated proteins, such as β-arrestins, have been identified (1–3). Although ubiquitination of these receptors is not required for endocytosis, ubiquitin-resistant mutant receptors show diminished postendocytic sorting to lysosomes and impaired down-regulation. However, despite of the reversible nature of this post-translational modification, little is known about the role of deubiquitinating proteases (DUBs) in the postendocytic trafficking and signaling of GPCRs.Our understanding of the role of DUBs in postendocytic receptor trafficking mostly derives from studies of receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR). Two endosomal DUBs, AMSH (associated molecule with the Src homology 3 domain of STAM (signal-transducing adapter molecule)) and UBPY (ubiquitin-specific protease Y) (also known as USP8), regulate deubiquitination and postendocytic trafficking of EGFR (4). AMSH belongs to the JAMM (JAB1/MPN/Mov34) family of metalloproteases and shows specificity for Lys⁶³- over Lys⁴⁸-linked ubiquitin chains (5, 6). UBPY is a cysteine protease of the ubiquitin-specific protease (USP) family and does not discriminate between Lys⁴⁸- and Lys⁶³-linked ubiquitin (7, 8). Activated EGFR recruits the E3 ligase c-Cbl, which ubiquitinates the receptor (9). Ubiquitinated EGFR then interacts with the Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate)-STAM complex in early endosomes (10). Hrs-STAM forms part of the ESCRT (endosomal sorting complex required for transport)-I, -II, -III complex that sorts ubiquitinated receptors in multivesicular bodies (MVBs) to intralumenal vesicles that eventually fuse with lysosomes, where degradation occurs (11). Before receptors are incorporated into the intralumenal vesicles, they are deubiquitinated, which serves to maintain levels of free ubiquitin (11). AMSH and UBPY interact directly with STAM through a common binding site within its Src homology 3 domain (12–14). The balance of EGFR ubiquitination by c-Cbl and deubiquitination by AMSH and UBPY controls the postendocytic trafficking and down-regulation of the EGFR. c-Cbl promotes lysosomal degradation of the EGFR (9), AMSH opposes c-Cbl action and promotes EGFR recycling (5), and UBPY is required for lysosomal sorting and degradation of EGFR (8, 15–17). The role of AMSH and UBPY in regulating deubiquitination, trafficking, and signaling of GPCRs in endosomes is largely unknown. A recent study has shown, however, that AMSH and UBPY regulate the down-regulation of the δ-opioid receptor (DOR), a GPCR that is ubiquitinated and degraded following activation (18). Expression of catalytically inactive mutants of AMSH or UBPY or knockdown of AMSH or UBPY levels using siRNA inhibits down-regulation of DOR. Interestingly, the roles of AMSH and UBPY in DOR down-regulation appear to be nonredundant, since depletion of either DUB produced comparable effects, and simultaneous depletion of both DUBs did not have additional consequences (18). Different DUBs, USP20 and -33, have been recently shown to reverse agonist-induced ubiquitination of the β₂AR (19).We examined the roles of AMSH and UBPY in the ubiquitination, postendocytic trafficking, and lysosomal degradation of PAR₂. We also determined whether AMSH and UBPY regulate PAR₂ association with β-arrestins in endosomes and control β-arrestin-mediated extracellular signal-regulated kinase (ERK) activation. PAR₂ is a receptor for multiple serine proteases that are generated during injury and inflammation (20). Activated PAR₂ promotes inflammation and pain, and PAR₂ contributes to inflammatory diseases of the airway, joints, and intestine. PAR₂ levels are elevated during inflammation, due to increased mRNA expression or perhaps decreased receptor degradation, which amplifies the proinflammatory actions of proteases (21). Given the irreversible nature of proteolytic activation, and since the internalized receptor probably signals by the β-arrestin-dependent recruitment of mitogen-activated protein kinase (MAPK) to endosomes (22), termination of PAR₂ signaling requires receptor degradation in lysosomes, which in turn is ubiquitination-dependent (3, 23). It is therefore important to understand mechanisms of PAR₂ ubiquitination and lysosomal targeting and also how these processes can be reversed. We have reported that activated PAR₂ is monoubiquitinated at multiple sites by the E3 ligase c-Cbl and targeted to lysosomes by an Hrs-dependent pathway (3, 24). Nothing is known about the mechanism and function of PAR₂ deubiquitination. Herein, we examined the role of AMSH and UBPY in regulating the deubiquitination, lysosomal trafficking, and degradation of PAR₂, the interaction of PAR₂ with β-arrestin2, and β-arrestin-mediated ERK2 activation. We demonstrate that endosomal DUBs are key regulators of GPCR down-regulation.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

The Wavelet-Based Cluster Analysis for Temporal Gene Expression Data

A variety of high-throughput methods have made it possible to generate detailed temporal expression data for a single gene or large numbers of genes. Common methods for analysis of these large data sets can be problematic. One challenge is the comparison of temporal expression data obtained from different growth conditions where the patterns of expression may be shifted in time. We propose the use of wavelet analysis to transform the data obtained under different growth conditions to permit comparison of expression patterns from experiments that have time shifts or delays. We demonstrate this approach using detailed temporal data for a single bacterial gene obtained under 72 different growth conditions. This general strategy can be applied in the analysis of data sets of thousands of genes under different conditions.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Multipattern Consensus Regions in Multiple Aligned Protein Sequences and Their Segmentation

Decomposing a biological sequence into its functional regions is an important prerequisite to understand the molecule. Using the multiple alignments of the sequences, we evaluate a segmentation based on the type of statistical variation pattern from each of the aligned sites. To describe such a more general pattern, we introduce multipattern consensus regions as segmented regions based on conserved as well as interdependent patterns. Thus the proposed consensus region considers patterns that are statistically significant and extends a local neighborhood. To show its relevance in protein sequence analysis, a cancer suppressor gene called p53 is examined. The results show significant associations between the detected regions and tendency of mutations, location on the 3D structure, and cancer hereditable factors that can be inferred from human twin studies.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]     

Ubiquitination Regulates Proteolytic Processing of G Protein-coupled Receptors after Their Sorting to Lysosomes

Ubiquitination is essential for the endocytic sorting of various G protein-coupled receptors to lysosomes. Here we identify a distinct function of this covalent modification in controlling the later proteolytic processing of receptors. Mutation of all cytoplasmic lysine residues in the murine δ-opioid receptor blocked receptor ubiquitination without preventing ligand-induced endocytosis of receptors or their subsequent delivery to lysosomes, as verified by proteolysis of extramembrane epitope tags and down-regulation of radioligand binding to the transmembrane helices. Surprisingly, a functional screen revealed that the E3 ubiquitin ligase AIP4 specifically controls down-regulation of wild type receptors measured by radioligand binding without detectably affecting receptor delivery to lysosomes defined both immunochemically and biochemically. This specific AIP4-dependent regulation required direct ubiquitination of receptors and was also regulated by two deubiquitinating enzymes, AMSH and UBPY, which localized to late endosome/lysosome membranes containing internalized δ-opioid receptor. These results identify a distinct function of AIP4-dependent ubiquitination in controlling the later proteolytic processing of G protein-coupled receptors, without detectably affecting their endocytic sorting to lysosomes. We propose that ubiquitination or ubiquitination/deubiquitination cycling specifically regulates later proteolytic processing events required for destruction of the receptor''s hydrophobic core.A fundamental cellular mechanism contributing to homeostatic regulation of receptor-mediated signal transduction involves ligand-induced endocytosis of receptors followed by proteolysis in lysosomes. The importance of such proteolytic down-regulation has been documented extensively for a number of seven-transmembrane or G protein-coupled receptors (GPCRs),³ which comprise the largest known family of signaling receptors expressed in animals, as well as for other important signaling receptors, such as the epidermal growth factor receptor tyrosine kinase (1–5).One GPCR that is well known to undergo endocytic trafficking to lysosomes is the δ-opioid peptide receptor (DOR or DOP-R) (6). Following endocytosis, DOR traffics efficiently to lysosomes in both neural and heterologous cell models (6–8), whereas many membrane proteins, including various GPCRs, recycle rapidly to the plasma membrane (9–12). Such molecular sorting of internalized receptors between divergent recycling and degradative pathways is thought to play a fundamental role in determining the functional consequences of regulated endocytosis (2, 3, 13, 14). The sorting process that directs internalized DOR to lysosomes is remarkably efficient and appears to occur rapidly (within several min) after receptor endocytosis (11). Nevertheless, biochemical mechanisms that control lysosomal trafficking and proteolysis of DOR remain poorly understood.A conserved mechanism that promotes lysosomal trafficking of a number of membrane proteins, including various signaling receptors, is mediated by covalent modification of cytoplasmic lysine residues with ubiquitin (4, 15–17). Ubiquitination was first identified as an endocytic sorting determinant in studies of vacuolar trafficking of the yeast GPCR Ste2p (18). Subsequent studies have established numerous examples of lysyl-ubiquitination being required for sorting endocytic cargo to lysosomes and have identified conserved machinery responsible for the targeting of ubiquitinated cargo to lysosomes (3, 17, 19–22).The CXCR4 chemokine receptor provides a clear example of ubiquitin-dependent lysosomal sorting of a mammalian GPCR. Ubiquitination of the carboxyl-terminal cytoplasmic domain of the CXCR4 receptor, mediated by the E3 ubiquitin ligase AIP4, is specifically required for the HRS- and VPS4-dependent trafficking of internalized receptors to lysosomes. Blocking this ubiquitination event by Lys → Arg mutation of the receptor specifically inhibits trafficking of internalized receptors to lysosomes, resulting in recycling rather than lysosomal proteolysis of receptors after ligand-induced endocytosis (23–25).Lysosomal trafficking of DOR, in contrast, is not prevented by mutation of cytoplasmic lysine residues (26) and can be regulated by ubiquitination-independent protein interaction(s) (27, 28). Nevertheless, both wild type and lysyl-mutant DORs traffic to lysosomes via a similar pathway as ubiquitin-dependent membrane cargo and require both HRS and active VPS4 to do so (29). These observations indicate that DOR engages the same core endocytic mechanism utilized by ubiquitination-directed membrane cargo but leave unresolved whether ubiquitination of DOR plays any role in this important cellular mechanism of receptor down-regulation.There is no doubt that DOR can undergo significant ubiquitination in mammalian cells, including HEK293 cells (30–32), where lysosomal trafficking of lysyl-mutant receptors was first observed (26). Ubiquitination was shown previously to promote proteolysis of DOR by proteasomes and to function in degrading misfolded receptors from the biosynthetic pathway (30, 31). A specific role of ubiquitination in promoting proteasome- but not lysosome-mediated proteolysis of DOR has been emphasized (32) and proposed to contribute to proteolytic down-regulation of receptors also from the plasma membrane (33).To our knowledge, no previous studies have determined if DOR ubiquitination plays any role in controlling receptor proteolysis mediated by lysosomes, although this represents a predominant pathway by which receptors undergo rapid down-regulation following ligand-induced endocytosis in a number of cell types, including HEK293 cells (8). In the present study, we have taken two approaches to addressing this fundamental question. First, we have investigated in greater detail the effects of lysyl-mutation on DOR ubiquitination and trafficking. Second, we have independently investigated the role of ubiquitination in controlling lysosomal proteolysis of wild type DOR. Our results clearly establish the ability of DOR to traffic efficiently to lysosomes in the absence of any detectable ubiquitination. Further, they identify a distinct and unanticipated function of AIP4-dependent ubiquitination in regulating the later proteolytic processing of receptors and show that this distinct ubiquitin-dependent regulatory mechanism operates effectively downstream of the sorting decision that commits internalized receptors for delivery to lysosomes.