Cell biology:Collagen secretion explained

Cells package proteins into vesicles for secretion to the extracellular milieu. A study shows that an enzyme modifies the packaging machinery to encapsulate unusually large proteins such as collagen

Screening of DUB activity and specificity by MALDI-TOF mass spectrometry

Deubiquitylases (DUBs) are key regulators of the ubiquitin system which cleave ubiquitin moieties from proteins and polyubiquitin chains. Several DUBs have been implicated in various diseases and are attractive drug targets. We have developed a sensitive and fast assay to quantify in vitro DUB enzyme activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Unlike other current assays, this method uses unmodified substrates, such as diubiquitin topoisomers. By analyzing 42 human DUBs against all diubiquitin topoisomers we provide an extensive characterization of DUB activity and specificity. Our results confirm the high specificity of many members of the OTU and JAMM DUB families and highlight that all USPs tested display low linkage selectivity. We also demonstrate that this assay can be deployed to assess the potency and specificity of DUB inhibitors by profiling 11 compounds against a panel of 32 DUBs

The tumor suppressor TMEM127 is a Nedd4-family E3 ligase adaptor required by Salmonella SteD to ubiquitinate and degrade MHC class II molecules

The Salmonella enterica effector SteD depletes mature MHC class II (mMHCII) molecules from the surface of infected antigen-presenting cells through ubiquitination of the cytoplasmic tail of the mMHCII β chain. Here, through a genome-wide mutant screen of human antigen-presenting cells, we show that the NEDD4 family HECT E3 ubiquitin ligase WWP2 and a tumor-suppressing transmembrane protein of unknown biochemical function, TMEM127, are required for SteD-dependent ubiquitination of mMHCII. Although evidently not involved in normal regulation of mMHCII, TMEM127 was essential for SteD to suppress both mMHCII antigen presentation in mouse dendritic cells and MHCII-dependent CD4+ T cell activation. We found that TMEM127 contains a canonical PPxY motif, which was required for binding to WWP2. SteD bound to TMEM127 and enabled TMEM127 to interact with and induce ubiquitination of mature MHCII. Furthermore, SteD also underwent TMEM127- and WWP2-dependent ubiquitination, which both contributed to its degradation and augmented its activity on mMHCII

Activation of the canonical IKK complex by K63/M1-linked hybrid ubiquitin chains

Polyubiquitin (pUb) chains formed between the C terminus of ubiquitin and lysine 63 (K63) or methionine 1 (M1) of another ubiquitin have been implicated in the activation of the canonical IκB kinase (IKK) complex. Here, we demonstrate that nearly all of the M1-pUb chains formed in response to interleukin-1, or the Toll-Like Receptors 1/2 agonist Pam(3)CSK(4), are covalently attached to K63-pUb chains either directly as K63-pUb/M1-pUb hybrids or indirectly by attachment to the same protein. Interleukin-1 receptor (IL-1R)-associated kinase (IRAK) 1 is modified first by K63-pUb chains to which M1-pUb linkages are added subsequently, and myeloid differentiation primary response gene 88 (MyD88) and IRAK4 are also modified by both K63-pUb and M1-pUb chains. We show that the heme-oxidized IRP2 ubiquitin ligase 1 interacting protein (HOIP) component of the linear ubiquitin assembly complex catalyzes the formation of M1-pUb chains in response to interleukin-1, that the formation of K63-pUb chains is a prerequisite for the formation of M1-pUb chains, and that HOIP interacts with K63-pUb but not M1-pUb linkages. These findings identify K63-Ub oligomers as a major substrate of HOIP in cells where the MyD88-dependent signaling network is activated. The TGF-beta–activated kinase 1 (TAK1)-binding protein (TAB) 2 and TAB3 components of the TAK1 complex and the NFκB Essential Modifier (NEMO) component of the canonical IKK complex bind to K63-pUb chains and M1-pUb chains, respectively. The formation of K63/M1-pUb hybrids may therefore provide an elegant mechanism for colocalizing both complexes to the same pUb chain, facilitating the TAK1-catalyzed activation of IKKα and IKKβ. Our study may help to resolve the debate about the relative importance of K63-pUb and M1-pUb chains in activating the canonical IKK complex

Recruitment of TBK1 to cytosol‐invading Salmonella induces WIPI2‐dependent antibacterial autophagy

Mammalian cells deploy autophagy to defend their cytosol against bacterial invaders. Anti-bacterial autophagy relies on the core autophagy machinery, cargo receptors, and "eat-me" signals such as galectin-8 and ubiquitin that label bacteria as autophagy cargo. Anti-bacterial autophagy also requires the kinase TBK1, whose role in autophagy has remained enigmatic. Here we show that recruitment of WIPI2, itself essential for anti-bacterial autophagy, is dependent on the localization of catalytically active TBK1 to the vicinity of cytosolic bacteria. Experimental manipulation of TBK1 recruitment revealed that engagement of TBK1 with any of a variety of Salmonella-associated "eat-me" signals, including host-derived glycans and K48- and K63-linked ubiquitin chains, suffices to restrict bacterial proliferation. Promiscuity in recruiting TBK1 via independent signals may buffer TBK1 functionality from potential bacterial antagonism and thus be of evolutionary advantage to the host

Analysis of the LKB1-STRAD-MO25 complex

Mutations in the LKB1 tumour suppressor threonine kinase cause the inherited Peutz-Jeghers cancer syndrome and are also observed in some sporadic cancers. Recent work indicates that LKB1 exerts effects on metabolism, polarity and proliferation by phosphorylating and activating protein kinases belonging to the AMPK subfamily. In vivo, LKB1 forms a complex with STRAD, an inactive pseudokinase, and MO25, an armadillo repeat scaffolding-like protein. Binding of LKB1 to STRAD-MO25 activates LKB1 and re-localises it from the nucleus to the cytoplasm. To learn more about the inherent properties of the LKB1-STRAD-MO25 complex, we first investigated the activity of 34 point mutants of LKB1 found in human cancers and their ability to interact with STRAD and MO25. Interestingly, 12 of these mutants failed to interact with STRAD-MO25. Performing mutagenesis analysis, we defined two binding sites located on opposite surfaces of MO25α, which are required for the assembly of MO25α into a complex with STRADα and LKB1. In addition, we demonstrate that LKB1 does not require phosphorylation of its own T-loop to be activated by STRADα-MO25α, and discuss the possibility that this unusual mechanism of regulation arises from LKB1 functioning as an upstream kinase. Finally, we establish that STRADα, despite being catalytically inactive, is still capable of binding ATP with high affinity, but that this is not required for activation of LKB1. Taken together, our findings reinforce the functional importance of the binding of LKB1 to STRAD, and provide a greater understanding of the mechanism by which LKB1 is regulated and activated through its interaction with STRAD and MO25

The molecular basis for ubiquitin and ubiquitin-like specificities in bacterial effector proteases

Pathogenic bacteria rely on secreted effector proteins to manipulate host signaling pathways, often in creative ways. CE clan proteases, specific hydrolases for ubiquitin-like modifications (SUMO and NEDD8) in eukaryotes, reportedly serve as bacterial effector proteins with deSUMOylase, deubiquitinase, or, even, acetyltransferase activities. Here, we characterize bacterial CE protease activities, revealing K63-linkage-specific deubiquitinases in human pathogens, such as Salmonella, Escherichia, and Shigella, as well as ubiquitin/ubiquitin-like cross-reactive enzymes in Chlamydia, Rickettsia, and Xanthomonas. Five crystal structures, including ubiquitin/ubiquitin-like complexes, explain substrate specificities and redefine relationships across the CE clan. Importantly, this work identifies novel family members and provides key discoveries among previously reported effectors, such as the unexpected deubiquitinase activity in Xanthomonas XopD, contributed by an unstructured ubiquitin binding region. Furthermore, accessory domains regulate properties such as subcellular localization, as exemplified by a ubiquitin-binding domain in Salmonella Typhimurium SseL. Our work both highlights and explains the functional adaptations observed among diverse CE clan proteins

Dissecting distinct proteolytic activities of FMDV Lpro implicates cleavage and degradation of RLR signaling proteins, not its deISGylase/DUB activity, in type I interferon suppression

Author summary Outbreaks of the picornavirus foot-and-mouth disease virus (FMDV) have significant consequences for animal health and product safety and place a major economic burden on the global livestock industry. Understanding how this notorious animal pathogen suppresses the antiviral type I interferon (IFN-alpha/beta) response may help to develop countermeasures to control FMDV infections. FMDV suppresses the IFN-alpha/beta response through the activity of its Leader protein (L-pro), a protease that can cleave host cell proteins. L(pro)was also shown to have deubiquitinase and deISGylase activity, raising the possibility that L(pro)suppresses IFN-alpha/beta by removing ubiquitin and/or ISG15, two posttranslational modifications that can regulate the activation, interactions and localization of (signaling) proteins. Here, we show that TBK1 and MAVS, two signaling proteins that are important for activation of IFN-alpha/beta gene transcription, are cleaved by L-pro. By generating L(pro)mutants lacking either of these two activities, we demonstrate that L-pro's ability to cleave signaling proteins, but not its deubiquitination/deISGylase activity, correlates with suppression of IFN-beta gene transcription. The type I interferon response is an important innate antiviral pathway. Recognition of viral RNA by RIG-I-like receptors (RLRs) activates a signaling cascade that leads to type I interferon (IFN-alpha/beta) gene transcription. Multiple proteins in this signaling pathway (e.g. RIG-I, MDA5, MAVS, TBK1, IRF3) are regulated by (de)ubiquitination events. Most viruses have evolved mechanisms to counter this antiviral response. The leader protease (L-pro) of foot-and-mouth-disease virus (FMDV) has been recognized to reduce IFN-alpha/beta gene transcription; however, the exact mechanism is unknown. The proteolytic activity of L(pro)is vital for releasing itself from the viral polyprotein and for cleaving and degrading specific host cell proteins, such as eIF4G and NF-kappa B. In addition, L(pro)has been demonstrated to have deubiquitination/deISGylation activity. L-pro's deubiquitination/deISGylation activity and the cleavage/degradation of signaling proteins have both been postulated to be important for reduced IFN-alpha/beta gene transcription. Here, we demonstrate that TBK1, the kinase that phosphorylates and activates the transcription factor IRF3, is cleaved by L(pro)in FMDV-infected cells as well as in cells infected with a recombinant EMCV expressing L-pro.In vitrocleavage experiments revealed that L(pro)cleaves TBK1 at residues 692-694. We also observed cleavage of MAVS in HeLa cells infected with EMCV-L-pro, but only observed decreasing levels of MAVS in FMDV-infected porcine LFPK alpha V beta 6 cells. We set out to dissect L-pro's ability to cleave RLR signaling proteins from its deubiquitination/deISGylation activity, to determine their relative contributions to the reduction of IFN-alpha/beta gene transcription. The introduction of specific mutations, of which several were based on the recently published structure of L(pro)in complex with ISG15, allowed us to identify specific amino acid substitutions that separate the different proteolytic activities of L-pro. Characterization of the effects of these mutations revealed that L-pro's ability to cleave RLR signaling proteins but not its deubiquitination/deISGylation activity correlates with the reduced IFN-beta gene transcription

Higher-Order Assembly of BRCC36-KIAA0157 Is Required for DUB Activity and Biological Function

BRCC36 is a Zn²⁺-dependent deubiquitinating enzyme (DUB) that hydrolyzes lysine-63-linked ubiquitin chains as part of distinct macromolecular complexes that participate in either interferon signaling or DNA-damage recognition. The MPN⁺ domain protein BRCC36 associates with pseudo DUB MPN⁻ proteins KIAA0157 or Abraxas, which are essential for BRCC36 enzymatic activity. To understand the basis for BRCC36 regulation, we have solved the structure of an active BRCC36-KIAA0157 heterodimer and an inactive BRCC36 homodimer. Structural and functional characterizations show how BRCC36 is switched to an active conformation by contacts with KIAA0157. Higher-order association of BRCC36 and KIAA0157 into a dimer of heterodimers (super dimers) was required for DUB activity and interaction with targeting proteins SHMT2 and RAP80. These data provide an explanation of how an inactive pseudo DUB allosterically activates a cognate DUB partner and implicates super dimerization as a new regulatory mechanism underlying BRCC36 DUB activity, subcellular localization, and biological function

The E3 ubiquitin ligase SCF(Fbxo7) mediates proteasomal degradation of UXT isoform 2 (UXT-V2) to inhibit the NF-κB signaling pathway.

BACKGROUND: Ubiquitously eXpressed Transcript isoform 2 (UXTV2) is a prefoldin-like protein involved in NF-κB signaling, apoptosis, and the androgen and estrogen response. UXT-V2 is a cofactor in the NF-κB transcriptional enhanceosome, and its knockdown inhibits TNF-α -induced NF-κB activation. Fbxo7 is an F-box protein that interacts with SKP1, Cullin1 and RBX1 proteins to form an SCF(Fbxo7) E3 ubiquitin ligase complex. Fbxo7 negatively regulates NF-κB signaling through TRAF2 and cIAP1 ubiquitination. METHODS: We combine co-immunoprecipitation, ubiquitination in vitro and in vivo, cycloheximide chase assay, ubiquitin chain restriction analysis and microscopy to investigate interaction between Fbxo7 and overexpressed UXT-V2-HA. RESULTS: The Ubl domain of Fbxo7 contributes to interaction with UXTV2. This substrate is polyubiquitinated by SCF(Fbxo7) with K48 and K63 ubiquitin chain linkages in vitro and in vivo. This post-translational modification decreases UXT-V2 stability and promotes its proteasomal degradation. We further show that UXTV1, an alternatively spliced isoform of UXT, containing 12 additional amino acids at the N-terminus as compared to UXTV2, also interacts with and is ubiquitinated by Fbxo7. Moreover, FBXO7 knockdown promotes UXT-V2 accumulation, and the overexpression of Fbxo7-ΔF-box protects UXT-V2 from proteasomal degradation and enhances the responsiveness of NF-κB reporter. We find that UXT-V2 colocalizes with Fbxo7 in the cell nucleus. CONCLUSIONS: Together, our study reveals that SCF(Fbxo7) mediates the proteasomal degradation of UXT-V2 causing the inhibition of the NF-κB signaling pathway. GENERAL SIGNIFICANCE: Discovering new substrates of E3 ubiquitin-ligase SCF(Fbxo7) contributes to understand its function in different diseases such as cancer and Parkinson.Biotechnology and Biological Science Research Council [BB/J007846/1]