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Background: Maternal iron deficiency (ID) is associated with poor pregnancy and fetal outcomes. The effect is thought to be mediated by the placenta but there is no comprehensive assessment of placental responses to maternal ID. Additionally, whether the influence of maternal ID on the placenta differs by fetal sex is unknown.Objectives: To identify gene and protein signatures of ID mouse placentas at mid-gestation. A secondary objective was to profile the expression of iron genes in mouse placentas across gestation.Methods: We used a real-time PCR-based array to determine the mRNA expression of all known iron genes in mouse placentas at embryonic day (E) 12.5, E14.5, E16.5, and E19.5 (n = 3 placentas/time point). To determine the effect of maternal ID, we performed RNA sequencing and proteomics in male and female placentas from ID and iron-adequate mice at E12.5 (n = 8 dams/diet).Results: In female placentas, 6 genes, including transferrin receptor (Tfrc) and solute carrier family 11 member 2, were significantly changed by maternal ID. An additional 154 genes were altered in male ID placentas. A proteomic analysis quantified 7662 proteins in the placenta. Proteins translated from iron-responsive element (IRE)-containing mRNA were altered in abundance; ferritin and ferroportin 1 decreased, while TFRC increased in ID placentas. Less than 4% of the significantly altered genes in ID placentas occurred both at the transcriptional and translational levels.Conclusions: Our data demonstrate that the impact of maternal ID on placental gene expression in mice is limited in scope and magnitude at mid-gestation. We provide strong evidence for IRE-based transcriptional and translational coordination of iron gene expression in the mouse placenta. Finally, we discover sexually dimorphic effects of maternal ID on placental gene expression, with more genes and pathways altered in male compared with female mouse placentas. [ABSTRACT FROM AUTHOR]

Coronavirus disease 2019 (COVID-19) remains a significant public health threat due to the ability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants to evade the immune system and cause breakthrough infections. Although pathogenic coronaviruses such as SARS-CoV-2 and Middle East respiratory syndrome (MERS)-CoV lead to severe respiratory infections, how these viruses affect the chromatin proteomic composition upon infection remains largely uncharacterized. Here, we use our recently developed integrative DNA And Protein Tagging methodology to identify changes in host chromatin accessibility states and chromatin proteomic composition upon infection with pathogenic coronaviruses. SARS-CoV-2 infection induces TP53 stabilization on chromatin, which contributes to its host cytopathic effect. We mapped this TP53 stabilization to the SARS-CoV-2 spike and its propensity to form syncytia, a consequence of cell-cell fusion. Differences in SARS-CoV-2 spike variant-induced syncytia formation modify chromatin accessibility, cellular senescence, and inflammatory cytokine release via TP53. Our findings suggest that differences in syncytia formation alter senescence-associated inflammation, which varies among SARS-CoV-2 variants. [Display omitted] • Global chromatin proteomics reveal TP53 stabilization in SARS-CoV-2 infection • Host cell spike expression drives TP53 stabilization and chromatin accessibility changes • Fusogenicity of variant spikes affects TP53, chromatin accessibility, and senescence Lee et al. find that spike-mediated cell-cell fusion triggers TP53 stabilization, chromatin accessibility changes, and activation of senescence. The extent of these effects differs based on the fusogenicity of various SARS-CoV-2 variant spike sequences. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Targeting MCL-1 induces DNA damage and impairs cell proliferation • MCL-1 regulates DNA integrity and cell-cycle progression independent from apoptosis • Replicative stress induces MCL-1 interaction with minichrosome maintenance complex • Noncanonical roles of MCL-1 expand the benefits and risks of therapeutic targeting Adhikary et al. report that MCL-1 regulates cell proliferation and DNA integrity independent from its role in apoptosis suppression. Genetic deletion or pharmacologic targeting has a striking anti-proliferative effect, informing opportunities to expand the utility of MCL-1 inhibitors in cancer as single agents and in combinations, but with heightened precautions. [ABSTRACT FROM AUTHOR]

Objectives Understanding how loci identified by genome wide association studies (GWAS) contribute to pathogenesis requires new mechanistic insights. Variants within CDKAL1 are strongly linked to an increased risk of developing type 2 diabetes and obesity. Investigations in mouse models have focused on the function of Cdkal1 as a tRNA Lys modifier and downstream effects of Cdkal1 loss on pro-insulin translational fidelity in pancreatic β−cells. However, Cdkal1 is broadly expressed in other metabolically relevant tissues, including adipose tissue. In addition, the Cdkal1 homolog Cdk5rap1 regulates mitochondrial protein translation and mitochondrial function in skeletal muscle. We tested whether adipocyte-specific Cdkal1 deletion alters systemic glucose homeostasis or adipose mitochondrial function independently of its effects on pro-insulin translation and insulin secretion. Methods We measured mRNA levels of type 2 diabetes GWAS genes, including Cdkal1 , in adipose tissue from lean and obese mice. We then established a mouse model with adipocyte-specific Cdkal1 deletion. We examined the effects of adipose Cdkal1 deletion using indirect calorimetry on mice during a cold temperature challenge, as well as by measuring cellular and mitochondrial respiration in vitro . We also examined brown adipose tissue (BAT) mitochondrial morphology by electron microscopy. Utilizing co-immunoprecipitation followed by mass spectrometry, we performed interaction mapping to identify new CDKAL1 binding partners. Furthermore, we tested whether Cdkal1 loss in adipose tissue affects total protein levels or accurate Lys incorporation by tRNA Lys using quantitative mass spectrometry. Results We found that Cdkal1 mRNA levels are reduced in adipose tissue of obese mice. Using adipose-specific Cdkal1 KO mice (A-KO), we demonstrated that mitochondrial function is impaired in primary differentiated brown adipocytes and in isolated mitochondria from A-KO brown adipose tissue. A-KO mice displayed decreased energy expenditure during 4 °C cold challenge. Furthermore, mitochondrial morphology was highly abnormal in A-KO BAT. Surprisingly, we found that lysine codon representation was unchanged in Cdkal1 A-KO adipose tissue. We identified novel protein interactors of CDKAL1, including SLC25A4/ANT1, an inner mitochondrial membrane ADP/ATP translocator. ANT proteins can account for the UCP1-independent basal proton leak in BAT mitochondria. Cdkal1 A-KO mice had increased ANT1 protein levels in their white adipose tissue. Conclusions Cdkal1 is necessary for normal mitochondrial morphology and function in adipose tissue. These results suggest that the type 2 diabetes susceptibility gene CDKAL1 has novel functions in regulating mitochondrial activity. [ABSTRACT FROM AUTHOR]

Summary 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. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • SENP3-mediated deSUMOylation regulates the association of the rixosome with PRC1 • The USP7 ubiquitin protease associates with canonical PRC1 • USP7 regulates PRC1 stability by deubiquitinating CBX2 and CBX4 • SENP3 and USP7 are required for silencing of Polycomb target genes Zhou et al. find that association of the rixosome, which degrades RNA, and Polycomb silencing complexes is regulated by enzymatic removal of ubiquitin and the ubiquitin-like moiety SUMO. The deSUMOylating and deubiquitinating enzymes are associated with the rixosome and Polycomb complexes, respectively, and are required for their silencing functions. [ABSTRACT FROM AUTHOR]

Summary Exercise provides many health benefits, including improved metabolism, cardiovascular health, and cognition. We have shown previously that FNDC5, a type I transmembrane protein, and its circulating form, irisin, convey some of these benefits in mice. However, recent reports questioned the existence of circulating human irisin both because human FNDC5 has a non-canonical ATA translation start and because of claims that many human irisin antibodies used in commercial ELISA kits lack required specificity. In this paper we have identified and quantitated human irisin in plasma using mass spectrometry with control peptides enriched with heavy stable isotopes as internal standards. This precise state-of-the-art method shows that human irisin is mainly translated from its non-canonical start codon and circulates at ∼3.6 ng/ml in sedentary individuals; this level is increased to ∼4.3 ng/ml in individuals undergoing aerobic interval training. These data unequivocally demonstrate that human irisin exists, circulates, and is regulated by exercise. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • MCL-1 controls fatty acid oxidation independent of its anti-apoptotic function • Genetic deletion of Mcl-1 induces global downregulation of the FAO pathway • MCL-1-driven hematologic cancer cells are dependent on FAO as a fuel source • MCL-1-dependent cancer cells are selectively susceptible to FAO inhibitors Prew et al. report that, independent of its anti-apoptotic function, MCL-1 enforces a programmatic dependency on fatty acid oxidation (FAO) in MCL-1-driven hematologic cancer cells, rendering them vulnerable to FAO inhibition. Mcl-1 deletion broadly downregulates the FAO pathway, revealing MCL-1 as a master regulator of fatty acid metabolism. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Proximity proteome of activated EGFR tracked by time-resolved APEX-mediated labeling • Multiple signaling proteins remain proximal to EGFR during its endocytosis • Trk-fused gene (TFG) is associated with early and sorting endosomes • TFG regulates endosomal sorting of EGFR Perez Verdaguer et al. use time-resolved APEX labeling of the proximity proteome of EGFR upon ligand activation to provide comprehensive information about the dynamics of the EGFR-associated protein networks involved in receptor endocytosis and signaling. [ABSTRACT FROM AUTHOR]

Rapid alterations in cellular metabolism allow tissues to maintain homeostasis during changes in energy availability. The central metabolic regulator acetyl-CoA carboxylase 2 (ACC2) is robustly phosphorylated during cellular energy stress by AMP-activated protein kinase (AMPK) to relieve its suppression of fat oxidation. While ACC2 can also be hydroxylated by prolyl hydroxylase 3 (PHD3), the physiological consequence thereof is poorly understood. We find that ACC2 phosphorylation and hydroxylation occur in an inverse fashion. ACC2 hydroxylation occurs in conditions of high energy and represses fatty acid oxidation. PHD3-null mice demonstrate loss of ACC2 hydroxylation in heart and skeletal muscle and display elevated fatty acid oxidation. Whole body or skeletal muscle-specific PHD3 loss enhances exercise capacity during an endurance exercise challenge. In sum, these data identify an unexpected link between AMPK and PHD3, and a role for PHD3 in acute exercise endurance capacity and skeletal muscle metabolism. • AMPK-mediated phosphorylation of ACC2 reduces PHD3-mediated hydroxylation of ACC2 • Loss of PHD3 or AMPK results in inverse effects on fat metabolism • Loss of PHD3 in skeletal muscle endows mice with increased endurance exercise capacity The mechanisms that maintain low fatty acid oxidation during energy-replete conditions are not completely understood. Here, Yoon et al. show that AMPK-mediated phosphorylation and PHD3-mediated hydroxylation modulate ACC2 under opposing energy states. Loss of PHD3 in skeletal muscle improves mitochondrial fat metabolism and muscle performance in response to exercise stress. [ABSTRACT FROM AUTHOR]

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. • SL-TMT strategy enables (phospho)proteome analysis of 110 yeast deletion strains • Complete interpretation of phosphorylation data requires protein normalization • Kinase and phosphatase relationships are characterized at multiple levels • Network analyses illuminate phosphorylation signaling pathway organizations Li et al. measure (phospho)proteomic responses to perturbations of phosphorylation signaling networks in 110 yeast deletion strains. The results emphasize that broad interpretation of phosphorylation data requires protein normalization. Kinase and phosphatase relationships are characterized at both proteome and phosphoproteome levels. Phosphorylation signaling pathway organizations are illuminated by network analyses. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) causes Burkitt, Hodgkin, and post-transplant B cell lymphomas. How EBV remodels metabolic pathways to support rapid B cell outgrowth remains largely unknown. To gain insights, primary human B cells were profiled by tandem-mass-tag-based proteomics at rest and at nine time points after infection; >8,000 host and 29 viral proteins were quantified, revealing mitochondrial remodeling and induction of one-carbon (1C) metabolism. EBV-encoded EBNA2 and its target MYC were required for upregulation of the central mitochondrial 1C enzyme MTHFD2, which played key roles in EBV-driven B cell growth and survival. MTHFD2 was critical for maintaining elevated NADPH levels in infected cells, and oxidation of mitochondrial NADPH diminished B cell proliferation. Tracing studies underscored contributions of 1C to nucleotide synthesis, NADPH production, and redox defense. EBV upregulated import and synthesis of serine to augment 1C flux. Our results highlight EBV-induced 1C as a potential therapeutic target and provide a new paradigm for viral onco-metabolism. • Global analysis of EBV proteome and metabolism remodeling in B cell transformation • EBV induces mitochondrial 1C metabolic enzyme expression and flux • EBNA2 is a viral master regulator of B cell metabolic reprogramming • 1C drives nucleotide, mitochondrial NADPH, and glutathione production Global unbiased proteomic analysis reveals key metabolic pathways induced by Epstein-Barr virus critical for B cell growth transformation. In this issue of Cell Metabolism, Wang et al. utilized multiplexed proteomics to identify key virus-induced metabolic pathways important for outgrowth of newly infected primary human B cells. The authors describe virus-activated mitochondrial one-carbon metabolism as being crucial for nucleotide and glutathione syntheses, as well as generation of intramitochondrial NADPH. [ABSTRACT FROM AUTHOR]

Summary 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. Graphical Abstract Highlights • The NAD+ salvage pathway is required for serine biosynthesis • PHGDHhigh cancer cells are highly sensitive to NAD+ salvage pathway inhibition • NAMPT and PHGDH correlate in ER-negative, basal-like breast cancer Subsets of breast cancers depend on the serine biosynthesis enzyme PHGDH. Murphy et al. show that NAD+ used for PHGDH function requires the NAD+ salvage pathway and that PHGDH-dependent cancers are, thus, sensitive to NAD+ salvage inhibitors. Serine biosynthesis and NAD+ salvage pathway enzymes are also commonly co-expressed in breast cancers. [ABSTRACT FROM AUTHOR]

Thioredoxin/glutathione reductase (TXNRD3) is a selenoprotein composed of thioredoxin reductase and glutaredoxin domains. This NADPH-dependent thiol oxidoreductase evolved through gene duplication within the Txnrd family, is expressed in the testes, and can reduce both thioredoxin and glutathione in vitro; however, the function of this enzyme remains unknown. To characterize the function of TXNRD3 in vivo, we generated a strain of mice bearing deletion of Txnrd3 gene. We show that these Txnrd3 knockout mice are viable and without discernable gross phenotypes, and also that TXNRD3 deficiency leads to fertility impairment in male mice. We found that Txnrd3 knockout animals exhibited a lower fertilization rate in vitro, a sperm movement phenotype, and an altered thiol redox status in sperm cells. Proteomic analyses further revealed a broad range of substrates reduced by TXNRD3 during sperm maturation, presumably as a part of sperm quality control. Taken together, these results show that TXNRD3 plays a critical role in male reproduction via the thiol redox control of spermatogenesis. [ABSTRACT FROM AUTHOR]

• 7,8 dihydroxyflavone protects inner retina against functional and structural losses in glaucoma. • Protective effects of 7,8 DHF in glaucoma are not affected by BDNF heterozygosity. • 7,8 DHF treatment leads to activation of TrkB signalling in the retina. • Amyloid β levels in the retina in glaucoma are modulated by 7,8 DHF treatment. Glaucoma is an age-related neurodegenerative disorder characterized by retinal ganglion cell (RGC) degeneration and excavation of the optic nerve head (ONH). It is associated with an increase in intraocular pressure (IOP) and progressive decline in the visual field. Reduction in the retrograde axonal transport of neurotrophic factors such as brain-derived neurotrophic factor (BDNF) from the brain to the neuronal cell bodies in retina, has been suggested as one of the key mechanisms underlying selective degeneration of ganglion cells and optic nerve in glaucoma. Multiple studies have indicated that BDNF and its high affinity receptor Tropomyosin receptor kinase B (TrkB) play crucial roles in survival of RGCs and that upregulating BDNF/TrkB signalling using gene therapy can protect the ganglion cells against degeneration. This study corroborates previous findings and demonstrates that glaucoma is associated with downregulation of TrkB downstream signalling and enhanced levels of amyloid β (Aβ 1–42) accumulation in the retina. 7,8 dihydroxyflavone (7,8 DHF) is a TrkB agonist and regular administration of this compound imparted significant protection against loss of GCL density and preserved inner retinal function in experimental glaucoma models. 7,8 DHF treatment stimulated activation of TrkB intracellular signalling as well as ameliorated the increase in the levels of soluble Aβ (1–42) in the retinas of rats and mice exposed to high IOP. The protective effects of 7,8 DHF were also evident in BDNF+/− mice indicating that TrkB agonist mediated activation of TrkB signalling was not altered upon BDNF allelic impairment. These data support BDNF/TrkB axis as a promising therapeutic target in glaucoma and highlight that the detrimental effects of high IOP exposure can be compensated by the exogenous administration of a TrkB agonist. [ABSTRACT FROM AUTHOR]

WWP2 is a HECT E3 ligase that targets protein Lys residues for ubiquitination and is comprised of an N-terminal C2 domain, four central WW domains, and a C-terminal catalytic HECT domain. The peptide segment between the middle WW domains, the 2,3-linker, is known to autoinhibit the catalytic domain, and this autoinhibition can be relieved by phosphorylation at Tyr369. Several protein substrates of WWP2 have been identified, including the tumor suppressor lipid phosphatase PTEN, but the full substrate landscape and biological functions of WWP2 remain to be elucidated. Here, we used protein microarray technology and the activated enzyme phosphomimetic mutant WWP2Y369E to identify potential WWP2 substrates. We identified 31 substrate hits for WWP2Y369E using protein microarrays, of which three were known autophagy receptors (NDP52, OPTN, and SQSTM1). These three hits were validated with in vitro and cell-based transfection assays and the Lys ubiquitination sites on these proteins were mapped by mass spectrometry. Among the mapped ubiquitin sites on these autophagy receptors, many had been previously identified in the endogenous proteins. Finally, we observed that WWP2 KO SH-SH5Y neuroblastoma cells using CRISPR-Cas9 showed a defect in mitophagy, which could be rescued by WWP2Y369E transfection. These studies suggest that WWP2-mediated ubiquitination of the autophagy receptors NDP52, OPTN, and SQSTM1 may positively contribute to the regulation of autophagy. [ABSTRACT FROM AUTHOR]

Since the discovery of the prolyl hydroxylases domain (PHD) proteins and their canonical hypoxia-inducible factor (HIF) substrate two decades ago, a number of in vitro hydroxylation (IVH) assays for PHD activity have been developed to measure the PHD-HIF interaction. However, most of these assays either require complex proteomics mass spectrometry methods that rely on the specific PHD-HIF interaction or require the handling of radioactive material, as seen in the most commonly used assay measuring [14C]O2 release from labeled [14C]a-ketoglutarate. Here, we report an alternative rapid, cost-effective assay in which the consumption of a-ketoglutarate is monitored by its derivatization with 2,4-dinitrophenylhydrazine (2,4-DNPH) followed by treatment with concentrated base. We extensively optimized this 2,4-DNPH a-ketoglutarate assay to maximize the signal-to-noise ratio and demonstrated that it is robust enough to obtain kinetic parameters of the well-characterized PHD2 isoform comparable with those in published literature. We further showed that it is also sensitive enough to detect and measure the IC50 values of pan-PHD inhibitors and several PHD2 inhibitors in clinical trials for chronic kidney disease (CKD)-induced anemia. Given the efficiency of this assay coupled with its multiwell format, the 2,4-DNPH a-KG assay may be adaptable to explore non-HIF substrates of PHDs and potentially to high-throughput assays. [ABSTRACT FROM AUTHOR]

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 retroelementderived 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. [ABSTRACT FROM AUTHOR]

Deubiquitinases are proteases with a wide functional diversity that profoundly impact multiple biological processes. Among them, the ubiquitin-specific protease 36 (USP36) has been implicated in the regulation of nucleolar activity. However, its functional relevance in vivo has not yet been fully described. Here, we report the generation of an Usp36-deficient mouse model to examine the function of this enzyme. We show that Usp36 depletion is lethal in preimplantation mouse embryos, where it blocks the transition from morula to blastocyst during embryonic development. USP36 reduces the ubiquitination levels and increases the stability of the DEAH-box RNA helicase DHX33, which is critically involved in ribosomal RNA synthesis and mRNA translation. In agreement with this finding, O-propargyl-puromycin incorporation experiments, Northern blot, and electron microscopy analyses demonstrated the role of USP36 in ribosomal RNA and protein synthesis. Finally, we show that USP36 down-regulation alters cell proliferation in human cancer cells by inducing both apoptosis and cell cycle arrest, and that reducing DHX33 levels through short hairpin RNA interference has the same effect. Collectively, these results support that Usp36 is essential for cell and organism viability because of its role in ribosomal RNA processing and protein synthesis, which is mediated, at least in part, by regulating DHX33 stability. [ABSTRACT FROM AUTHOR]