Accelerating inhibitor discovery for deubiquitinating enzymes.

Deubiquitinating enzymes (DUBs) are an emerging drug target class of ~100 proteases that cleave ubiquitin from protein substrates to regulate many cellular processes. A lack of selective chemical probes impedes pharmacologic interrogation of this important gene family. DUBs engage their cognate ligands through a myriad of interactions. We embrace this structural complexity to tailor a chemical diversification strategy for a DUB-focused covalent library. Pairing our library with activity-based protein profiling as a high-density primary screen, we identify selective hits against 23 endogenous DUBs spanning four subfamilies. Optimization of an azetidine hit yields a probe for the understudied DUB VCPIP1 with nanomolar potency and in-family selectivity. Our success in identifying good chemical starting points as well as structure-activity relationships across the gene family from a modest but purpose-build library challenges current paradigms that emphasize ultrahigh throughput in vitro or virtual screens against an ever-increasing scope of chemical space.

Small molecules as tools for functional assessment of deubiquitinating enzyme function.

Deubiquitinating enzymes (DUBs) are a largely understudied and untapped resource in the toolkit of protein degradation functionalities. They comprise a large repertoire of enzymes that remove ubiquitin from substrates in a variety of cellular and pathophysiological contexts, and have enormous potential for research and clinical use. It is only within the last 5 years that potent, selective, and well-characterized small-molecule inhibitors of DUBs have been described. These compounds are now being used to study the biological roles of DUBs. Here, we describe downstream applications of small-molecule inhibitors for studying DUBs and provide a framework for future studies. We highlight recent examples of using these inhibitors to confirm and explore the role of these enzymes in both normal and pathological contexts. These studies represent the first steps in the burgeoning field of pharmacological and chemoproteomic studies of DUBs, which will be critical for the continued advancement of DUB field.

Development of IsoTaG, a Chemical Glycoproteomics Technique for Profiling Intact N- and O-Glycopeptides from Whole Cell Proteomes.

Protein glycosylation can have an enormous variety of biological consequences, reflecting the molecular diversity encoded in glycan structures. This same structural diversity has imposed major challenges on the development of methods to study the intact glycoproteome. We recently introduced a method termed isotope-targeted glycoproteomics (IsoTaG), which utilizes isotope recoding to characterize azidosugar-labeled glycopeptides bearing fully intact glycans. Here, we describe the broad application of the method to analyze glycoproteomes from a collection of tissue-diverse cell lines. The effort was enabled by a new high-fidelity pattern-searching and glycopeptide validation algorithm termed IsoStamp v2.0, as well as by novel stable isotope probes. Application of the IsoTaG platform to 15 cell lines metabolically labeled with Ac4GalNAz or Ac4ManNAz revealed 1375 N- and 2159 O-glycopeptides, variously modified with 74 discrete glycan structures. Glycopeptide-bound glycans observed by IsoTaG were found to be comparable to released N-glycans identified by permethylation analysis. IsoTaG is therefore positioned to enhance structural understanding of the glycoproteome.

Isotope-targeted glycoproteomics (IsoTaG) analysis of sialylated N- and O-glycopeptides on an Orbitrap Fusion Tribrid using azido and alkynyl sugars.

Protein glycosylation is a post-translational modification (PTM) responsible for many aspects of proteomic diversity and biological regulation. Assignment of intact glycan structures to specific protein attachment sites is a critical step towards elucidating the function encoded in the glycome. Previously, we developed isotope-targeted glycoproteomics (IsoTaG) as a mass-independent mass spectrometry method to characterize azide-labeled intact glycopeptides from complex proteomes. Here, we extend the IsoTaG approach with the use of alkynyl sugars as metabolic labels and employ new probes in analysis of the sialylated glycoproteome from PC-3 cells. Using an Orbitrap Fusion Tribrid mass spectrometer, we identified 699 intact glycopeptides from 192 glycoproteins. These intact glycopeptides represent a total of eight sialylated glycan structures across 126 N- and 576 O-glycopeptides. IsoTaG is therefore an effective platform for identification of intact glycopeptides labeled by alkynyl or azido sugars and will facilitate further studies of the glycoproteome.

Antigenic diversity and distribution of rabies virus in Mexico.

Rabies remains a public health problem in the Americas because of the great diversity of wild reservoirs that maintain the virus in nature. Here we report the antigenic characterization of 254 rabies viruses isolated from 148 nonreservoir and 106 reservoir hosts collected in 27 states of Mexico. Nine out of 11 antigenic variants previously reported in the United States were detected in Mexico by using the limited panel of monoclonal antibodies donated by the Centers for Disease Control and Prevention. Some rabies virus variants were isolated from their natural reservoirs, which were also taxonomically identified. Terrestrial reservoirs included stray dogs with V1, Urocyon cineroargenteus (gray foxes) with V7, and two subspecies of Spilogale putorius (spotted skunks) with different viral variants (V8 and V10). Aerial hosts included Tadarida brasiliensis mexicana and Desmodus rotundus, which harbored V9 and V4 and harbored V11, respectively. All variants, with the exception of V9, were isolated from nonreservoir hosts, while V3, V4, and V5 were not isolated from their natural reservoirs but only from livestock. Rabies virus antigenic typing allowed us to determine rabies reservoirs and their distribution in Mexico, data which will probably improve prevention and control of the illness in humans and in the reservoir hosts.