Binding and dynamic studies of the human ubiquitin conjugating enzyme UBE2G2 by nuclear magnetic resonance spectroscopy

Ubiquitination is the covalent attachment of the 76-residue protein ubiquitin to another protein. Ubiquitin forms polyubiquitin, which are linkages formed by isopeptide bonds between a lysine ϵ-amine of one ubiquitin to the C-terminus of another ubiquitin. The particular biological role of polyubiquitination is dictated by the ubiquitin lysine that is involved in the isopeptide linkage. The K48-linkage dictates the proteasomal degradation of the protein to which it is attached. Regulation of protein degradation is important for a variety of biological processes, notably controlling degradation of cyclins that dictate the growth and replication of cells. Ubiquitination has been implicated in many illnesses, including several forms of cancer, Alzheimer's disease, and Parkinson's disease. Ubiquitin and ubiquitin chains form through a hierarchical pathway. Ubiquitin is first activated by a ubiquitin activating enzyme (E1), which transfers the activated ubiquitin to a ubiquitin conjugating enzyme (E2). Ubiquitin is attached to E2 by a thioester bond between the ubiquitin C-terminus and an E2 active site cysteine. A third group of proteins, ubiquitin ligases (E3s), mediate the transfer of ubiquitin from E2 to a target protein. One E2 that specifically catalyzes the formation of K48-linked polyubiquitin is Ube2g2. In order for the E2 and E3 to form an isopeptide linkage, they must bring together two ubiquitins and link them in a specific fashion. If the E2/E3 complex is to extend a chain, the complex must recognize the linkages already present in this chain in order to ensure the fidelity of chain elongation. We studied the binding specificity of Ube2g2 to monoubiquitin and both K48-linked and K63-linked ubiquitin dimers by Nuclear Magnetic Resonance Spectroscopic (NMR) techniques of Chemical Shift Perturbation (CSP) and Paramagnetic Relaxation Enhancement (PRE). We found that Ube2g2 bound the distal subunits of both ubiquitin dimers with a weaker affinity to the proximal ubiquitin subunits. PRE experiments also suggested that ubiquitin and ubiquitin dimers bind Ube2g2 with different conformations. One characteristic that distinguishes Ube2g2 from other E2s is the inclusion of a thirteen-residue sequence insertion near its active site cysteine, known to be a loop from structural studies. Through collection of relaxation parameters and subsequent analysis, it was established that this insertion loop and another loop spanning residues 130–135 are mobile. The active site cysteine sits between these two loops, suggesting that the loops play a role in the catalytic mechanism of ubiquitin transfer. Additional relaxation studies suggest that these residues remain mobile when ubiquitin binds noncovalently, indicating that loop mobility is not affected by ubiquitin binding at a remote site

Conformational Dynamics Modulate Activation of the Ubiquitin Conjugating Enzyme Ube2g2

The ubiquitin conjugating enzyme Ube2g2 together with its cognate E3 ligase gp78 catalyzes the synthesis of lysine-48 polyubiquitin chains constituting signals for the proteasomal degradation of misfolded proteins in the endoplasmic reticulum. Here, we employ NMR spectroscopy in combination with single-turnover diubiquitin formation assays to examine the role of the RING domain from gp78 in the catalytic activation of Ube2g2∼Ub conjugates. We find that approximately 60% of the Ube2g2∼Ub conjugates occupy a closed conformation in the absence of gp78-RING, with the population increasing to 82% upon gp78-RING binding. As expected, strong mutations in the hydrophobic patch residues of the ∼Ub moiety result in Ube2g2∼Ub populating only open states with corresponding loss of the ubiquitin conjugation activity. Less disruptive mutations introduced into the hydrophobic patch of the ∼Ub moiety also destabilize the closed conformational state, yet the corresponding effect on the ubiquitin conjugation activity ranges from complete loss to an enhancement of the catalytic activity. These results present a picture in which Ube2g2’s active site is in a state of continual dynamic flux with the organization of the active site into a catalytically viable conformation constituting the rate-limiting step for a single ubiquitin ligation event. Ube2g2’s function as a highly specific K48-polyubiquitin chain elongator leads us to speculate that this may be a strategy by which Ube2g2 reduces the probability of nonproductive catalytic outcomes in the absence of available substrate

Evaluation of NCI‑7 Cell Line Panel as a Reference Material for Clinical Proteomics

Reference materials are vital to benchmarking the reproducibility of clinical tests and essential for monitoring laboratory performance for clinical proteomics. The reference material utilized for mass spectrometric analysis of the human proteome would ideally contain enough proteins to be suitably representative of the human proteome, as well as exhibit a stable protein composition in different batches of sample regeneration. Previously, The Clinical Proteomic Tumor Analysis Consortium (CPTAC) utilized a PDX-derived comparative reference (CompRef) materials for the longitudinal assessment of proteomic performance; however, inherent drawbacks of PDX-derived material, including extended time needed to grow tumors and high level of expertise needed, have resulted in efforts to identify a new source of CompRef material. In this study, we examined the utility of using a panel of seven cancer cell lines, NCI-7 Cell Line Panel, as a reference material for mass spectrometric analysis of human proteome. Our results showed that not only is the NCI-7 material suitable for benchmarking laboratory sample preparation methods, but also NCI-7 sample generation is highly reproducible at both the global and phosphoprotein levels. In addition, the predicted genomic and experimental coverage of the NCI-7 proteome suggests the NCI-7 material may also have applications as a universal standard proteomic reference

Evaluation of NCI‑7 Cell Line Panel as a Reference Material for Clinical Proteomics

Reference materials are vital to benchmarking the reproducibility of clinical tests and essential for monitoring laboratory performance for clinical proteomics. The reference material utilized for mass spectrometric analysis of the human proteome would ideally contain enough proteins to be suitably representative of the human proteome, as well as exhibit a stable protein composition in different batches of sample regeneration. Previously, The Clinical Proteomic Tumor Analysis Consortium (CPTAC) utilized a PDX-derived comparative reference (CompRef) materials for the longitudinal assessment of proteomic performance; however, inherent drawbacks of PDX-derived material, including extended time needed to grow tumors and high level of expertise needed, have resulted in efforts to identify a new source of CompRef material. In this study, we examined the utility of using a panel of seven cancer cell lines, NCI-7 Cell Line Panel, as a reference material for mass spectrometric analysis of human proteome. Our results showed that not only is the NCI-7 material suitable for benchmarking laboratory sample preparation methods, but also NCI-7 sample generation is highly reproducible at both the global and phosphoprotein levels. In addition, the predicted genomic and experimental coverage of the NCI-7 proteome suggests the NCI-7 material may also have applications as a universal standard proteomic reference

Targeted mass spectrometry-based assays enable multiplex quantification of receptor tyrosine kinase, MAP Kinase, and AKT signaling.

SummaryA primary goal of the US National Cancer Institute's Ras initiative at the Frederick National Laboratory for Cancer Research is to develop methods to quantify RAS signaling to facilitate development of novel cancer therapeutics. We use targeted proteomics technologies to develop a community resource consisting of 256 validated multiple reaction monitoring (MRM)-based, multiplexed assays for quantifying protein expression and phosphorylation through the receptor tyrosine kinase, MAPK, and AKT signaling networks. As proof of concept, we quantify the response of melanoma (A375 and SK-MEL-2) and colorectal cancer (HCT-116 and HT-29) cell lines to BRAF inhibition by PLX-4720. These assays replace over 60 Western blots with quantitative mass spectrometry-based assays of high molecular specificity and quantitative precision, showing the value of these methods for pharmacodynamic measurements and mechanism of action studies. Methods, fit-for-purpose validation, and results are publicly available as a resource for the community at assays.cancer.gov.MotivationA lack of quantitative, multiplexable assays for phosphosignaling limits comprehensive investigation of aberrant signaling in cancer and evaluation of novel treatments. To alleviate this limitation, we sought to develop assays using targeted mass spectrometry for quantifying protein expression and phosphorylation through the receptor tyrosine kinase, MAPK, and AKT signaling networks. The resulting assays provide a resource for replacing over 60 Western blots in examining cancer signaling and tumor biology with high molecular specificity and quantitative rigor

Mechanism of Polyubiquitin Chain Recognition by the Human Ubiquitin Conjugating Enzyme Ube2g2*

Ube2g2 is a human ubiquitin conjugating (E2) enzyme involved in the endoplasmic reticulum-associated degradation pathway, which is responsible for the identification and degradation of unfolded and misfolded proteins in the endoplasmic reticulum compartment. The Ube2g2-specific role is the assembly of Lys-48-linked polyubiquitin chains, which constitutes a signal for proteasomal degradation when attached to a substrate protein. NMR chemical shift perturbation and paramagnetic relaxation enhancement approaches were employed to characterize the binding interaction between Ube2g2 and ubiquitin, Lys-48-linked diubiquitin, and Lys-63-linked diubiquitin. Results demonstrate that ubiquitin binds to Ube2g2 with an affinity of 90 μm in two different orientations that are rotated by 180° in models generated by the RosettaDock modeling suite. The binding of Ube2g2 to Lys-48- and Lys-63-linked diubiquitin is primarily driven by interactions with individual ubiquitin subunits, with a clear preference for the subunit containing the free Lys-48 or Lys-63 side chain (i.e. the distal subunit). This preference is particularly striking in the case of Lys-48-linked diubiquitin, which exhibits an ∼3-fold difference in affinities between the two ubiquitin subunits. This difference can be attributed to the partial steric occlusion of the subunit whose Lys-48 side chain is involved in the isopeptide linkage. As such, these results suggest that Lys-48-linked polyubiquitin chains may be designed to bind certain proteins like Ube2g2 such that the terminal ubiquitin subunit carrying the reactive Lys-48 side chain can be positioned properly for chain elongation regardless of chain length

Proteogenomic characterization of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor patient survival. Toward understanding the underlying molecular alterations that drive PDAC oncogenesis, we conducted comprehensive proteogenomic analysis of 140 pancreatic cancers, 67 normal adjacent tissues, and 9 normal pancreatic ductal tissues. Proteomic, phosphoproteomic, and glycoproteomic analyses were used to characterize proteins and their modifications. In addition, whole-genome sequencing, whole-exome sequencing, methylation, RNA sequencing (RNA-seq), and microRNA sequencing (miRNA-seq) were performed on the same tissues to facilitate an integrated proteogenomic analysis and determine the impact of genomic alterations on protein expression, signaling pathways, and post-translational modifications. To ensure robust downstream analyses, tumor neoplastic cellularity was assessed via multiple orthogonal strategies using molecular features and verified via pathological estimation of tumor cellularity based on histological review. This integrated proteogenomic characterization of PDAC will serve as a valuable resource for the community, paving the way for early detection and identification of novel therapeutic targets. [Display omitted] •Proteogenomic characterization reveals the functional impact of genomic alterations•Phosphoproteomics uncovers putative therapeutic targets downstream of KRAS•Multiomics links endothelial cell remodeling and glycolysis to immune exclusion•Proteomics and glycoproteomics reveal candidates for early detection or intervention Comparative multiomic analyses of pancreatic ductal adenocarcinoma tumors with normal adjacent and pancreatic ductal tissues provide insight into genomic, proteomic, and immune dysregulation in driving disease

Proteogenomic insights into the biology and treatment of HPV-negative head and neck squamous cell carcinoma

We present a proteogenomic study of 108 human papilloma virus (HPV)-negative head and neck squamous cell carcinomas (HNSCCs). Proteomic analysis systematically catalogs HNSCC-associated proteins and phosphosites, prioritizes copy number drivers, and highlights an oncogenic role for RNA processing genes. Proteomic investigation of mutual exclusivity between FAT1 truncating mutations and 11q13.3 amplifications reveals dysregulated actin dynamics as a common functional consequence. Phosphoproteomics characterizes two modes of EGFR activation, suggesting a new strategy to stratify HNSCCs based on EGFR ligand abundance for effective treatment with inhibitory EGFR monoclonal antibodies. Widespread deletion of immune modulatory genes accounts for low immune infiltration in immune-cold tumors, whereas concordant upregulation of multiple immune checkpoint proteins may underlie resistance to anti-programmed cell death protein 1 monotherapy in immune-hot tumors. Multi-omic analysis identifies three molecular subtypes with high potential for treatment with CDK inhibitors, anti-EGFR antibody therapy, and immunotherapy, respectively. Altogether, proteogenomics provides a systematic framework to inform HNSCC biology and treatment.[Display omitted]•A systematic inventory of HNSCC-associated proteins, phosphosites, and pathways•Three multi-omic subtypes linked to targeted treatment approaches and immunotherapy•Widespread deletion of immune modulatory genes accounts for loss of immunogenicity•Two modes of EGFR activation inform response to anti-EGFR monoclonal antibodiesHuang et al. report a proteogenomic study on 108 HPV-negative head and neck squamous cell carcinomas (HNSCCs). In addition to creating a comprehensive resource for pathogenic insights, multi-omic analysis identifies therapeutic hypotheses that may inform more precise approaches to treatment