Ligand binding to the EGF receptor (EGFR) triggers multiple signal-transduction processes and promotes endocytosis of the receptor. The mechanisms of EGFR endocytosis and its cross-talk with signaling are poorly understood. Here, we combine peroxidase-catalyzed proximity labeling, isobaric peptide tagging, and quantitative mass spectrometry to define the dynamics of the proximity proteome of ligand-activated EGFR. Using this approach, we identify a network of signaling proteins, which remain associated with the receptor during its internalization and trafficking through the endosomal system. We show that Trk-fused gene (TFG), a protein known to function at the endoplasmic reticulum exit sites, is enriched in the proximity proteome of EGFR in early/sorting endosomes and localized in these endosomes and demonstrate that TFG regulates endosomal sorting of EGFR. This study provides a comprehensive resource of time-dependent nanoscale environment of EGFR, thus opening avenues to discovering new regulatory mechanisms of signaling and intracellular trafficking of receptor tyrosine kinases.

Elucidating the mechanisms of resistance to immunotherapy and developing strategies to improve its efficacy are challenging goals. Bioinformatics analysis demonstrates that high CDK6 expression in melanoma is associated with poor progression-free survival of patients receiving single-agent immunotherapy. Depletion of CDK6 or cyclin D3 (but not of CDK4, cyclin D1, or D2) in cells of the tumor microenvironment inhibits tumor growth. CDK6 depletion reshapes the tumor immune microenvironment, and the host anti-tumor effect depends on cyclin D3/CDK6-expressing CD8+ and CD4+ T cells. This occurs by CDK6 phosphorylating and increasing the activities of PTP1B and T cell protein tyrosine phosphatase (TCPTP), which, in turn, decreases tyrosine phosphorylation of CD3ζ, reducing the signal transduction for T cell activation. Administration of a PTP1B and TCPTP inhibitor prove more efficacious than using a CDK6 degrader in enhancing T cell-mediated immunotherapy. Targeting protein tyrosine phosphatases (PTPs) might be an effective strategy for cancer patients who resist immunotherapy treatment.

Ribosome-associated quality control (RQC) disassembles aberrantly stalled translation complexes to recycle or degrade the constituent parts. A key step of RQC is the cleavage of P-site tRNA by the endonuclease ANKZF1 (Vms1 in yeast) to release incompletely synthesized polypeptides from ribosomes for degradation. Re-use of the cleaved tRNA for translation requires re-addition of the universal 3'CCA nucleotides removed by ANKZF1. Here, we show that ELAC1 is both necessary and sufficient to remove the 2',3'-cyclic phosphate on ANKZF1-cleaved tRNAs to permit CCA re-addition by TRNT1. ELAC1 activity is optimized for tRNA recycling, whereas ELAC2, the essential RNase Z isoform in eukaryotes, is required to remove 3' trailers during tRNA biogenesis. Cells lacking ELAC1 specifically accumulate unrepaired tRNA intermediates upon the induction of ribosome stalling. Thus, optimal recycling of ANKZF1-cleaved tRNAs in vertebrates is achieved through the duplication and specialization of a conserved tRNA biosynthesis enzyme.

NAD+ is a key metabolic redox cofactor that is regenerated from nicotinamide through the NAD+ salvage pathway. Here, we find that inhibiting the NAD+ salvage pathway depletes serine biosynthesis from glucose by impeding the NAD+-dependent protein, 3-phosphoglycerate dehydrogenase (PHGDH). Importantly, we find that PHGDHhigh breast cancer cell lines are exquisitely sensitive to inhibition of the NAD+ salvage pathway. Further, we find that PHGDH protein levels and those of the rate-limiting enzyme of NAD+ salvage, NAMPT, correlate in ER-negative, basal-like breast cancers. Although NAD+ salvage pathway inhibitors are actively being pursued in cancer treatment, their efficacy has been poor, and our findings suggest that they may be effective for PHGDH-dependent cancers.

MCL-1 is a high-priority target due to its dominant role in the pathogenesis and chemoresistance of cancer, yet clinical trials of MCL-1 inhibitors are revealing toxic side effects. MCL-1 biology is complex, extending beyond apoptotic regulation and confounded by its multiple isoforms, its domains of unresolved structure and function, and challenges in distinguishing noncanonical activities from the apoptotic response. We find that, in the presence or absence of an intact mitochondrial apoptotic pathway, genetic deletion or pharmacologic targeting of MCL-1 induces DNA damage and retards cell proliferation. Indeed, the cancer cell susceptibility profile of MCL-1 inhibitors better matches that of anti-proliferative than pro-apoptotic drugs, expanding their potential therapeutic applications, including synergistic combinations, but heightening therapeutic window concerns. Proteomic profiling provides a resource for mechanistic dissection and reveals the minichromosome maintenance DNA helicase as an interacting nuclear protein complex that links MCL-1 to the regulation of DNA integrity and cell-cycle progression.

Hexanucleotide repeat expansion in the C9ORF72 gene results in production of dipeptide repeat (DPR) proteins that may disrupt pre-mRNA splicing in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. At present, the mechanisms underlying this mis-splicing are not understood. Here, we show that addition of proline-arginine (PR) and glycine-arginine (GR) toxic DPR peptides to nuclear extracts blocks spliceosome assembly and splicing, but not other types of RNA processing. Proteomic and biochemical analyses identified the U2 small nuclear ribonucleoprotein particle (snRNP) as a major interactor of PR and GR peptides. In addition, U2 snRNP, but not other splicing factors, mislocalizes from the nucleus to the cytoplasm both in C9ORF72 patient induced pluripotent stem cell (iPSC)-derived motor neurons and in HeLa cells treated with the toxic peptides. Bioinformatic studies support a specific role for U2-snRNP-dependent mis-splicing in C9ORF72 patient brains. Together, our data indicate that DPR-mediated dysfunction of U2 snRNP could account for as much as ∼44% of the mis-spliced cassette exons in C9ORF72 patient brains.

Maintenance of protein homeostasis degrades with age, contributing to aging-related decline and disease. Previous studies have primarily surveyed transcriptional aging changes. To define the effects of age directly at the protein level, we perform discovery-based proteomics in 10 tissues from 20 C57BL/6J mice, representing both sexes at adult and late midlife ages (8 and 18 months). Consistent with previous studies, age-related changes in protein abundance often have no corresponding transcriptional change. Aging results in increases in immune proteins across all tissues, consistent with a global pattern of immune infiltration with age. Our protein-centric data reveal tissue-specific aging changes with functional consequences, including altered endoplasmic reticulum and protein trafficking in the spleen. We further observe changes in the stoichiometry of protein complexes with important roles in protein homeostasis, including the CCT/TriC complex and large ribosomal subunit. These data provide a foundation for understanding how proteins contribute to systemic aging across tissues.

The rixosome and PRC1 silencing complexes are associated with deSUMOylating and deubiquitinating enzymes, SENP3 and USP7, respectively. How deSUMOylation and deubiquitylation contribute to rixosome- and Polycomb-mediated silencing is not fully understood. Here, we show that the enzymatic activities of SENP3 and USP7 are required for silencing of Polycomb target genes. SENP3 deSUMOylates several rixosome subunits, and this activity is required for association of the rixosome with PRC1. USP7 associates with canonical PRC1 (cPRC1) and deubiquitinates the chromodomain subunits CBX2 and CBX4, and inhibition of USP activity results in disassembly of cPRC1. Finally, both SENP3 and USP7 are required for Polycomb- and rixosome-dependent silencing at an ectopic reporter locus. These findings demonstrate that SUMOylation and ubiquitination regulate the assembly and activities of the rixosome and Polycomb complexes and raise the possibility that these modifications provide regulatory mechanisms that may be utilized during development or in response to environmental challenges.

Selenof (15-kDa selenoprotein; Sep15) is an endoplasmic reticulum (ER)-resident thioredoxin-like oxidoreductase that occurs in a complex with UDP-glucose:glycoprotein glucosyltransferase. We found that Selenof deficiency in mice leads to elevated levels of non-functional circulating plasma immunoglobulins and increased secretion of IgM during in vitro splenic B cell differentiation. However, Selenof knockout animals show neither enhanced bacterial killing capacity nor antigen-induced systemic IgM activity, suggesting that excess immunoglobulins are not functional. In addition, ER-to-Golgi transport of a target glycoprotein was delayed in Selenof knockout embryonic fibroblasts, and proteomic analyses revealed that Selenof deficiency is primarily associated with antigen presentation and ER-to-Golgi transport. Together, the data suggest that Selenof functions as a gatekeeper of immunoglobulins and, likely, other client proteins that exit the ER, thereby supporting redox quality control of these proteins.

The Fanconi anemia (FA) pathway is critical for the cellular response to toxic DNA interstrand crosslinks (ICLs). Using a biochemical purification strategy, we identified UHRF1 as a protein that specifically interacts with ICLs in vitro and in vivo. Reduction of cellular levels of UHRF1 by RNAi attenuates the FA pathway and sensitizes cells to mitomycin C. Knockdown cells display a drastic reduction in FANCD2 foci formation. Using live-cell imaging, we observe that UHRF1 is rapidly recruited to chromatin in response to DNA crosslinking agents and that this recruitment both precedes and is required for the recruitment of FANCD2 to ICLs. Based on these results, we describe a mechanism of ICL sensing and propose that UHRF1 is a critical factor that binds to ICLs. In turn, this binding is necessary for the subsequent recruitment of FANCD2, which allows the DNA repair process to initiate.

Epstein-Barr virus (EBV) replication contributes to multiple human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, B cell lymphomas, and oral hairy leukoplakia. We performed systematic quantitative analyses of temporal changes in host and EBV proteins during lytic replication to gain insights into virus-host interactions, using conditional Burkitt lymphoma models of type I and II EBV infection. We quantified profiles of >8,000 cellular and 69 EBV proteins, including >500 plasma membrane proteins, providing temporal views of the lytic B cell proteome and EBV virome. Our approach revealed EBV-induced remodeling of cell cycle, innate and adaptive immune pathways, including upregulation of the complement cascade and proteasomal degradation of the B cell receptor complex, conserved between EBV types I and II. Cross-comparison with proteomic analyses of human cytomegalovirus infection and of a Kaposi-sarcoma-associated herpesvirus immunoevasin identified host factors targeted by multiple herpesviruses. Our results provide an important resource for studies of EBV replication.

Governance of protein phosphorylation by kinases and phosphatases constitutes an essential regulatory network in eukaryotic cells. Network dysregulation leads to severe consequences and is often a key factor in disease pathogenesis. Previous studies revealed multiple roles for protein phosphorylation and pathway structures in cellular functions from different perspectives. We seek to understand the roles of kinases and phosphatases from a protein homeostasis point of view. Using a streamlined tandem mass tag (SL-TMT) strategy, we systematically measure proteomic and phosphoproteomic responses to perturbations of phosphorylation signaling networks in yeast deletion strains. Our results emphasize the requirement for protein normalization for more complete interpretation of phosphorylation data. Functional relationships between kinases and phosphatases were characterized at both proteome and phosphoproteome levels in three ways: (1) Gene Ontology enrichment analysis, (2) Δgene-Δgene correlation networks, and (3) molecule covariance networks. This resource illuminates kinase and phosphatase functions and pathway organizations.

The Fanconi anemia (FA) pathway repairs DNA interstrand crosslinks (ICLs). Many FA proteins are recruited to ICLs in a timely fashion so that coordinated repair can occur. However, the mechanism of this process is poorly understood. Here, we report the purification of a FANCD2-containing protein complex with multiple subunits, including WRNIP1. Using live-cell imaging, we show that WRNIP1 is recruited to ICLs quickly after their appearance, promoting repair. The observed recruitment facilitates subsequent recruitment of the FANCD2/FANCI complex. Depletion of WRNIP1 sensitizes cells to ICL-forming drugs. We find that ubiquitination of WRNIP1 and the activity of its UBZ domain are required to facilitate recruitment of FANCD2/FANCI and promote repair. Altogether, we describe a mechanism by which WRNIP1 is recruited rapidly to ICLs, resulting in chromatin loading of the FANCD2/FANCI complex in an unusual process entailing ubiquitination of WRNIP1 and the activity of its integral UBZ domain.

MCL-1 is an anti-apoptotic BCL-2 family protein essential for survival of diverse cell types and is a major driver of cancer and chemoresistance. The mechanistic basis for the oncogenic supremacy of MCL-1 among its anti-apoptotic homologs is unclear and implicates physiologic roles of MCL-1 beyond apoptotic suppression. Here we find that MCL-1-dependent hematologic cancer cells specifically rely on fatty acid oxidation (FAO) as a fuel source because of metabolic wiring enforced by MCL-1 itself. We demonstrate that FAO regulation by MCL-1 is independent of its anti-apoptotic activity, based on metabolomic, proteomic, and genomic profiling of MCL-1-dependent leukemia cells lacking an intact apoptotic pathway. Genetic deletion of Mcl-1 results in transcriptional downregulation of FAO pathway proteins such that glucose withdrawal triggers cell death despite apoptotic blockade. Our data reveal that MCL-1 is a master regulator of FAO, rendering MCL-1-driven cancer cells uniquely susceptible to treatment with FAO inhibitors.

Proteasome-mediated degradation occurs with proteins principally modified with lysine-48 polyubiquitin chains. Whether the proteasome also can bind atypical ubiquitin chains, including those linked by lysine-11, has not been well established. This is critically important, as lysine-11 polyubiquitination has been implicated in both proteasome-mediated degradation and non-degradative outcomes. Here we demonstrate that pure homotypic lysine-11-linked chains do not bind strongly to the mammalian proteasome. By contrast, heterotypic polyubiquitin chains, containing lysine-11 and lysine-48 linkages, not only bind to the proteasome but also stimulate the proteasomal degradation of the cell-cycle regulator cyclin B1. Thus, while heterotypic lysine-11-linked chains facilitate proteasomal degradation, homotypic lysine-11 linkages adopt conformations that prevent association with the proteasome. Our data demonstrate the capacity of the proteasome to bind ubiquitin chains of distinct topology, with implications for the recognition and diverse biological functions of mixed ubiquitin chains.

The NFkB/Rel family of proteins play critical roles in a variety of cellular processes. Thus, their physiological activation is tightly controlled. Recently, the NFkB2/p100 precursor has been characterized as the fourth IkB type of suppressor for NFkB. However, the molecular mechanism(s) underlying regulated destruction of NFkB2 remains largely unknown. Here, we report that, unlike other IkBs, ubiquitination and destruction of NFkB2 are governed by SCF(Fbw7) in a GSK3-dependent manner. In Fbw(7-/-) cells, elevated expression of NFkB2/p100 leads to a subsequent reduction in NFkB signaling pathways and elevated sensitivity to TNFa-induced cell death. Reintroducing wild-type Fbw7, but not disease-derived mutant forms of Fbw7, rescues NFkB activity. Furthermore, T cell-specific depletion of Fbw7 also leads to reduced NFkB activity and perturbed T cell differentiation. Therefore, our work identifies Fbw7 as a physiological E3 ligase controlling NFkB20s stability. It further implicates that Fbw7 might exert its tumor-suppressor function by regulating NFkB activity.

Interstrand crosslinks (ICLs) of the DNA helix are a deleterious form of DNA damage. ICLs can be repaired by the Fanconi anemia pathway. At the center of the pathway is the FANCD2/FANCI complex, recruitment of which to DNA is a critical step for repair. After recruitment, monoubiquitination of both FANCD2 and FANCI leads to their retention on chromatin, ensuring subsequent repair. However, regulation of recruitment is poorly understood. Here, we report a cluster of phosphosites on FANCD2 whose phosphorylation by CK2 inhibits both FANCD2 recruitment to ICLs and its monoubiquitination in vitro and in vivo. We have found that phosphorylated FANCD2 possesses reduced DNA binding activity, explaining the previous observations. Thus, we describe a regulatory mechanism operating as a molecular switch, where in the absence of DNA damage, the FANCD2/FANCI complex is prevented from loading onto DNA, effectively suppressing the FA pathway.