A Translation-Activating Function of MIWI/piRNA during Mouse Spermiogenesis.

Spermiogenesis is a highly orchestrated developmental process during which chromatin condensation decouples transcription from translation. Spermiogenic mRNAs are transcribed earlier and stored in a translationally inert state until needed for translation; however, it remains largely unclear how such repressed mRNAs become activated during spermiogenesis. We previously reported that the MIWI/piRNA machinery is responsible for mRNA elimination during late spermiogenesis in preparation for spermatozoa production. Here we unexpectedly discover that the same machinery is also responsible for activating translation of a subset of spermiogenic mRNAs to coordinate with morphological transformation into spermatozoa. Such action requires specific base-pairing interactions of piRNAs with target mRNAs in their 3' UTRs, which activates translation through coupling with cis-acting AU-rich elements to nucleate the formation of a MIWI/piRNA/eIF3f/HuR super-complex in a developmental stage-specific manner. These findings reveal a critical role of the piRNA system in translation activation, which we show is functionally required for spermatid development.

SHARPIN controls regulatory T cells by negatively modulating the T cell antigen receptor complex.

SHARPIN forms a linear-ubiquitin-chain-assembly complex that promotes signaling via the transcription factor NF-κB. SHARPIN deficiency leads to progressive multi-organ inflammation and immune system malfunction, but how SHARPIN regulates T cell responses is unclear. Here we found that SHARPIN deficiency resulted in a substantial reduction in the number of and defective function of regulatory T cells (Treg cells). Transfer of SHARPIN-sufficient Treg cells into SHARPIN-deficient mice considerably alleviated their systemic inflammation. SHARPIN-deficient T cells displayed enhanced proximal signaling via the T cell antigen receptor (TCR) without an effect on the activation of NF-κB. SHARPIN conjugated with Lys63 (K63)-linked ubiquitin chains, which led to inhibition of the association of TCRζ with the signaling kinase Zap70; this affected the generation of Treg cells. Our study therefore identifies a role for SHARPIN in TCR signaling whereby it maintains immunological homeostasis and tolerance by regulating Treg cells.

HDAC4 governs a transcriptional program essential for synaptic plasticity and memory.

Neuronal activity influences genes involved in circuit development and information processing. However, the molecular basis of this process remains poorly understood. We found that HDAC4, a histone deacetylase that shuttles between the nucleus and cytoplasm, controls a transcriptional program essential for synaptic plasticity and memory. The nuclear import of HDAC4 and its association with chromatin is negatively regulated by NMDA receptors. In the nucleus, HDAC4 represses genes encoding constituents of central synapses, thereby affecting synaptic architecture and strength. Furthermore, we show that a truncated form of HDAC4 encoded by an allele associated with mental retardation is a gain-of-function nuclear repressor that abolishes transcription and synaptic transmission despite the loss of the deacetylase domain. Accordingly, mice carrying a mutant that mimics this allele exhibit deficits in neurotransmission, spatial learning, and memory. These studies elucidate a mechanism of experience-dependent plasticity and define the biological role of HDAC4 in the brain.

A proteomic classifier panel for early screening of colorectal cancer: a case control study.

BACKGROUND: Diagnosis of colorectal cancer (CRC) during early stages can greatly improve patient outcome. Although technical advances in the field of genomics and proteomics have identified a number of candidate biomarkers for non-invasive screening and diagnosis, developing more sensitive and specific methods with improved cost-effectiveness and patient compliance has tremendous potential to help combat the disease. METHODS: We enrolled three cohorts of 479 subjects, including 226 CRC cases, 197 healthy controls, and 56 advanced precancerous lesions (APC). In the discovery cohort, we used quantitative mass spectrometry to measure the expression profile of plasma proteins and applied machine-learning to select candidate proteins. We then developed a targeted mass spectrometry assay to measure plasma concentrations of seven proteins and a logistic regression classifier to distinguish CRC from healthy subjects. The classifier was further validated using two independent cohorts. RESULTS: The seven-protein panel consisted of leucine rich alpha-2-glycoprotein 1 (LRG1), complement C9 (C9), insulin-like growth factor binding protein 2 (IGFBP2), carnosine dipeptidase 1 (CNDP1), inter-alpha-trypsin inhibitor heavy chain 3 (ITIH3), serpin family A member 1 (SERPINA1), and alpha-1-acid glycoprotein 1 (ORM1). The panel classified CRC and healthy subjects with high accuracy, since the area under curve (AUC) of the training and testing cohort reached 0.954 and 0.958. The AUC of the two independent validation cohorts was 0.905 and 0.909. In one validation cohort, the panel had an overall sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 89.9%, 81.8%, 89.2%, and 82.9%, respectively. In another blinded validation cohort, the panel classified CRC from healthy subjects with a sensitivity of 81.5%, specificity of 97.9%, and overall accuracy of 92.0%. Finally, the panel was able to detect APC with a sensitivity of 49%. CONCLUSIONS: This seven-protein classifier is a clear improvement compared to previously published blood-based protein biomarkers for detecting early-stage CRC, and is of translational potential to develop into a clinically useful assay.

The ubiquitination of rag A GTPase by RNF152 negatively regulates mTORC1 activation.

mTORC1 is essential for regulating cell growth and metabolism in response to various environmental stimuli. Heterodimeric Rag GTPases are required for amino-acid-mediated mTORC1 activation at the lysosome. However, the mechanism by which amino acids regulate Rag activation remains not fully understood. Here, we identified the lysosome-anchored E3 ubiquitin ligase RNF152 as an essential negative regulator of the mTORC1 pathway by targeting RagA for K63-linked ubiquitination. RNF152 interacts with and ubiquitinates RagA in an amino-acid-sensitive manner. The mutation of RagA ubiquitination sites abolishes this effect of RNF152 and enhances the RagA-mediated activation of mTORC1. Ubiquitination by RNF152 generates an anchor on RagA to recruit its inhibitor GATOR1, a GAP complex for Rag GTPases. RNF152 knockout results in the hyperactivation of mTORC1 and protects cells from amino-acid-starvation-induced autophagy. Thus, this study reveals a mechanism for regulation of mTORC1 signaling by RNF152-mediated K63-linked polyubiquitination of RagA.

Linking nuclear matrix-localized PIAS1 to chromatin SUMOylation via direct binding of histones H3 and H2A.Z.

As a conserved posttranslational modification, SUMOylation has been shown to play important roles in chromatin-related biological processes including transcription. However, how the SUMOylation machinery associates with chromatin is not clear. Here, we present evidence that multiple SUMOylation machinery components, including SUMO E1 proteins SAE1 and SAE2 and the PIAS (protein inhibitor of activated STAT) family SUMO E3 ligases, are primarily associated with the nuclear matrix rather than with chromatin. We show using nuclease digestion that all PIAS family proteins maintain nuclear matrix association in the absence of chromatin. Of importance, we identify multiple histones including H3 and H2A.Z as directly interacting with PIAS1 and demonstrate that this interaction requires the PIAS1 SAP (SAF-A/B, Acinus, and PIAS) domain. We demonstrate that PIAS1 promotes SUMOylation of histones H3 and H2B in both a SAP domain- and an E3 ligase activity-dependent manner. Furthermore, we show that PIAS1 binds to heat shock-induced genes and represses their expression and that this function also requires the SAP domain. Altogether, our study reveals for the first time the nuclear matrix as the compartment most enriched in SUMO E1 and PIAS family E3 ligases. Our finding that PIAS1 interacts directly with histone proteins also suggests a molecular mechanism as to how nuclear matrix-associated PIAS1 is able to regulate transcription and other chromatin-related processes.

High-throughput tandem-microwell assay for ammonia repositions FDA-Approved drugs to inhibit Helicobacter pylori urease.

To date, little attempt has been made to develop new treatments for Helicobacter pylori (H. pylori), although the community is aware of the shortage of treatments for H. pylori. In this study, we developed a 192-tandem-microwell-based high-throughput assay for ammonia that is a known virulence factor of H. pylori and a product of urease. We could identify few drugs, that is, panobinostat, dacinostat, ebselen, captan, and disulfiram, to potently inhibit the activity of ureases from bacterial or plant species. These inhibitors suppress the activity of urease via substrate-competitive or covalent-allosteric mechanism, but all except captan prevent the antibiotic-resistant H. pylori strain from killing human gastric cells, with a more pronounced effect than acetohydroxamic acid, a well-known urease inhibitor and clinically used drug for the treatment of bacterial infection. This study offers several bases for the development of new treatments for urease-containing pathogens and to study the mechanism responsible for the regulation of urease activity.

PINK1 phosphorylates Drp1S616 to regulate mitophagy-independent mitochondrial dynamics.

Impairment of PINK1/parkin-mediated mitophagy is currently proposed to be the molecular basis of mitochondrial abnormality in Parkinson's disease (PD). We here demonstrate that PINK1 directly phosphorylates Drp1 on S616. Drp1S616 phosphorylation is significantly reduced in cells and mouse tissues deficient for PINK1, but unaffected by parkin inactivation. PINK1-mediated mitochondrial fission is Drp1S616 phosphorylation dependent. Overexpression of either wild-type Drp1 or of the phosphomimetic mutant Drp1S616D , but not a dephosphorylation-mimic mutant Drp1S616A , rescues PINK1 deficiency-associated phenotypes in Drosophila. Moreover, Drp1 restores PINK1-dependent mitochondrial fission in ATG5-null cells and ATG7-null Drosophila. Reduced Drp1S616 phosphorylation is detected in fibroblasts derived from 4 PD patients harboring PINK1 mutations and in 4 out of 7 sporadic PD cases. Taken together, we have identified Drp1 as a substrate of PINK1 and a novel mechanism how PINK1 regulates mitochondrial fission independent of parkin and autophagy. Our results further link impaired PINK1-mediated Drp1S616 phosphorylation with the pathogenesis of both familial and sporadic PD.

UBE3A-mediated PTPA ubiquitination and degradation regulate PP2A activity and dendritic spine morphology.

Deficiency in the E3 ubiquitin ligase UBE3A leads to the neurodevelopmental disorder Angelman syndrome (AS), while additional dosage of UBE3A is linked to autism spectrum disorder. The mechanisms underlying the downstream effects of UBE3A gain or loss of function in these neurodevelopmental disorders are still not well understood, and effective treatments are lacking. Here, using stable-isotope labeling of amino acids in mammals and ubiquitination assays, we identify PTPA, an activator of protein phosphatase 2A (PP2A), as a bona fide ubiquitin ligase substrate of UBE3A. Maternal loss of Ube3a (Ube3am-/p+) increased PTPA level, promoted PP2A holoenzyme assembly, and elevated PP2A activity, while maternal 15q11-13 duplication containing Ube3a down-regulated PTPA level and lowered PP2A activity. Reducing PTPA level in vivo restored the defects in dendritic spine maturation in Ube3am-/p+ mice. Moreover, pharmacological inhibition of PP2A activity with the small molecule LB-100 alleviated both reduction in excitatory synaptic transmission and motor impairment in Ube3am-/p+ mice. Together, our results implicate a critical role of UBE3A-PTPA-PP2A signaling in the pathogenesis of UBE3A-related disorders and suggest that PP2A-based drugs could be potential therapeutic candidates for treatment of UBE3A-related disorders.

SET8 prevents excessive DNA methylation by methylation-mediated degradation of UHRF1 and DNMT1.

Faithful inheritance of DNA methylation across cell division requires DNMT1 and its accessory factor UHRF1. However, how this axis is regulated to ensure DNA methylation homeostasis remains poorly understood. Here we show that SET8, a cell-cycle-regulated protein methyltransferase, controls protein stability of both UHRF1 and DNMT1 through methylation-mediated, ubiquitin-dependent degradation and consequently prevents excessive DNA methylation. SET8 methylates UHRF1 at lysine 385 and this modification leads to ubiquitination and degradation of UHRF1. In contrast, LSD1 stabilizes both UHRF1 and DNMT1 by demethylation. Importantly, SET8 and LSD1 oppositely regulate global DNA methylation and do so most likely through regulating the level of UHRF1 than DNMT1. Finally, we show that UHRF1 downregulation in G2/M by SET8 has a role in suppressing DNMT1-mediated methylation on post-replicated DNA. Altogether, our study reveals a novel role of SET8 in promoting DNA methylation homeostasis and identifies UHRF1 as the hub for tuning DNA methylation through dynamic protein methylation.

Comprehensive Map of the Artemisia annua Proteome and Quantification of Differential Protein Expression in Chemotypes Producing High versus Low Content of Artemisinin.

Artemisia annua is well known for biosynthesizing the antimalarial drug artemisinin. Here, a global proteomic profiling of A. annua is conducted with identification of a total of 13 403 proteins based on the genome sequence annotation database. Furthermore, a spectral library is generated to perform quantitative proteomic analysis using data independent acquisition mass spectrometry. Specifically, proteins between two chemotypes that produce high (HAP) and low (LAP) artemisinin content, respectively, are comprehensively quantified and compared. 182 proteins are identified with abundance significantly different between these two chemotypes means after the statistic use the p-value and fold change it is found 182 proteins can reach the demand conditions which represent the expression are significantly different between the high artemisnin content plants (HAPs) and the low artemisnin content plants (LAPs). Data are available via ProteomeXchange with identifier PXD015547. Overall, this current study globally identifies the proteome of A. annua and quantitatively compares the targeted sub-proteomes between the two cultivars of HAP and LAP, providing systematic information on metabolic pathways of A. annua.

Protein Kinase C Lambda Mediates Acid-Sensing Ion Channel 1a-Dependent Cortical Synaptic Plasticity and Pain Hypersensitivity.

Chronic pain is a serious debilitating disease for which effective treatment is still lacking. Acid-sensing ion channel 1a (ASIC1a) has been implicated in nociceptive processing at both peripheral and spinal neurons. However, whether ASIC1a also contributes to pain perception at the supraspinal level remains elusive. Here, we report that ASIC1a in ACC is required for thermal and mechanical hypersensitivity associated with chronic pain. ACC-specific genetic deletion or pharmacological blockade of ASIC1a reduced the probability of cortical LTP induction and attenuated inflammatory thermal hyperalgesia and mechanical allodynia in male mice. Using cell type-specific manipulations, we demonstrate that ASIC1a in excitatory neurons of ACC is a major player in cortical LTP and pain behavior. Mechanistically, we show that ASIC1a tuned pain-related cortical plasticity through protein kinase C λ-mediated increase of membrane trafficking of AMPAR subunit GluA1 in ACC. Importantly, postapplication of ASIC1a inhibitors in ACC reversed previously established nociceptive hypersensitivity in both chronic inflammatory pain and neuropathic pain models. These results suggest that ASIC1a critically contributes to a higher level of pain processing through synaptic potentiation in ACC, which may serve as a promising analgesic target for treatment of chronic pain.SIGNIFICANCE STATEMENT Chronic pain is a debilitating disease that still lacks effective therapy. Ion channels are good candidates for developing new analgesics. Here, we provide several lines of evidence to support an important role of cortically located ASIC1a channel in pain hypersensitivity through promoting long-term synaptic potentiation in the ACC. Our results indicate a promising translational potential of targeting ASIC1a to treat chronic pain.

PAQR3 controls autophagy by integrating AMPK signaling to enhance ATG14L-associated PI3K activity.

The Beclin1-VPS34 complex is recognized as a central node in regulating autophagy via interacting with diverse molecules such as ATG14L for autophagy initiation and UVRAG for autophagosome maturation. However, the underlying molecular mechanism that coordinates the timely activation of VPS34 complex is poorly understood. Here, we identify that PAQR3 governs the preferential formation and activation of ATG14L-linked VPS34 complex for autophagy initiation via two levels of regulation. Firstly, PAQR3 functions as a scaffold protein that facilitates the formation of ATG14L- but not UVRAG-linked VPS34 complex, leading to elevated capacity of PI(3)P generation ahead of starvation signals. Secondly, AMPK phosphorylates PAQR3 at threonine 32 and switches on PI(3)P production to initiate autophagosome formation swiftly after glucose starvation. Deletion of PAQR3 leads to reduction of exercise-induced autophagy in mice, accompanied by a certain degree of disaggregation of ATG14L-associated VPS34 complex. Together, this study uncovers that PAQR3 can not only enhance the capacity of pro-autophagy class III PI3K due to its scaffold function, but also integrate AMPK signal to activation of ATG14L-linked VPS34 complex upon glucose starvation.

Reactivation of nonsense-mediated mRNA decay protects against C9orf72 dipeptide-repeat neurotoxicity.

Amyotrophic lateral sclerosis is a deleterious neurodegenerative disease without effective treatment options. Recent studies have indicated the involvement of the dysregulation of RNA metabolism in the pathogenesis of amyotrophic lateral sclerosis. Among the various RNA regulatory machineries, nonsense-mediated mRNA decay (NMD) is a stress responsive cellular surveillance system that degrades selected mRNA substrates to prevent the translation of defective or harmful proteins. Whether this pathway is affected in neurodegenerative diseases is unclear. Here we report the inhibition of NMD by arginine-rich dipeptide repeats derived from C9orf72 hexanucleotide repeat expansion, the most common cause of familial amyotrophic lateral sclerosis. Bioinformatic analysis of multiple transcriptome profiles revealed significant overlap of upregulated genes in NMD-defective cells with those in the brain tissues, micro-dissected motor neurons, or induced pluripotent stem cell-derived motor neurons specifically from amyotrophic lateral sclerosis patients carrying C9orf72 hexanucleotide repeat expansion, suggesting the suppression of NMD pathway in these patients. Using Drosophila as a model, we have validated that the C9orf72 hexanucleotide repeat expansion products could lead to the accumulation of the NMD substrates and identified arginine-rich dipeptide repeats, including poly glycine-arginine and poly proline-arginine, as the main culprits of NMD inhibition. Furthermore, in human SH-SY5Y neuroblastoma cells and in mouse brains, expression of glycine-arginine with 36 repeats (GR36) was sufficient to cause NMD inhibition. In cells expressing GR36, stress granule accumulation was accompanied by decreased processing body formation, which contributed to the inhibition of NMD. Remarkably, expression of UPF1, a core gene in the NMD pathway, efficiently blocked neurotoxicity caused by arginine-rich dipeptide repeats in both cellular and Drosophila models. Although not as effective as UPF1, expression of another NMD gene UPF2 also ameliorated the degenerative phenotypes in dipeptide repeat-expressing flies, indicating that genetically reactivating the NMD pathway could suppress dipeptide repeat toxicity. Finally, after validating tranilast as an NMD-activating drug, we demonstrated the therapeutic potential of this asthma drug in cellular and Drosophila models of C9orf72 dipeptide repeat neurotoxicity. Therefore, our study has revealed a cellular mechanism whereby arginine-rich C9orf72 dipeptide repeats could inhibit NMD activities by reducing the abundance of processing bodies. Furthermore, our results suggested that activation of the NMD pathway could be a potential therapeutic strategy for amyotrophic lateral sclerosis with defective RNA metabolism.

Differential Sensitivity of Target Genes to Translational Repression by miR-17~92.

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5'UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes.

HAP1 is an in vivo UBE3A target that augments autophagy in a mouse model of Angelman syndrome.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by maternal mutation and paternal imprinting of the gene encoding UBE3A, an E3 ubiquitin ligase. Although several potential target proteins of UBE3A have been reported, how these proteins regulate neuronal development remains unclear. We performed a large-scale quantitative proteomic analysis using stable-isotope labeling of amino acids in mammals (SILAM) in mice with maternal Ube3a mutation. We identified huntingtin (Htt)-associated protein (HAP1), a protein that is involved in Huntington's disease (HD), as a new target of UBE3A. We demonstrate that HAP1 regulates autophagy at the initiation stage by promoting PtdIns3K complex formation and enhancing its activity. HAP1 also co-localized with MAP1LC3 (LC3) and other proteins involved in autophagosome expansion. As a result, HAP1 increased autophagy flux. Strikingly, knocking down of HAP1 alleviated aberrant autophagy in primary neurons from AS mice. Concordantly, treatment of AS neurons with an autophagy inhibitor alleviated the reduction in density of dendritic spines. Furthermore, autophagy inhibition in AS mice partially alleviated a social interaction deficit as shown in open field test. Thus, our results identify HAP1 as an in vivo UBE3A target that contributes to deregulated autophagy and synaptic dysfunction in the central nervous system of AS mouse.

Hepatic PPARα function is controlled by polyubiquitination and proteasome-mediated degradation through the coordinated actions of PAQR3 and HUWE1.

Peroxisome proliferator-activated receptor α (PPARα) is a key transcriptional factor that regulates hepatic lipid catabolism by stimulating fatty acid oxidation and ketogenesis in an adaptive response to nutrient starvation. However, how PPARα is regulated by posttranslational modification is poorly understood. In this study, we identified that progestin and adipoQ receptor 3 (PAQR3) promotes PPARα ubiquitination through the E3 ubiquitin ligase HUWE1, thereby negatively modulating PPARα functions both in vitro and in vivo. Adenovirus-mediated Paqr3 knockdown and liver-specific deletion of the Paqr3 gene reduced hepatic triglyceride levels while increasing fatty acid oxidation and ketogenesis upon fasting. PAQR3 deficiency enhanced the fasting-induced expression of PPARα target genes, including those involved in fatty acid oxidation and fibroblast growth factor 21, a key molecule that mediates the metabolism-modulating effects of PPARα. PAQR3 directly interacted with PPARα and increased the polyubiquitination and proteasome-mediated degradation of PPARα. Furthermore, the E3 ubiquitin ligase HUWE1 was identified to mediate PPARα polyubiquitination. Additionally, PAQR3 enhanced the interaction between HUWE1 and PPARα. CONCLUSION: Ubiquitination modification through the coordinated action of PAQR3 with HUWE1 plays a crucial role in regulating the activity of PPARα in response to starvation. (Hepatology 2018;68:289-303).

K33-linked polyubiquitination of T cell receptor-zeta regulates proteolysis-independent T cell signaling.

Tagging the cell surface receptor with ubiquitin is believed to provide a signal for the endocytic pathway. E3 ubiquitin ligases such as Cbl-b and Itch have been implicated in T cell activation and tolerance induction. However, the underlying mechanisms remain unclear. We describe that in mice deficient in the E3 ubiquitin ligases Cbl-b and Itch, T cell activation was augmented, accompanied by spontaneous autoimmunity. The double-mutant T cells exhibited increased phosphorylation of the T cell receptor-zeta (TCR-zeta) chain, whereas the endocytosis and stability of the TCR complex were not affected. TCR-zeta was polyubiquitinated via a K33-linkage, which affected its phosphorylation and association with the zeta chain-associated protein kinase Zap-70. The juxtamembrane K54 residue in TCR-zeta was identified to be a primary ubiquitin conjugation site, whose mutation increased its phosphorylation and association of TCR-zeta and Zap-70. Thus, the present study reveals unconventional K33-linked polyubiquitination in nonproteolytic regulation of cell-surface-receptor-mediated signal transduction.

The butterfly effect in cancer: a single base mutation can remodel the cell.

We have compared the proteome, transcriptome, and metabolome of two cell lines: the human breast epithelial line MCF-10A and its mutant descendant MCF-10A-H1047R. These cell lines are derived from the same parental stock and differ by a single amino acid substitution (H1047R) caused by a single nucleotide change in one allele of the PIK3CA gene, which encodes the catalytic subunit p110α of PI3K (phosphatidylinositol 3-kinase). They are considered isogenic. The H1047R mutation of PIK3CA is one of the most frequently encountered somatic cancer-specific mutations. In MCF-10A, this mutation induces an extensive cellular reorganization that far exceeds the known signaling activities of PI3K. The changes are highly diverse, with examples in structural protein levels, the DNA repair machinery, and sterol synthesis. Gene set enrichment analysis reveals a highly significant concordance of the genes differentially expressed in MCF-10A-H1047R cells and the established protein and RNA signatures of basal breast cancer. No such concordance was found with the specific gene signatures of other histological types of breast cancer. Our data document the power of a single base mutation, inducing an extensive remodeling of the cell toward the phenotype of a specific cancer.

Fmr1 deficiency promotes age-dependent alterations in the cortical synaptic proteome.

Fragile X syndrome (FXS) is an X-linked neurodevelopmental disorder characterized by severe intellectual disability and other symptoms including autism. Although caused by the silencing of a single gene, Fmr1 (fragile X mental retardation 1), the complexity of FXS pathogenesis is amplified because the encoded protein, FMRP, regulates the activity-dependent translation of numerous mRNAs. Although the mRNAs that associate with FMRP have been extensively studied, little is known regarding the proteins whose expression levels are altered, directly or indirectly, by loss of FMRP during brain development. Here we systematically measured protein expression in neocortical synaptic fractions from Fmr1 knockout (KO) and wild-type (WT) mice at both adolescent and adult stages. Although hundreds of proteins are up-regulated in the absence of FMRP in young mice, this up-regulation is largely diminished in adulthood. Up-regulated proteins included previously unidentified as well as known targets involved in synapse formation and function and brain development and others linked to intellectual disability and autism. Comparison with putative FMRP target mRNAs and autism susceptibility genes revealed substantial overlap, consistent with the idea that the autism endophenotype of FXS is due to a "multiple hit" effect of FMRP loss, particularly within the PSD95 interactome. Through studies of de novo protein synthesis in primary cortical neurons from KO and WT mice, we found that neurons lacking FMRP produce nascent proteins at higher rates, many of which are synaptic proteins and encoded by FMRP target mRNAs. Our results provide a greatly expanded view of protein changes in FXS and identify age-dependent effects of FMRP in shaping the neuronal proteome.