USP14 is a major regulator of the proteasome and one of three proteasome-associated deubiquitinating enzymes. Its effects on protein turnover are substrate-specific, for unknown reasons. We report that USP14 shows a marked preference for ubiquitin-cyclin B conjugates that carry more than one ubiquitin modification or chain. This specificity is conserved from yeast to humans and is independent of chain linkage type. USP14 has been thought to cleave single ubiquitin groups from the distal tip of a chain, but we find that it removes chains from cyclin B en bloc, proceeding until a single chain remains. The suppression of degradation by USP14's catalytic activity reflects its capacity to act on a millisecond time scale, before the proteasome can initiate degradation of the substrate. In addition, single-molecule studies showed that the dwell time of ubiquitin conjugates at the proteasome was reduced by USP14-dependent deubiquitination. In summary, the specificity of the proteasome can be regulated by rapid ubiquitin chain removal, which resolves substrates based on a novel aspect of ubiquitin conjugate architecture.

System-wide quantitative analysis of ubiquitylomes has proven to be a valuable tool for elucidating targets and mechanisms of the ubiquitin-driven signaling systems, as well as gaining insights into neurodegenerative diseases and cancer. Current mass spectrometry methods for ubiquitylome detection require large amounts of starting material and rely on stochastic data collection to increase replicate analyses. We describe a method compatible with cell line and tissue samples for large-scale quantification of 5,000-9,000 ubiquitylation forms across ten samples simultaneously. Using this method, we reveal site-specific ubiquitylation in mammalian brain and liver tissues, as well as in cancer cells undergoing proteasome inhibition. To demonstrate the power of the approach for signal-dependent ubiquitylation, we examined protein and ubiquitylation dynamics for mitochondria undergoing PARKIN- and PINK1-dependent mitophagy. This analysis revealed the largest collection of PARKIN- and PINK1-dependent ubiquitylation targets to date in a single experiment, and it also revealed a subset of proteins recruited to the mitochondria during mitophagy.

Tumor Necrosis Factor (TNF) interacts with two receptors known as TNFR1 and TNFR2. TNFR1 activation may result in either cell proliferation or cell death. TNFR2 activates Nuclear Factor-kappaB (NF-kB) and c-Jun N-terminal kinase (JNK) which lead to transcriptional activation of genes related to cell proliferation and survival. This depends on the binding of TNF Receptor Associated Factor 2 (TRAF2) to the receptor. TNFR2 also induces TRAF2 degradation. In this work we have investigated the structural features of TNFR2 responsible for inducing TRAF2 degradation and have studied the biological consequences of this activity. We show that when TNFR1 and TNFR2 are co-expressed, TRAF2 depletion leads to an enhanced TNFR1 cytotoxicity which correlates with the inhibition of NF-kB. NF-kB activation and TRAF2 degradation depend of different regions of the receptor since TNFR2 mutants at amino acids 343-349 fail to induce TRAF2 degradation and have lost their ability to enhance TNFR1-mediated cell death but are still able to activate NF-kB. Moreover, whereas NF-kB activation requires TRAF2 binding to the receptor, TRAF2 degradation appears independent of TRAF2 binding. Thus, TNFR2 mutants unable to bind TRAF2 are still able to induce its degradation and to enhance TNFR1-mediated cytotoxicity. To test further this receptor crosstalk we have developed a system stably expressing in cells carrying only endogenous TNFR1 the chimeric receptor RANK-TNFR2, formed by the extracellular region of RANK (Receptor activator of NF-kB) and the intracellular region of TNFR2.This has made possible to study independently the signals triggered by TNFR1 and TNFR2. In these cells TNFR1 is selectively activated by soluble TNF (sTNF) while RANK-TNFR2 is selectively activated by RANKL. Treatment of these cells with sTNF and RANKL leads to an enhanced cytotoxicity.

Mitochondrial stress activates a mitonuclear response to safeguard and repair mitochondrial function and to adapt cellular metabolism to stress. Using a multiomics approach in mammalian cells treated with four types of mitochondrial stressors, we identify activating transcription factor 4 (ATF4) as the main regulator of the stress response. Surprisingly, canonical mitochondrial unfolded protein response genes mediated by ATF5 are not activated. Instead, ATF4 activates the expression of cytoprotective genes, which reprogram cellular metabolism through activation of the integrated stress response (ISR). Mitochondrial stress promotes a local proteostatic response by reducing mitochondrial ribosomal proteins, inhibiting mitochondrial translation, and coupling the activation of the ISR with the attenuation of mitochondrial function. Through a trans-expression quantitative trait locus analysis, we provide genetic evidence supporting a role for Fh1 in the control of Atf4 expression in mammals. Using gene expression data from mice and humans with mitochondrial diseases, we show that the ATF4 pathway is activated in vivo upon mitochondrial stress. Our data illustrate the value of a multiomics approach to characterize complex cellular networks and provide a versatile resource to identify new regulators of mitochondrial-related diseases.

Ubiquilins (Ubqlns) are a family of ubiquitin receptors that promote the delivery of hydrophobic and aggregated ubiquitinated proteins to the proteasome for degradation. We carried out a proteomic analysis of a B cell lymphoma-derived cell line, BJAB, that requires UBQLN1 for survival to identify UBQLN1 client proteins. When UBQLN1 expression was acutely inhibited, 120 mitochondrial proteins were enriched in the cytoplasm, suggesting that the accumulation of mitochondrial client proteins in the absence of UBQLN1 is cytostatic. Using a Ubqln1-/- mouse strain, we found that B cell receptor (BCR) ligation of Ubqln1-/- B cells led to a defect in cell cycle entry. As in BJAB cells, mitochondrial proteins accumulated in BCR-stimulated cells, leading to protein synthesis inhibition and cell cycle block. Thus, UBQLN1 plays an important role in clearing mislocalized mitochondrial proteins upon cell stimulation, and its absence leads to suppression of protein synthesis and cell cycle arrest.

Myeloproliferative neoplasms (MPNs) are characterized by the activated JAK2/STAT pathway. Pleckstrin-2 (Plek2) is a downstream target of the JAK2/STAT5 pathway and is overexpressed in patients with MPNs. We previously revealed that Plek2 plays critical roles in the pathogenesis of JAK2-mutated MPNs. The nonessential roles of Plek2 under physiologic conditions make it an ideal target for MPN therapy. Here, we identified first-in-class Plek2 inhibitors through an in silico high-throughput screening approach and cell-based assays, followed by the synthesis of analogs. Plek2-specific small-molecule inhibitors showed potent inhibitory effects on cell proliferation. Mechanistically, Plek2 interacts with and enhances the activity of Akt through the recruitment of downstream effector proteins. The Plek2-signaling complex also includes Hsp72, which protects Akt from degradation. These functions were blocked by Plek2 inhibitors via their direct binding to the Plek2 dishevelled, Egl-10 and pleckstrin (DEP) domain. The role of Plek2 in activating Akt signaling was further confirmed in vivo using a hematopoietic-specific Pten-knockout mouse model. We next tested Plek2 inhibitors alone or in combination with an Akt inhibitor in various MPN mouse models, which showed significant therapeutic efficacies similar to that seen with the genetic depletion of Plek2. The Plek2 inhibitor was also effective in reducing proliferation of CD34-positive cells from MPN patients. Our studies reveal a Plek2/Akt complex that drives cell proliferation and can be targeted by a class of antiproliferative compounds for MPN therapy.

Chromatin organization is a highly orchestrated process that influences gene expression, in part by modulating access of regulatory factors to DNA and nucleosomes. Here, we report that the chromatin accessibility regulator HMGN1, a target of recurrent DNA copy gains in leukemia, controls myeloid differentiation. HMGN1 amplification is associated with increased accessibility, expression, and histone H3K27 acetylation of loci important for hematopoietic stem cells (HSCs) and leukemia, such as HoxA cluster genes. In vivo, HMGN1 overexpression is linked to decreased quiescence and increased HSC activity in bone marrow transplantation. HMGN1 overexpression also cooperates with the AML-ETO9a fusion oncoprotein to impair myeloid differentiation and enhance leukemia stem cell (LSC) activity. Inhibition of histone acetyltransferases CBP/p300 relieves the HMGN1-associated differentiation block. These data nominate factors that modulate chromatin accessibility as regulators of HSCs and LSCs, and suggest that targeting HMGN1 or its downstream effects on histone acetylation could be therapeutically active in AML.

RNA alternative splicing (AS) expands the regulatory potential of eukaryotic genomes. The mechanisms regulating liver-specific AS profiles and their contribution to liver function are poorly understood. Here, we identify a key role for the splicing factor RNA-binding Fox protein 2 (RBFOX2) in maintaining cholesterol homeostasis in a lipogenic environment in the liver. Using enhanced individual-nucleotide-resolution ultra-violet cross-linking and immunoprecipitation, we identify physiologically relevant targets of RBFOX2 in mouse liver, including the scavenger receptor class B type I (Scarb1). RBFOX2 function is decreased in the liver in diet-induced obesity, causing a Scarb1 isoform switch and alteration of hepatocyte lipid homeostasis. Our findings demonstrate that specific AS programmes actively maintain liver physiology, and underlie the lipotoxic effects of obesogenic diets when dysregulated. Splice-switching oligonucleotides targeting this network alleviate obesity-induced inflammation in the liver and promote an anti-atherogenic lipoprotein profile in the blood, underscoring the potential of isoform-specific RNA therapeutics for treating metabolism-associated diseases.

Familial neurodegenerative diseases commonly involve mutations that result in either aberrant proteins or dysfunctional components of the proteolytic machinery that act on aberrant proteins. UBQLN2 is a ubiquitin receptor of the UBL/UBA family that binds the proteasome through its ubiquitin-like domain and is thought to deliver ubiquitinated proteins to proteasomes for degradation. UBQLN2 mutations result in familial amyotrophic lateral sclerosis (ALS)/frontotemporal dementia in humans through an unknown mechanism. Quantitative multiplexed proteomics was used to provide for the first time an unbiased and global analysis of the role of Ubqln2 in controlling the composition of the proteome. We studied several murine models of Ubqln2-linked ALS and also generated Ubqln2 null mutant mice. We identified impacts of Ubqln2 on diverse physiological pathways, most notably serotonergic signaling. Interestingly, we observed an upregulation of proteasome subunits, suggesting a compensatory response to diminished proteasome output. Among the specific proteins whose abundance is linked to UBQLN2 function, the strongest hits were the ubiquitin ligase TRIM32 and two retroelement-derived proteins, PEG10 and CXX1B. Cycloheximide chase studies using induced human neurons and HEK293 cells suggested that PEG10 and TRIM32 are direct clients. Although UBQLN2 directs the degradation of multiple proteins via the proteasome, it surprisingly conferred strong protection from degradation on the Gag-like protein CXX1B, which is expressed from the same family of retroelement genes as PEG10. In summary, this study charts the proteomic landscape of ALS-related Ubqln2 mutants and identifies candidate client proteins that are altered in vivo in disease models and whose degradation is promoted by UBQLN2.