Phosphorylation of the nuclear poly(A) binding protein (PABPN1) during mitosis protects mRNA from hyperadenylation and maintains transcriptome dynamics.

Polyadenylation controls mRNA biogenesis, nucleo-cytoplasmic export, translation and decay. These processes are interdependent and coordinately regulated by poly(A)-binding proteins (PABPs), yet how PABPs are themselves regulated is not fully understood. Here, we report the discovery that human nuclear PABPN1 is phosphorylated by mitotic kinases at four specific sites during mitosis, a time when nucleoplasm and cytoplasm mix. To understand the functional consequences of phosphorylation, we generated a panel of stable cell lines inducibly over-expressing PABPN1 with point mutations at these sites. Phospho-inhibitory mutations decreased cell proliferation, highlighting the importance of PABPN1 phosphorylation in cycling cells. Dynamic regulation of poly(A) tail length and RNA stability have emerged as important modes of gene regulation. We therefore employed long-read sequencing to determine how PABPN1 phospho-site mutants affected poly(A) tails lengths and TimeLapse-seq to monitor mRNA synthesis and decay. Widespread poly(A) tail lengthening was observed for phospho-inhibitory PABPN1 mutants. In contrast, expression of phospho-mimetic PABPN1 resulted in shorter poly(A) tails with increased non-A nucleotides, in addition to increased transcription and reduced stability of a distinct cohort of mRNAs. Taken together, PABPN1 phosphorylation remodels poly(A) tails and increases mRNA turnover, supporting the model that enhanced transcriptome dynamics reset gene expression programs across the cell cycle.

Mechanosensing through talin 1 contributes to tissue mechanical homeostasis.

It is widely believed that tissue mechanical properties, determined mainly by the extracellular matrix (ECM), are actively maintained. However, despite its broad importance to biology and medicine, tissue mechanical homeostasis is poorly understood. To explore this hypothesis, we developed mutations in the mechanosensitive protein talin1 that alter cellular sensing of ECM stiffness. Mutation of a novel mechanosensitive site between talin1 rod domain helix bundles 1 and 2 (R1 and R2) shifted cellular stiffness sensing curves, enabling cells to spread and exert tension on compliant substrates. Opening of the R1-R2 interface promotes binding of the ARP2/3 complex subunit ARPC5L, which mediates the altered stiffness sensing. Ascending aortas from mice bearing these mutations show increased compliance, less fibrillar collagen, and rupture at lower pressure. Together, these results demonstrate that cellular stiffness sensing regulates ECM mechanical properties. These data thus directly support the mechanical homeostasis hypothesis and identify a novel mechanosensitive interaction within talin that contributes to this mechanism.

Dual regulation of SLC25A39 by AFG3L2 and iron controls mitochondrial glutathione homeostasis.

Organelle transporters define metabolic compartmentalization, and how this metabolite transport process can be modulated is poorly explored. Here, we discovered that human SLC25A39, a mitochondrial transporter critical for mitochondrial glutathione uptake, is a short-lived protein under dual regulation at the protein level. Co-immunoprecipitation mass spectrometry and CRISPR knockout (KO) in mammalian cells identified that mitochondrial m-AAA protease AFG3L2 is responsible for degrading SLC25A39 through the matrix loop 1. SLC25A39 senses mitochondrial iron-sulfur cluster using four matrix cysteine residues and inhibits its degradation. SLC25A39 protein regulation is robust in developing and mature neurons. This dual transporter regulation, by protein quality control and metabolic sensing, allows modulating mitochondrial glutathione level in response to iron homeostasis, opening avenues for exploring regulation of metabolic compartmentalization. Neuronal SLC25A39 regulation connects mitochondrial protein quality control, glutathione, and iron homeostasis, which were previously unrelated biochemical features in neurodegeneration.

Natural isoaspartyl protein modification of ZAP70 alters T cell responses in lupus.

Protein posttranslational modifications (PTMs) arise in a number of normal cellular biological pathways and in response to pathology caused by inflammation and/or infection. Indeed, a number of PTMs have been identified and linked to specific autoimmune responses and metabolic pathways. One particular PTM, termed isoaspartyl (isoAsp or isoD) modification, is among the most common spontaneous PTM occurring at physiological pH and temperature. Herein, we demonstrate that isoAsp modifications arise within the ZAP70 protein tyrosine kinase upon T-cell antigen receptor (TCR) engagement. The enzyme protein L-isoaspartate O-methyltransferase (PCMT1, or PIMT, EC 2.1.1.77) evolved to repair isoaspartyl modifications in cells. In this regard, we observe that increased levels of isoAsp modification that arise under oxidative stress are correlated with reduced PIMT activity in patients with systemic lupus erythematosus (SLE). PIMT deficiency leads to T cell hyper-proliferation and hyper-phosphorylation through ZAP70 signaling. We demonstrate that inducing the overexpression of PIMT can correct the hyper-responsive phenotype in lupus T cells. Our studies reveal a phenotypic role of isoAsp modification and phosphorylation of ZAP70 in lupus T cell autoimmunity and provide a potential therapeutic target through the repair of isoAsp modification.

Widespread association of the Argonaute protein AGO2 with meiotic chromatin suggests a distinct nuclear function in mammalian male reproduction.

Argonaute 2 (AGO2) is a ubiquitously expressed protein critical for regulation of mRNA translation and vital to animal development. AGO2 protein is found in both cytoplasmic and nuclear compartments, and although its cytoplasmic role is well studied, the biological relevance of nuclear AGO2 is unclear. Here, we address this problem in vivo using spermatogenic cells as a model. We find that AGO2 transiently binds both chromatin and nucleus-specific mRNA transcripts of hundreds of genes required for sperm production during male meiosis in mice, and that germline conditional knockout (cKO) of Ago2 causes depletion of the encoded proteins. Correspondingly, Ago2 cKO males show abnormal sperm head morphology and reduced sperm count, along with reduced postnatal viability of offspring. Together, our data reveal an unexpected nuclear role for AGO2 in enhancing expression of developmentally important genes during mammalian male reproduction.

Carbonyl Posttranslational Modification Associated With Early-Onset Type 1 Diabetes Autoimmunity.

Inflammation and oxidative stress in pancreatic islets amplify the appearance of various posttranslational modifications to self-proteins. In this study, we identified a select group of carbonylated islet proteins arising before the onset of hyperglycemia in NOD mice. Of interest, we identified carbonyl modification of the prolyl-4-hydroxylase ß subunit (P4Hb) that is responsible for proinsulin folding and trafficking as an autoantigen in both human and murine type 1 diabetes. We found that carbonylated P4Hb is amplified in stressed islets coincident with decreased glucose-stimulated insulin secretion and altered proinsulin-to-insulin ratios. Autoantibodies against P4Hb were detected in prediabetic NOD mice and in early human type 1 diabetes prior to the onset of anti-insulin autoimmunity. Moreover, we identify autoreactive CD4+ T-cell responses toward carbonyl-P4Hb epitopes in the circulation of patients with type 1 diabetes. Our studies provide mechanistic insight into the pathways of proinsulin metabolism and in creating autoantigenic forms of insulin in type 1 diabetes.

A versatile new tool derived from a bacterial deubiquitylase to detect and purify ubiquitylated substrates and their interacting proteins.

Protein ubiquitylation is an important posttranslational modification affecting a wide range of cellular processes. Due to the low abundance of ubiquitylated species in biological samples, considerable effort has been spent on methods to purify and detect ubiquitylated proteins. We have developed and characterized a novel tool for ubiquitin detection and purification based on OtUBD, a high-affinity ubiquitin-binding domain (UBD) derived from an Orientia tsutsugamushi deubiquitylase (DUB). We demonstrate that OtUBD can be used to purify both monoubiquitylated and polyubiquitylated substrates from yeast and human tissue culture samples and compare their performance with existing methods. Importantly, we found conditions for either selective purification of covalently ubiquitylated proteins or co-isolation of both ubiquitylated proteins and their interacting proteins. As proof of principle for these newly developed methods, we profiled the ubiquitylome and ubiquitin-associated proteome of the budding yeast Saccharomyces cerevisiae. Combining OtUBD affinity purification with quantitative proteomics, we identified potential substrates for the E3 ligases Bre1 and Pib1. OtUBD provides a versatile, efficient, and economical tool for ubiquitin research with specific advantages over certain other methods, such as in efficiently detecting monoubiquitylation or ubiquitin linkages to noncanonical sites.

Citrullination of glucokinase is linked to autoimmune diabetes.

Inflammation, including reactive oxygen species and inflammatory cytokines in tissues amplify various post-translational modifications of self-proteins. A number of post-translational modifications have been identified as autoimmune biomarkers in the initiation and progression of Type 1 diabetes. Here we show the citrullination of pancreatic glucokinase as a result of inflammation, triggering autoimmunity and affecting glucokinase biological functions. Glucokinase is expressed in hepatocytes to regulate glycogen synthesis, and in pancreatic beta cells as a glucose sensor to initiate glycolysis and insulin signaling. We identify autoantibodies and autoreactive CD4+ T cells to glucokinase epitopes in the circulation of Type 1 diabetes patients and NOD mice. Finally, citrullination alters glucokinase biologic activity and suppresses glucose-stimulated insulin secretion. Our study define glucokinase as a Type 1 diabetes biomarker, providing new insights of how inflammation drives post-translational modifications to create both neoautoantigens and affect beta cell metabolism.

Mycobacterium tuberculosis encodes a YhhN family membrane protein with lysoplasmalogenase activity that protects against toxic host lysolipids.

The pathogen Mycobacterium tuberculosis (M.tb) resides in human macrophages, wherein it exploits host lipids for survival. However, little is known about the interaction between M.tb and macrophage plasmalogens, a subclass of glycerophospholipids with a vinyl ether bond at the sn-1 position of the glycerol backbone. Lysoplasmalogens, produced from plasmalogens by hydrolysis at the sn-2 carbon by phospholipase A2, are potentially toxic but can be broken down by host lysoplasmalogenase, an integral membrane protein of the YhhN family that hydrolyzes the vinyl ether bond to release a fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. Curiously, M.tb encodes its own YhhN protein (MtbYhhN), despite having no endogenous plasmalogens. To understand the purpose of this protein, the gene for MtbYhhN (Rv1401) was cloned and expressed in Mycobacterium smegmatis (M.smeg). We found the partially purified protein exhibited abundant lysoplasmalogenase activity specific for lysoplasmenylethanolamine or lysoplasmenylcholine (pLPC) (Vmax∼15.5 µmol/min/mg; Km∼83 µM). Based on cell density, we determined that lysoplasmenylethanolamine, pLPC, lysophosphatidylcholine, and lysophosphatidylethanolamine were not toxic to M.smeg cells, but pLPC and LPC were highly toxic to M.smeg spheroplasts, which are cell wall-deficient mycobacterial forms. Importantly, spheroplasts prepared from M.smeg cells overexpressing MtbYhhN were protected from membrane disruption/lysis by pLPC, which was rapidly depleted from the media. Finally, we found that overexpression of full-length MtbYhhN in M.smeg increased its survival within human macrophages by 2.6-fold compared to vector controls. These data support the hypothesis that MtbYhhN protein confers a growth advantage for mycobacteria in macrophages by cleaving toxic host pLPC into potentially energy-producing products.

Simple, Single-Shot Phosphoproteomic Analysis of Heat-Stable Tau Identifies Age-Related Changes in pS235- and pS396-Tau Levels in Non-human Primates.

Age is the most significant risk factor for Alzheimer's disease (AD), and understanding its role in specific aspects of AD pathology will be critical for therapeutic development. Neurofibrillary tangles composed of hyperphosphorylated tau are a quintessential hallmark of AD. To study age-related changes in tau phosphorylation, we developed a simple, antibody-free approach for single shot analysis of tau phosphorylation across the entire protein by liquid-chromatography tandem mass spectrometry. This methodology is species independent; thus, while initially developed in a rodent model, we utilized this technique to analyze 36 phosphorylation sites on rhesus monkey tau from the prefrontal cortex (PFC), a region vulnerable to AD-linked degeneration. Data are available via ProteomeXchange with identifier PXD027971. We identified novel, age-related changes in tau phosphorylation in the rhesus monkey PFC and analyzed patterns of phosphorylation change across domains of the protein. We confirmed a significant increase and positive correlation with age of phosphorylated serine 235 tau and phosphorylated serine 396 tau levels in an expanded cohort of 14 monkeys. Histology showed robust labeling for tau phosphorylated at these sites in vulnerable layer III pyramidal cells in the PFC. The results presented in this study suggest an important role of the natural aging process in tau phosphorylation in rhesus monkey.

Bioactive Plasma Mitochondrial DNA Is Associated With Disease Progression in Scleroderma-Associated Interstitial Lung Disease.

OBJECTIVE: Systemic sclerosis-associated interstitial lung disease (SSc-ILD) is characterized by variable clinical outcomes, activation of innate immune pattern-recognition receptors (PRRs), and accumulation of α-smooth muscle actin (α-SMA)-expressing myofibroblasts. The aim of this study was to identify an association between these entities and mitochondrial DNA (mtDNA), an endogenous ligand for the intracellular DNA-sensing PRRs Toll-like receptor 9 (TLR-9) and cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING), which has yet to be determined. METHODS: Human lung fibroblasts (HLFs) from normal donors and SSc-ILD explants were treated with synthetic CpG DNA and assayed for α-SMA expression and extracellular mtDNA using quantitative polymerase chain reaction for the human MT-ATP6 gene. Plasma MT-ATP6 concentrations were evaluated in 2 independent SSc-ILD cohorts and demographically matched controls. The ability of SSc-ILD and control plasma to induce TLR-9 and cGAS/STING activation was evaluated with commercially available HEK 293 reporter cells. Plasma concentrations of type I interferons (IFNs), interleukin-6 (IL-6), and oxidized DNA were measured using electrochemiluminescence and enzyme-linked immunosorbent assay-based methods. Extracellular vesicles (EVs) precipitated from plasma were evaluated for MT-ATP6 concentrations and proteomics via liquid chromatography mass spectrometry. RESULTS: Normal HLFs and SSc-ILD fibroblasts developed increased α-SMA expression and MT-ATP6 release following CpG stimulation. Plasma mtDNA concentrations were increased in the 2 SSc-ILD cohorts, reflective of ventilatory decline, and were positively associated with both TLR-9 and cGAS/STING activation as well as type I IFN and IL-6 expression. Plasma mtDNA was not oxidized and was conveyed by EVs displaying a proteomics profile consistent with a multicellular origin. CONCLUSION: These findings demonstrate a previously unrecognized connection between EV-encapsulated mtDNA, clinical outcomes, and intracellular DNA-sensing PRR activation in SSc-ILD. Further study of these interactions could catalyze novel mechanistic and therapeutic insights into SSc-ILD and related disorders.

Phosphorylation of human placental aromatase CYP19A1.

Aromatase CYP19A1 catalyzes the synthesis of estrogens in endocrine, reproductive and central nervous systems. Higher levels of 17ß-estradiol (E2) are associated with malignancies and diseases of the breast, ovary and endometrium, while low E2 levels increase the risk for osteoporosis, cardiovascular diseases and cognitive disorders. E2, the transcriptional activator of the estrogen receptors, is also known to be involved in non-genomic signaling as a neurotransmitter/neuromodulator, with recent evidence for rapid estrogen synthesis (RES) within the synaptic terminal. Although regulation of brain aromatase activity by phosphorylation/dephosphorylation has been suggested, it remains obscure in the endocrine and reproductive systems. RES and overabundance of estrogens could stimulate the genomic and non-genomic signaling pathways, and genotoxic effects of estrogen metabolites. Here, by utilizing biochemical, cellular, mass spectrometric, and structural data we unequivocally demonstrate phosphorylation of human placental aromatase and regulation of its activity. We report that human aromatase has multiple phosphorylation sites, some of which are consistently detectable. Phosphorylation of the residue Y361 at the reductase-coupling interface significantly elevates aromatase activity. Other sites include the active site residue S478 and several at the membrane interface. We present the evidence that two histidine residues are phosphorylated. Furthermore, oxidation of two proline residues near the active site may have implications in regulation. Taken together, the results demonstrate that aromatase activity is regulated by phosphorylation and possibly other post-translational modifications. Protein level regulation of aromatase activity not only represents a paradigm shift in estrogen-mediated biology, it could also explain unresolved clinical questions such as aromatase inhibitor resistance.

Preincubation With Everolimus and Sirolimus Reduces Organic Anion-Transporting Polypeptide (OATP)1B1- and 1B3-Mediated Transport Independently of mTOR Kinase Inhibition: Implication in Assessing OATP1B1- and OATP1B3-Mediated Drug-Drug Interactions.

Organic anion transporting polypeptides (OATP)1B1 and OATP1B3 mediate hepatic uptake of many drugs including lipid-lowering statins. Current studies determined the OATP1B1/1B3-mediated drug-drug interaction (DDI) potential of mammalian target of rapamycin (mTOR) inhibitors, everolimus and sirolimus, using R-value and physiologically based pharmacokinetic models. Preincubation with everolimus and sirolimus significantly decreased OATP1B1/1B3-mediated transport even after washing and decreased inhibition constant values up to 8.3- and 2.9-fold for OATP1B1 and both 2.7-fold for OATP1B3, respectively. R-values of everolimus, but not sirolimus, were greater than the FDA-recommended cutoff value of 1.1. Physiologically based pharmacokinetic models predict that everolimus and sirolimus have low OATP1B1/1B3-mediated DDI potential against pravastatin. OATP1B1/1B3-mediated transport was not affected by preincubation with INK-128 (10 µM, 1 h), which does however abolish mTOR kinase activity. The preincubation effects of everolimus and sirolimus on OATP1B1/1B3-mediated transport were similar in cells before preincubation with vehicle control or INK-128, suggesting that inhibition of mTOR activity is not a prerequisite for the preincubation effects observed for everolimus and sirolimus. Nine potential phosphorylation sites of OATP1B1 were identified by phosphoproteomics; none of these are the predicted mTOR phosphorylation sites. We report the everolimus/sirolimus-preincubation-induced inhibitory effects on OATP1B1/1B3 and relatively low OATP1B1/1B3-mediated DDI potential of everolimus and sirolimus.

Reaction products of hexamethylene diisocyanate vapors with "self" molecules in the airways of rabbits exposed via tracheostomy.

1. Hexamethylenediisocyanate (HDI) is a widely used aliphatic diisocyanate and a well-recognized cause of occupational asthma. 2. "Self" molecules (peptides/proteins) in the lower airways, susceptible to chemical reactivity with HDI, have been hypothesized to play a role in asthma pathogenesis and/or chemical metabolism, but remain poorly characterized. 3. This study employed unique approaches to identify and characterize "self" targets of HDI reactivity in the lower airways. Anesthetized rabbits free breathed through a tracheostomy tube connected to chambers containing either, O2, or O2 plus ∼200 ppb HDI vapors. Following 60 minutes of exposure, the airways were lavaged and the fluid was analyzed by LC-MS and LC-MS/MS. 4. The low-molecular weight (<3 kDa) fraction of HDI exposed, but not control rabbit bronchoalveolar lavage (BAL) fluid identified 783.26 and 476.18 m/z [M+H]+ ions with high energy collision-induced dissociation (HCD) fragmentation patterns consistent with bis glutathione (GSH)-HDI and mono(GSH)-HDI. Proteomic analyses of the high molecular weight (>3 kDa) fraction of exposed rabbit BAL fluid identified HDI modification of specific lysines in uteroglobin (aka clara cell protein) and albumin. 5. In summary, this study utilized a unique approach to chemical vapor exposure in rabbits, to identify HDI reaction products with "self" molecules in the lower airways.

A multiregional proteomic survey of the postnatal human brain.

Detailed observations of transcriptional, translational and post-translational events in the human brain are essential to improving our understanding of its development, function and vulnerability to disease. Here, we exploited label-free quantitative tandem mass-spectrometry to create an in-depth proteomic survey of regions of the postnatal human brain, ranging in age from early infancy to adulthood. Integration of protein data with existing matched whole-transcriptome sequencing (RNA-seq) from the BrainSpan project revealed varied patterns of protein-RNA relationships, with generally increased magnitudes of protein abundance differences between brain regions compared to RNA. Many of the differences amplified in protein data were reflective of cytoarchitectural and functional variation between brain regions. Comparing structurally similar cortical regions revealed significant differences in the abundances of receptor-associated and resident plasma membrane proteins that were not readily observed in the RNA expression data.

USP7-Specific Inhibitors Target and Modify the Enzyme's Active Site via Distinct Chemical Mechanisms.

USP7 is a deubiquitinating enzyme that plays a pivotal role in multiple oncogenic pathways and therefore is a desirable target for new anti-cancer therapies. However, the lack of structural information about the USP7-inhibitor interactions has been a critical gap in the development of potent inhibitors. USP7 is unique among USPs in that its active site is catalytically incompetent, and is postulated to rearrange into a productive conformation only upon binding to ubiquitin. Surprisingly, we found that ubiquitin alone does not induce an active conformation in solution. Using a combination of nuclear magnetic resonance, mass spectrometry, computational modeling, and cell-based assays, we found that DUB inhibitors P22077 and P50429 covalently modify the catalytic cysteine of USP7 and induce a conformational switch in the enzyme associated with active site rearrangement. This work represents the first experimental insights into USP7 activation and inhibition and provides a structural basis for rational development of potent anti-cancer therapeutics.

Reciprocal regulation of ARPP-16 by PKA and MAST3 kinases provides a cAMP-regulated switch in protein phosphatase 2A inhibition.

ARPP-16, ARPP-19, and ENSA are inhibitors of protein phosphatase PP2A. ARPP-19 and ENSA phosphorylated by Greatwall kinase inhibit PP2A during mitosis. ARPP-16 is expressed in striatal neurons where basal phosphorylation by MAST3 kinase inhibits PP2A and regulates key components of striatal signaling. The ARPP-16/19 proteins were discovered as substrates for PKA, but the function of PKA phosphorylation is unknown. We find that phosphorylation by PKA or MAST3 mutually suppresses the ability of the other kinase to act on ARPP-16. Phosphorylation by PKA also acts to prevent inhibition of PP2A by ARPP-16 phosphorylated by MAST3. Moreover, PKA phosphorylates MAST3 at multiple sites resulting in its inhibition. Mathematical modeling highlights the role of these three regulatory interactions to create a switch-like response to cAMP. Together, the results suggest a complex antagonistic interplay between the control of ARPP-16 by MAST3 and PKA that creates a mechanism whereby cAMP mediates PP2A disinhibition.

Netrin-1 Regulates Fibrocyte Accumulation in the Decellularized Fibrotic Sclerodermatous Lung Microenvironment and in Bleomycin-Induced Pulmonary Fibrosis.

OBJECTIVE: Fibrocytes are collagen-producing leukocytes that accumulate in patients with systemic sclerosis (SSc; scleroderma)-related interstitial lung disease (ILD) via unknown mechanisms that have been associated with altered expression of neuroimmune proteins. The extracellular matrix (ECM) influences cellular phenotypes. However, a relationship between the lung ECM and fibrocytes in SSc has not been explored. The aim of this study was to use a novel translational platform based on decellularized human lungs to determine whether the lung ECM of patients with scleroderma controls the development of fibrocytes from peripheral blood mononuclear cells. METHODS: We performed biomechanical evaluation of decellularized scaffolds prepared from lung explants from healthy control subjects and patients with scleroderma, using tensile testing and biochemical and proteomic analysis. Cells obtained from healthy controls and patients with SSc-related ILD were cultured on these scaffolds, and CD45+pro-ColIα1+ cells meeting the criteria for fibrocytes were quantified. The contribution of the neuromolecule netrin-1 to fibrosis was assessed using neutralizing antibodies in this system and by administering bleomycin via inhalation to netrin-1(+/-) mice. RESULTS: Compared with control lung scaffolds, lung scaffolds from patients with SSc-related ILD showed aberrant anatomy, enhanced stiffness, and abnormal ECM composition. Culture of control cells in lung scaffolds from patients with SSc-related ILD increased production of pro-ColIα1+ cells, which was stimulated by enhanced stiffness and abnormal ECM composition. Cells from patients with SSc-related ILD demonstrated increased pro-ColIα1 responsiveness to lung scaffolds from scleroderma patients but not enhanced stiffness. Enhanced detection of netrin-1-expressing CD14(low) cells in patients with SSc-related ILD was observed, and antibody-mediated netrin-1 neutralization attenuated detection of CD45+pro-ColIα1+ cells in all settings. Netrin-1(+/-) mice were protected against bleomycin-induced lung fibrosis and fibrocyte accumulation. CONCLUSION: Factors present in the lung matrices of patients with scleroderma regulate fibrocyte accumulation via a netrin-1-dependent pathway. Netrin-1 regulates bleomycin-induced pulmonary fibrosis in mice. Netrin-1 might be a novel therapeutic target in SSc-related ILD.

MAP4K4 Is a Threonine Kinase That Phosphorylates FARP1.

Mitogen-activated protein kinase 4 (MAP4K4) regulates the MEK kinase cascade and is implicated in cytoskeletal rearrangement and migration; however, identifying MAP4K4 substrates has remained a challenge. To ascertain MAP4K4-dependent phosphorylation events, we combined phosphoproteomic studies of MAP4K4 inhibition with in vitro assessment of its kinase specificity. We identified 235 phosphosites affected by MAP4K4 inhibition in cells and found that pTP and pSP motifs were predominant among them. In contrast, in vitro assessment of kinase specificity showed that MAP4K4 favors a pTL motif. We showed that MAP4K4 directly phosphorylates and coimmunoprecipitates with FERM, RhoGEF, and pleckstrin domain-containing protein 1 (FARP1). MAP4K4 inhibition in SH-SY5Y cells increases neurite outgrowth, a process known to involve FARP1. As FARP1 and MAP4K4 both contribute to cytoskeletal rearrangement, the results suggest that MAP4K4 exerts some of its effects on the cytoskeleton via phosphorylation of FARP1.

Inhibitor of the tyrosine phosphatase STEP reverses cognitive deficits in a mouse model of Alzheimer's disease.

STEP (STriatal-Enriched protein tyrosine Phosphatase) is a neuron-specific phosphatase that regulates N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking, as well as ERK1/2, p38, Fyn, and Pyk2 activity. STEP is overactive in several neuropsychiatric and neurodegenerative disorders, including Alzheimer's disease (AD). The increase in STEP activity likely disrupts synaptic function and contributes to the cognitive deficits in AD. AD mice lacking STEP have restored levels of glutamate receptors on synaptosomal membranes and improved cognitive function, results that suggest STEP as a novel therapeutic target for AD. Here we describe the first large-scale effort to identify and characterize small-molecule STEP inhibitors. We identified the benzopentathiepin 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (known as TC-2153) as an inhibitor of STEP with an IC50 of 24.6 nM. TC-2153 represents a novel class of PTP inhibitors based upon a cyclic polysulfide pharmacophore that forms a reversible covalent bond with the catalytic cysteine in STEP. In cell-based secondary assays, TC-2153 increased tyrosine phosphorylation of STEP substrates ERK1/2, Pyk2, and GluN2B, and exhibited no toxicity in cortical cultures. Validation and specificity experiments performed in wild-type (WT) and STEP knockout (KO) cortical cells and in vivo in WT and STEP KO mice suggest specificity of inhibitors towards STEP compared to highly homologous tyrosine phosphatases. Furthermore, TC-2153 improved cognitive function in several cognitive tasks in 6- and 12-mo-old triple transgenic AD (3xTg-AD) mice, with no change in beta amyloid and phospho-tau levels.