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.

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.

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.

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.