USP15: a review of its implication in immune and inflammatory processes and tumor progression.

The covalent post-translational modification of proteins by ubiquitination not only influences protein stability and half-life, but also several aspects of protein function including enzymatic activity, sub-cellular localization, and interactions with binding partners. Protein ubiquitination status is determined by the action of large families of ubiquitin ligases and deubiquitinases, whose combined activities regulate many physiological and cellular pathways. The Ubiquitin Specific Protease (USP) family is one of 8 subfamilies of deubiquitinating enzymes composed of more than 50 members. Recent studies have shown that USP15 plays a critical role in regulating many aspects of immune and inflammatory function of leukocytes in response to a broad range of infectious and autoimmune insults and following tissue damage. USP15 regulated pathways reviewed herein include TLR signaling, RIG-I signaling, NF-kB, and IRF3/IRF7-dependent transcription for production of pro-inflammatory cytokines and type I interferons. In addition, USP15 has been found to regulate pathways implicated in tumor onset and progression such as p53, and TGF-ß signaling, but also influences the leukocytes-determined immune and inflammatory microenvironment of tumors to affect progression and outcome. Hereby reviewed are recent studies of USP15 in model cell lines in vitro, and in mutant mice in vivo with reference to available human clinical datasets.

ZBTB7B (ThPOK) Is Required for Pathogenesis of Cerebral Malaria and Protection against Pulmonary Tuberculosis.

We used a genome-wide screen in N-ethyl-N-nitrosourea (ENU)-mutagenized mice to identify genes in which recessive loss-of-function mutations protect against pathological neuroinflammation. We identified an R367Q mutation in the ZBTB7B (ThPOK) protein in which homozygosity causes protection against experimental cerebral malaria (ECM) caused by infection with Plasmodium berghei ANKA. Zbtb7bR367Q homozygous mice show a defect in the lymphoid compartment expressed as severe reduction in the number of single-positive CD4 T cells in the thymus and in the periphery, reduced brain infiltration of proinflammatory leukocytes in P. berghei ANKA-infected mice, and reduced production of proinflammatory cytokines by primary T cells ex vivo and in vivo Dampening of proinflammatory immune responses in Zbtb7bR367Q mice is concomitant to increased susceptibility to infection with avirulent (Mycobacterium bovis BCG) and virulent (Mycobacterium tuberculosis H37Rv) mycobacteria. The R367Q mutation maps to the first DNA-binding zinc finger domain of ThPOK and causes loss of base contact by R367 in the major groove of the DNA, which is predicted to impair DNA binding. Global immunoprecipitation of ThPOK-containing chromatin complexes coupled to DNA sequencing (ChIP-seq) identified transcriptional networks and candidate genes likely to play key roles in CD4+ CD8+ T cell development and in the expression of lineage-specific functions of these cells. This study highlights ThPOK as a global regulator of immune function in which alterations may affect normal responses to infectious and inflammatory stimuli.

Crystal Structure of USP7 Ubiquitin-like Domains with an ICP0 Peptide Reveals a Novel Mechanism Used by Viral and Cellular Proteins to Target USP7.

Herpes simplex virus-1 immediate-early protein ICP0 activates viral genes during early stages of infection, affects cellular levels of multiple host proteins and is crucial for effective lytic infection. Being a RING-type E3 ligase prone to auto-ubiquitination, ICP0 relies on human deubiquitinating enzyme USP7 for protection against 26S proteasomal mediated degradation. USP7 is involved in apoptosis, epigenetics, cell proliferation and is targeted by several herpesviruses. Several USP7 partners, including ICP0, GMPS, and UHRF1, interact through its C-terminal domain (CTD), which contains five ubiquitin-like (Ubl) structures. Despite the fact that USP7 has emerged as a drug target for cancer therapy, structural details of USP7 regulation and the molecular mechanism of interaction at its CTD have remained elusive. Here, we mapped the binding site between an ICP0 peptide and USP7 and determined the crystal structure of the first three Ubl domains bound to the ICP0 peptide, which showed that ICP0 binds to a loop on Ubl2. Sequences similar to the USP7-binding site in ICP0 were identified in GMPS and UHRF1 and shown to bind USP7-CTD through Ubl2. In addition, co-immunoprecipitation assays in human cells comparing binding to USP7 with and without a Ubl2 mutation, confirmed the importance of the Ubl2 binding pocket for binding ICP0, GMPS and UHRF1. Therefore we have identified a novel mechanism of USP7 recognition that is used by both viral and cellular proteins. Our structural information was used to generate a model of near full-length USP7, showing the relative position of the ICP0/GMPS/UHRF1 binding pocket and the structural basis by which it could regulate enzymatic activity.

Proteomics methods for discovering viral-host interactions.

The functions of many viral proteins involve direct interactions with specific host proteins. Therefore considerable insight into the functions of a viral protein and its mechanisms of action can come from applying proteomics approaches to viral proteins in order to identify their cellular binding partners. In this chapter we describe proteomics approaches that have proven to be the most useful in identifying host interactions of viral proteins in human cells. Caveats and potential alternatives for each step are also discussed.

Deubiquitinase catalytic activity of MYSM1 is essential in vivo for hematopoiesis and immune cell development.

Myb-like SWIRM and MPN domains 1 (MYSM1) is a chromatin binding protein with deubiquitinase (DUB) catalytic activity. Rare MYSM1 mutations in human patients result in an inherited bone marrow failure syndrome, highlighting the biomedical significance of MYSM1 in the hematopoietic system. We and others characterized Mysm1-knockout mice as a model of this disorder and established that MYSM1 regulates hematopoietic function and leukocyte development in such models through different mechanisms. It is, however, unknown whether the DUB catalytic activity of MYSM1 is universally required for its many functions and for the maintenance of hematopoiesis in vivo. To test this, here we generated a new mouse strain carrying a Mysm1D660N point mutation (Mysm1DN) and demonstrated that the mutation renders MYSM1 protein catalytically inactive. We characterized Mysm1DN/DN and Mysm1fl/DN CreERT2 mice, against appropriate controls, for constitutive and inducible loss of MYSM1 catalytic function. We report a profound similarity in the developmental, hematopoietic, and immune phenotypes resulting from the loss of MYSM1 catalytic function and the full loss of MYSM1 protein. Overall, our work for the first time establishes the critical role of MYSM1 DUB catalytic activity in vivo in hematopoiesis, leukocyte development, and other aspects of mammalian physiology.

Author Correction: Identification and Characterization of USP7 Targets in Cancer Cells.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

USP7 Regulates Cytokinesis through FBXO38 and KIF20B.

The ubiquitin specific protease 7 (USP7 or HAUSP) is known to regulate a variety of cellular processes by binding and deubiquitylating specific target proteins. To gain a more comprehensive understanding of its interactions and functions, we used affinity purification coupled to mass spectrometry to profile USP7 interactions. This revealed a novel interaction with FBXO38, a poorly characterized F-box protein. We showed that USP7 stabilizes FBXO38 dependent on its catalytic activity by protecting FBXO38 from proteasomal degradation. We used a BioID approach to profile the protein interactions (and putative functions) of FBXO38, revealing an interaction with KIF20B, a Kinesin-6 protein required for efficient cytokinesis. FBXO38 was shown to function independently from an SCF complex to stabilize KIF20B. Consequently, depletion of either FBXO38 or USP7 led to dramatic decreases in KIF20B levels and KIF20B at the midbody, which were manifested in cytokinetic defects. Furthermore, cytokinetic defects associated with USP7 silencing were rescued by restoring FBXO38 or KIF20B. The results indicate a novel mechanism of regulating cytokinesis through USP7 and FBXO38.

Identification and Characterization of USP7 Targets in Cancer Cells.

The ubiquitin specific protease, USP7, regulates multiple cellular pathways relevant for cancer through its ability to bind and sometimes stabilize specific target proteins through deubiquitylation. To gain a more complete profile of USP7 interactions in cancer cells, we performed affinity purification coupled to mass spectrometry to identify USP7 binding targets in gastric carcinoma cells. This confirmed reported associations of USP7 with USP11, PPM1G phosphatase and TRIP12 E3 ubiquitin ligase as well as identifying novel interactions with two DEAD/DEAH-box RNA helicases, DDX24 and DHX40. Using USP7 binding pocket mutants, we show that USP11, PPM1G, TRIP12 and DDX24 bind USP7 through its TRAF domain binding pocket, while DHX40 interacts with USP7 through a distinct binding pocket in the Ubl2 domain. P/A/ExxS motifs in USP11 and DDX24 that are critical for USP7 binding were also identified. Modulation of USP7 expression levels and inhibition of USP7 catalytic activity in multiple cells lines showed that USP7 consistently stabilizes DDX24, DHX40 and TRIP12 dependent on its catalytic activity, while USP11 and PPM1G levels were not consistently affected. Our study better defines the mechanisms of USP7 interaction with known targets and identifies DDX24 and DHX40 as new targets that are specifically bound and regulated by USP7.

Affinity Purification-Mass Spectroscopy Methods for Identifying Epstein-Barr Virus-Host Interactions.

Considerable insight into the function and mechanism of action of viral proteins has come from identifying the cellular proteins with which they interact. In recent years, mass spectrometry-based methods have emerged as the method of choice for protein interaction discovery due to their comprehensive and unbiased nature. Methods involving single affinity purifications of epitope-tagged viral proteins (AP-MS) and tandem affinity purifications of viral proteins with two purification tags (TAP tagging) have both been used to identify novel host interactions with EBV proteins. However, to date these methods have only been applied to a small number of EBV proteins. Here we provide detailed methods of AP-MS and TAP tagging approaches that can be applied to any EBV protein in order to discover its host interactions.

Metaproteomic analysis of a winter to spring succession in coastal northwest Atlantic Ocean microbial plankton.

In this study, we used comparative metaproteomics to investigate the metabolic activity of microbial plankton inhabiting a seasonally hypoxic basin in the Northwest Atlantic Ocean (Bedford Basin). From winter to spring, we observed a seasonal increase in high-affinity membrane transport proteins involved in scavenging of organic substrates; Rhodobacterales transporters were strongly associated with the spring phytoplankton bloom, whereas SAR11 transporters were abundant in the underlying waters. A diverse array of transporters for organic compounds were similar to the SAR324 clade, revealing an active heterotrophic lifestyle in coastal waters. Proteins involved in methanol oxidation (from the OM43 clade) and carbon monoxide (from a wide variety of bacteria) were identified throughout Bedford Basin. Metabolic niche partitioning between the SUP05 and ARCTIC96BD-19 clades, which together comprise the Gamma-proteobacterial sulfur oxidizers group was apparent. ARCTIC96BD-19 proteins involved in the transport of organic compounds indicated that in productive coastal waters this lineage tends toward a heterotrophic metabolism. In contrast, the identification of sulfur oxidation proteins from SUP05 indicated the use of reduced sulfur as an energy source in hypoxic bottom water. We identified an abundance of Marine Group I Thaumarchaeota proteins in the hypoxic deep layer, including proteins for nitrification and carbon fixation. No transporters for organic compounds were detected among the thaumarchaeal proteins, suggesting a reliance on autotrophic carbon assimilation. In summary, our analyses revealed the spatiotemporal structure of numerous metabolic activities in the coastal ocean that are central to carbon, nitrogen and sulfur cycling in the sea.