Phosphotyrosine Binding Domain Research Articles

Anti-CRISPR AcrIIC5 is a dsDNA mimic that inhibits type II-C Cas9 effectors by blocking PAM recognition.

Anti-CRISPR proteins are encoded by phages to inhibit the CRISPR-Cas systems of the hosts. AcrIIC5 inhibits several naturally high-fidelity type II-C Cas9 enzymes, including orthologs from Neisseria meningitidis (Nme1Cas9) and Simonsiella muelleri (SmuCas9). Here, we solve the structure of AcrIIC5 in complex with Nme1Cas9 and sgRNA. We show that AcrIIC5 adopts a novel fold to mimic the size and charge distribution of double-stranded DNA, and uses its negatively charged grooves to bind and occlude the protospacer adjacent motif (PAM) binding site in the target DNA cleft of Cas9. AcrIIC5 is positioned into the crevice between the WED and PI domains of Cas9, and one end of the anti-CRISPR interacts with the phosphate lock loop and a linker between the RuvC and BH domains. We employ biochemical and mutational analyses to build a model for AcrIIC5's mechanism of action, and identify residues on both the anti-CRISPR and Cas9 that are important for their interaction and inhibition. Together, the structure and mechanism of AcrIIC5 reveal convergent evolution among disparate anti-CRISPR proteins that use a DNA-mimic strategy to inhibit diverse CRISPR-Cas surveillance complexes, and provide new insights into a tool for potent inhibition of type II-C Cas9 orthologs.

Full-Length Model of SaCas9-sgRNA-DNA Complex in Cleavage State.

Staphylococcus aureus Cas9 (SaCas9) is a widely used genome editing tool. Understanding its molecular mechanisms of DNA cleavage could effectively guide the engineering optimization of this system. Here, we determined the first cryo-electron microscopy structure of the SaCas9-sgRNA-DNA ternary complex. This structure reveals that the HNH nuclease domain is tightly bound to the cleavage site of the target DNA strand, and is in close contact with the WED and REC domains. Moreover, it captures the complete structure of the sgRNA, including the previously unresolved stem-loop 2. Based on this structure, we build a full-length model for the ternary complex in cleavage state. This model enables identification of the residues for the interactions between the HNH domain and the WED and REC domains. Moreover, we found that the stem-loop 2 of the sgRNA tightly binds to the PI and RuvC domains and may also regulate the position shift of the RuvC domain. Further mutagenesis and molecular dynamics simulations supported the idea that the interactions of the HNH domain with the WED and REC domains play an important role in the DNA cleavage. Thus, this study provides new mechanistic insights into the DNA cleavage of SaCas9 and is also useful for guiding the future engineering of SaCas9-mediated gene editing systems.

Discovery of a small molecule ligand of FRS2 that inhibits invasion and tumor growth

PurposeAberrant activation of the fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases drives oncogenic signaling through its proximal adaptor protein FRS2. Precise disruption of this disease-causing signal transmission in metastatic cancers could stall tumor growth and progression. The purpose of this study was to identify a small molecule ligand of FRS2 to interrupt oncogenic signal transmission from activated FGFRs.MethodsWe used pharmacophore-based computational screening to identify potential small molecule ligands of the PTB domain of FRS2, which couples FRS2 to FGFRs. We confirmed PTB domain binding of molecules identified with biophysical binding assays and validated compound activity in cell-based functional assays in vitro and in an ovarian cancer model in vivo. We used thermal proteome profiling to identify potential off-targets of the lead compound.ResultsWe describe a small molecule ligand of the PTB domain of FRS2 that prevents FRS2 activation and interrupts FGFR signaling. This PTB-domain ligand displays on-target activity in cells and stalls FGFR-dependent matrix invasion in various cancer models. The small molecule ligand is detectable in the serum of mice at the effective concentration for prolonged time and reduces growth of the ovarian cancer model in vivo. Using thermal proteome profiling, we furthermore identified potential off-targets of the lead compound that will guide further compound refinement and drug development.ConclusionsOur results illustrate a phenotype-guided drug discovery strategy that identified a novel mechanism to repress FGFR-driven invasiveness and growth in human cancers. The here identified bioactive leads targeting FGF signaling and cell dissemination provide a novel structural basis for further development as a tumor agnostic strategy to repress FGFR- and FRS2-driven tumors.

Insulin/IGF signaling regulates presynaptic glutamate release in aversive olfactory learning.

Insulin/insulin-like growth factor (IGF) receptor signaling (IIS) supports context-dependent learning in vertebrates and invertebrates. Here, we identify cell-specific mechanisms of IIS that integrate sensory information with food context to drive synaptic plasticity and learning. In the nematode Caenorhabditis elegans, pairing food deprivation with an odor such as butanone suppresses attraction to that odor. We find that aversive olfactory learning requires the insulin receptor substrate (IRS) protein IST-1 and atypical signaling through the insulin/IGF-1 receptor DAF-2. Cell-specific knockout and rescue demonstrate that DAF-2 acts in the AWCON sensory neuron, which detects butanone, and that learning preferentially depends upon the axonally localized DAF-2c isoform. Acute food deprivation increases DAF-2 levels in AWCON post-transcriptionally through an insulin- and insulin receptor substrate-1 (ist-1)-dependent process. Aversive learning alters the synaptic output of AWCON by suppressing odor-regulated glutamate release in wild-type animals, but not in ist-1 mutants, suggesting that axonal insulin signaling regulates synaptic transmission to support aversive memory.

Aberrant protein expression of Appl1, Sortilin and Syndecan-1 during the biological progression of prostate cancer

Diagnosis and assessment of patients with prostate cancer is dependent on accurate interpretation and grading of histopathology. However, morphology does not necessarily reflect the complex biological changes occurring in prostate cancer disease progression, and current biomarkers have demonstrated limited clinical utility in patient assessment. This study aimed to develop biomarkers that accurately define prostate cancer biology by distinguishing specific pathological features that enable reliable interpretation of pathology for accurate Gleason grading of patients. Online gene expression databases were interrogated and a pathogenic pathway for prostate cancer was identified. The protein expression of key genes in the pathway, including adaptor protein containing a pleckstrin homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif 1 (Appl1), Sortilin and Syndecan-1, was examined by immunohistochemistry (IHC) in a pilot study of 29 patients with prostate cancer, using monoclonal antibodies designed against unique epitopes. Appl1, Sortilin, and Syndecan-1 expression was first assessed in a tissue microarray cohort of 112 patient samples, demonstrating that the monoclonal antibodies clearly illustrate gland morphologies. To determine the impact of a novel IHC-assisted interpretation (the utility of Appl1, Sortilin, and Syndecan-1 labelling as a panel) of Gleason grading, versus standard haematoxylin and eosin (H&E) Gleason grade assignment, a radical prostatectomy sample cohort comprising 114 patients was assessed. In comparison to H&E, the utility of the biomarker panel reduced subjectivity in interpretation of prostate cancer tissue morphology and improved the reliability of pathology assessment, resulting in Gleason grade redistribution for 41% of patient samples. Importantly, for equivocal IHC-assisted labelling and H&E staining results, the cancer morphology interpretation could be more accurately applied upon re-review of the H&E tissue sections. This study addresses a key issue in the field of prostate cancer pathology by presenting a novel combination of three biomarkers and has the potential to transform clinical pathology practice by standardising the interpretation of the tissue morphology.

TBIO-08. The molecular basis for rational targeting of FGFR-driven growth and invasiveness in pediatric brain tumors

Abstract The oncogenic activation of receptor tyrosine kinases (RTK) promotes growth, survival and dissemination in pediatric tumors including glioma, ependymoma and medulloblastoma (MB). Direct targeting of either the RTK or of downstream kinases can effectively block tumor promoting pathway functions. However, emergence of resistance is common. We hypothesized that alternative interference strategies that target protein-protein interactions (PPIs) instead of enzymatic activities could overcome the emergence of resistance. We characterized the molecular interactions downstream of the FGFR that regulate relevant growth and invasion-promoting mechanisms in MB cells, to identify potentially druggable PPIs. We found that the FRS2 protein is an essential up-stream effector of FGFR signaling towards invasiveness. Using a proteomics approach, we furthermore identified the Striatin 3 protein as a novel oncogenic effector of the FGFR pathway downstream of FRS2, as it integrates antagonistic growth and invasion signals downstream of FGFR. Mechanistically, Striatin 3 interacts with the Ser/Thr kinase MAP4K4, couples it to the protein phosphatase 2A, and thereby inactivates growth repressing activities of MAP4K4. In parallel, Striatin 3 enables MAP4K4-mediated phosphorylation of PKC-theta and VASP, which combined are necessary to promote tissue invasion. To selectively repress pro-invasive FGFR functions, we identified and functionally validated small molecule ligands of FRS2, that prevent FRS2 activation and downstream signaling. We demonstrate efficacy of these compounds in inhibiting invasion and growth promoting activities in vitro and in vivo, and identified potential off-target activities of the ligand using a proteome-wide interaction analysis. We propose inhibition of FRS2 by a small molecular PTB domain ligand as a strategy to repress FGF signaling in FGFR-driven tumors. The development of this ligand, and the de novo design of functional analogs thereof bear promise for further pre-clinical evaluation of these structures as anti-growth promoting and anti-metastatic therapeutics applicable to FGFR-driven tumors.

FER-mediated phosphorylation and PIK3R2 recruitment on IRS4 promotes AKT activation and tumorigenesis in ovarian cancer cells.

Tyrosine phosphorylation, orchestrated by tyrosine kinases and phosphatases, modulates a multi-layered signaling network in a time- and space-dependent manner. Dysregulation of this post-translational modification is inevitably associated with pathological diseases. Our previous work has demonstrated that non-receptor tyrosine kinase FER is upregulated in ovarian cancer, knocking down which attenuates metastatic phenotypes. However, due to the limited number of known substrates in the ovarian cancer context, the molecular basis for its pro-proliferation activity remains enigmatic. Here, we employed mass spectrometry and biochemical approaches to identify insulin receptor substrate 4 (IRS4) as a novel substrate of FER. FER engaged its kinase domain to associate with the PH and PTB domains of IRS4. Using a proximity-based tagging system in ovarian carcinoma-derived OVCAR-5 cells, we determined that FER-mediated phosphorylation of Tyr779 enables IRS4 to recruit PIK3R2/p85β, the regulatory subunit of PI3K, and activate the PI3K-AKT pathway. Rescuing IRS4-null ovarian tumor cells with phosphorylation-defective mutant, but not WT IRS4 delayed ovarian tumor cell proliferation both in vitro and in vivo. Overall, we revealed a kinase-substrate mode between FER and IRS4, and the pharmacological inhibition of FER kinase may be beneficial for ovarian cancer patients with PI3K-AKT hyperactivation.

Pro-oxidant vitamin C mechanistically exploits p66Shc/Rac1 GTPase pathway in inducing cytotoxicity.

P66Shc is the master regulator of oxidative stress whose pro-oxidant functioning is governed by ser36 phosphorylation. Phosphorylated p66Shc via Rac1 GTPase activation modulates ROS levels which in turn influence its pro-oxidative functions. Vitamin C at higher concentrations exhibits cytotoxic activity in various cancers, inducing ROS mediated cell death via pro-apoptotic mechanisms. Here we show a novel role of p66Shc in mediating pro-oxidant activity of vitamin C. Effect of vitamin C on the viability of breast cancer and normal cells was studied. High doses of vitamin C decreased viability of cancerous cells but not normal cells. Docking study displayed significant binding affinity of vitamin C with p66Shc PTB domain. Western blot results suggest that vitamin C not only enhances p66Shc expression but also induces its ser36 phosphorylation. Vitamin C at high doses was also found to activate Rac1, enhance ROS production and induce apoptosis. Interestingly, ser36 phosphorylation mutant transfection and pretreatment with antioxidant N-acetylcysteine results indicate that vitamin C induced Rac1 activation, ROS production and apoptosis is p66Shc ser36 phosphorylation dependent. Overall, results highlight that vitamin C mechanistically explores p66Shc/Rac1 pathway in inducing apoptosis and thus can pave a way to use this pathway as a potential therapeutic target in breast cancers.

Exercise ameliorates high-fat diet-induced insulin resistance accompanied by changes in protein levels of hepatic ATF3-related signaling in rats

PurposeExercise is an effective way to alleviate insulin resistance (IR). However, the underlying mechanisms remain to be elucidated. Previous studies demonstrated that cardiolipin synthase 1 (CRLS1)/interferon-regulatory factor-2 binding protein 2 (IRF2bp2)-activating transcription factor 3 (ATF3)-adiponectin receptor 2 (AdipoR2)-adaptor protein containing pH domain, PTB domain and leucine zipper motif 1 (APPL1)-protein kinase B (AKT/PKB)-related signaling was closely associated with obesity-induced IR-related diseases, but the correlation between exercise training alleviating obesity-induced IR and the protein levels of hepatic CRLS1/IRF2bp2-ATF3-AdipoR2-APPL1-AKT-related signaling in rats is unknown. Therefore, We want to investigate the effect of exercise training on IR and the protein levels of hepatic CRLS1/IRF2bp2-ATF3-AdipoR2-APPL1-AKT-related signaling in rat. MethodsThe male healthy Sprague-Dawley rats were divided into four groups: normal control group (NCG, n = 10), diet-induced obesity-sedentary group (DIO-SG, n = 10), diet-induced obesity-chronic exercise group (DIOCEG, n = 10) received chronic swim exercise training and diet-induced obesity-acute exercise group (DIO-AEG, n = 10) received acute swim exercise training. We measured the levels of IR-related indicators and the protein levels of hepatic CRLS1/IRF2bp2-ATF3-AdipoR2-APPL1-AKT-related signaling in NCG, DIO-SG, DIOCEG and DIO-AEG. ResultsWe found that high-fat diet (HFD)-induced obesity decreased insulin sensitivity in rats accompanied by decreased protein levels of hepatic CRLS1, IRF2bp2, AdipoR2, APPL1, p-AKT and increased protein level of hepatic ATF3. The acute exercise and the chronic exercise both increased insulin sensitivity in rats. The chronic exercise decreased hepatic ATF3 protein level and increased CRLS1, IRF2bp2, AdipoR2, APPL1, p-AKT protein levels in HFD-fed rats. The acute exercise decreased hepatic ATF3 protein level and increased hepatic IRF2bp2, APPL1 and p-AKT protein levels in HFD-fed rats. The acute exercise had no significant effect on hepatic CRLS1 and AdipoR2 protein levels in HFD-fed rats. ConclusionOur current findings indicated that exercise alleviated obesity-induced IR accompanied by changes in protein levels of hepatic ATF3-related signaling in rats. Our results are meaningful for exploring the molecular mechanism of exercise alleviating IR symptoms.

In-silico analysis of nonsynonymous genomic variants within CCM2 gene reaffirm the existence of dual cores within typical PTB domain

PurposeThe objective of this study is to validate the existence of dual cores within the typical phosphotyrosine binding (PTB) domain and to identify potentially damaging and pathogenic nonsynonymous coding single nuclear polymorphisms (nsSNPs) in the canonical PTB domain of the CCM2 gene that causes cerebral cavernous malformations (CCMs). MethodsThe nsSNPs within the coding sequence for PTB domain of human CCM2 gene, retrieved from exclusive database searches, were analyzed for their functional and structural impact using a series of bioinformatic tools. The effects of mutations on the tertiary structure of the PTB domain in human CCM2 protein were predicted to examine the effect of nsSNPs on the tertiary structure of PTB Cores. ResultsOur mutation analysis, through alignment of protein structures between wildtype CCM2 and mutant, predicted that the structural impacts of pathogenic nsSNPs is biophysically limited to only the spatially adjacent substituted amino acid site with minimal structural influence on the adjacent core of the PTB domain, suggesting both cores are independently functional and essential for proper CCM2 PTB function. ConclusionUtilizing a combination of protein conservation and structure-based analysis, we analyzed the structural effects of inherited pathogenic mutations within the CCM2 PTB domain. Our results predicted that the pathogenic amino acid substitutions lead to only subtle changes locally, confined to the surrounding tertiary structure of the PTB core within which it resides, while no structural disturbance to the neighboring PTB core was observed, reaffirming the presence of independently functional dual cores in the CCM2 typical PTB domain.

Contribution of protein-protein interactions to the endothelial-barrier-stabilizing function of KRIT1.

ABSTRACTKrev-interaction trapped protein 1 (KRIT1) is an endothelial scaffold protein that promotes adherens junction (AJ) stability. The precise mechanism by which KRIT1 promotes barrier stabilization is unclear. We tested the ability of a panel of KRIT1 constructs containing mutations that inhibit Rap1 binding, ICAP1α binding, disrupt KRIT1's phosphotyrosine-binding (PTB) domain, or direct KRIT1 to the plasma membrane, either alone or in combination, to restore barrier function in KRIT1-deficient endothelial cells. We found that ablating the 192NPAY195 motif or disrupting the PTB domain was sufficient to restore AJ protein localization and barrier function to control levels, irrespective of the junctional localization of KRIT1 or Rap1 binding. The ability of our KRIT1 constructs to rescue AJ and barrier function in KRIT1-depleted endothelial cells correlated with decreased β1 integrin activity and maintenance of cortical actin fibers. Taken together, our findings indicate that Rap1 binding, ICAP1α binding and junctional localization are not required for the ability of KRIT1 to stabilize endothelial contacts, and suggest that the ability of KRIT1 to limit integrin activity could be involved in barrier stabilization.

Impact of metabolic disorders on endometrial receptivity in patients with polycystic ovary syndrome.

The present study investigated the expression of endometrial receptivity-related molecules in patients with polycystic ovary syndrome (PCOS) and different androgen status, insulin resistance (IR) levels, and body mass indexes (BMI) to identify the mechanism underlying their effects on pregnancy outcomes. The present study recruited 43 participants from November 2020 to January 2021, which were classified into five groups: i) Hyperandrogenemia (HA) combined with impaired glucose tolerance group (n=8); ii) HA combined with diabetes mellitus group (n=8); iii) HA combined with non-IR (NIR) group (n=10); iv) non-HA (NHA) androgen combined with IR group (n=8); and v) NHA combined with NIR group (n=9). In addition, according to their BMIs, patients were sub-grouped into lean/normal (n=27), overweight (n=8) or obese (n=8) groups. The mRNA expression levels of endometrial receptivity-related molecules were detected using reverse transcription-quantitative PCR. In addition, flow cytometry was used to determine the phenotype and percentage of uterine natural killer cells (uNK). According to the results, patients with PCOS and IR status, HA and obesity (BMI ≥24 kg/m2) demonstrated significantly decreased mRNA expression levels of adiponectin, adiponectin receptor (AdipoR)1, AdipoR2, adapter protein containing PH domain, PTB domain and leucine zipper motif 1, estrogen receptor (ER) α, ERβ, progesterone receptor (PR), IL-15, integrin β3 avβ3, and insulin-like growth factor binding protein-1, but increased mRNA expression levels of IL-6 and IL-8 compared with NHA + NIR group or lean/normal group, respectively. In addition, obese patients with PCOS demonstrated increased mRNA expression levels of PR compared with overweight patients. This suggested that insulin resistant status, HA, and obesity could alter the endometrial receptivity of patients with PCOS, which may explain poorer embryo implantation and pregnancy outcomes in clinics.

Identification of an Immunogenic Medulloblastoma-Specific Fusion Involving EPC2 and GULP1

Simple SummaryMedulloblastoma is the most common malignant childhood brain tumor and it is considered poor immunogenic because of its low mutational burden. Nevertheless, several clinical trials are currently evaluating immunotherapy for medulloblastoma patients, since new treatment strategies for this entity are a matter of utmost urgency. Tumor specific antigens resulting from gene fusions are potentially highly immunogenic. In our study, we identified a new medulloblastoma-specific fusion transcript EPC2-GULP1.The resulting protein sequence produced a neoantigen, which was able to activate CD8+ T cells. Thus, our data indicate an immunotherapeutic approach for pediatric medulloblastoma patients carrying the EPC2-GULP1 fusion or other fusions generating immunogenic neoantigens.Medulloblastoma is the most common malignant brain tumor in children. Immunotherapy is yet to demonstrate dramatic results in medulloblastoma, one reason being the low rate of mutations creating new antigens in this entity. In tumors with low mutational burden, gene fusions may represent a source of tumor-specific neoantigens. Here, we reviewed the landscape of fusions in medulloblastoma and analyzed their predicted immunogenicity. Furthermore, we described a new in-frame fusion protein identified by RNA-Seq. The fusion involved two genes on chromosome 2 coding for the enhancer of polycomb homolog 2 (EPC2) and GULP PTB domain containing engulfment adaptor 1 (GULP1) respectively. By qRT-PCR analysis, the fusion was detected in 3 out of 11 medulloblastoma samples, whereby 2 samples were from the same patients obtained at 2 different time points (initial diagnosis and relapse), but not in other pediatric brain tumor entities. Cloning of the full-length sequence indicated that the fusion protein contains the N-terminal enhancer of polycomb-like domain A (EPcA) of EPC2 and the coiled-coil domain of GULP1. In silico analyses predicted binding of the neoantigen-derived peptide to HLA-A*0201. A total of 50% of the fusions described in the literature were also predicted to produce an immunogenic peptide. The EPC2-GULP1 fusion peptide was able to induce a de novo T cell response characterized by interferon gamma release of CD8+ cytotoxic T cells in vitro. While the functional relevance of this fusion in medulloblastoma biology remains to be clarified, our data support an immunotherapeutic approach for pediatric medulloblastoma patients carrying the EPC2-GULP1 fusion and other immunogenic fusions.

Abstract 13589: The Mechano-Signaling Roles of the Adaptor Protein Nck1 in Atherogenic Endothelial Activation

Rational: Alterations in hemodynamic shear stress (SS) at atherosclerosis-prone sites promotes endothelial activation, characterized by nuclear factor-κB (NF-κB)-driven expression of cell adhesion molecules that mediate leukocyte homing. In addition, proinflammatory cytokines (e.g. IL-1β) promote NF-κB-dependent endothelial activation. While the recent CANTOS trial using an IL-1β antagonist highlights the potential for treating atherogenic inflammation beyond lipid lowering therapies alone, our understanding of how endothelial activation contributes to this effect remains limited. Methodology & Results: We identified the signaling adaptor Nck1 as a critical regulator of atherogenic endothelial activation. Endothelial cells deficient in Nck1 lack SS-induced NF-κB activation and proinflammatory gene expression. Additionally, we demonstrated an interaction between Nck1 and IL-1β signaling in response to SS, showing that SS activates the IL-1β receptor (IL1R1) signaling partner IRAK-1 in a Nck1-dependent manner both in vitro and in vivo . IRAK-1 depletion inhibited the proinflammatory response to SS. Mechanistically, point mutation analysis suggest a critical role for the Nck1 SH2 domain (phosphotyrosine binding domain) in mediating these effects. Consistent with our cell culture studies, Global Nck1 knockout mice show reduced atherosclerosis characterized by diminished macrophage and smooth muscle incorporation and bone marrow chimeras suggest non-hematopoietic Nck1 mediates this response. Conclusions: A recent GWAS analysis linked Nck1 to coronary artery disease, underscoring its importance to human disease. Therefore, our data suggest that Nck1 mediates the formation of IL-1-related signaling complexes to induce atherogenic endothelial activation, and selective inhibition of Nck1 or its critical domains will allow efficient inhibition of atherosclerosis in humans.

Abstract MP06: The Signaling Roles Of The Adaptor Protein Nck1 In Atherosclerosis

Rational: Alterations in hemodynamic shear stress (SS) at atherosclerosis-prone sites promotes endothelial activation, characterized by nuclear factor-κB (NF-κB)-driven expression of cell adhesion molecules that mediate leukocyte homing. In addition, proinflammatory cytokines (e.g. IL-1β) promote NF-κB-dependent endothelial activation. While the recent CANTOS trial using an IL-1β antagonist highlights the potential for treating atherogenic inflammation beyond lipid lowering therapies alone, our understanding of how endothelial activation contributes to this effect remains limited. Methodology & Results: We identified the signaling adaptor Nck1 as a critical regulator of atherogenic endothelial activation. Endothelial cells deficient in Nck1 lack SS-induced NF-κB activation and proinflammatory gene expression. Additionally, we demonstrated an interaction between Nck1 and IL-1β signaling in response to SS, showing that SS activates the IL-1β receptor (IL1R1) signaling partner IRAK-1 in a Nck1-dependent manner both in vitro and in vivo . Mechanistically, point mutation analysis suggest a critical role for the Nck1 SH2 domain (phosphotyrosine binding domain) in mediating these effects. Nck1 affinity pulling down and mass spectrometry identified multiple downstream effectors of IL-1 pathway. In vivo, Global Nck1 knockout mice show reduced atherosclerosis characterized by diminished macrophage and smooth muscle incorporation and bone marrow chimeras suggest non-hematopoietic Nck1 mediates this response. Conclusions: A recent GWAS analysis linked Nck1 to coronary artery disease, underscoring its importance to human disease. Therefore, our data suggest that Nck1 mediates the formation of IL-1-related signaling complexes to induce atherogenic endothelial activation, and selective inhibition of Nck1 or its critical domains will allow efficient inhibition of atherosclerosis in humans.

Flexible Nanoporous Materials by Matrix Removal from Cylinder-Forming Diblock Copolymers.

We describe a straightforward self-assembly route to nanoporous materials derived from a hexagonally-packed cylinder (HEX) morphology of a polyisoprene-block-polylactide (PI-b-PLA) diblock copolymer, by thermal cross-linking of the minority PI domains followed by selective chemical etching of the PLA matrix. The resulting mechanically stable and porous samples defy the expectation that the remaining cylinders cannot yield a robust, integrated material upon matrix removal. Scanning electron microscopy imaging reveals that this unexpected structural integrity stems from the interconnected nanofibrils therein, reflecting topological defects at the grain boundaries of the parent polydomain HEX nanostructure. Hydrodynamic radius-dependent poly(ethylene oxide) (Mn = 0.4-35 kg/mol) permeation behavior through these monoliths directly demonstrated the continuity and size selectivity of the nanoporous material. The ready accessibility of block copolymer HEX morphologies of varied chemistries suggests that this matrix etching strategy will enable the future design of functional, size-selective nanofiltration membrane materials.

The phosphoinositide code is read by a plethora of protein domains

Introduction The proteins that decipher nucleic acid- and protein-based information are well known, however, those that read membrane-encoded information remain understudied. Here we report 70 different human, microbial and viral protein folds that recognize phosphoinositides (PIs), comprising the readers of a vast membrane code. Areas covered Membrane recognition is best understood for FYVE, PH and PX domains, which exemplify hundreds of PI code readers. Comparable lipid interaction mechanisms may be mediated by kinases, adjacent C1 and C2 domains, trafficking arrestin, GAT and VHS modules, membrane-perturbing annexin, BAR, CHMP, ENTH, HEAT, syntaxin and Tubby helical bundles, multipurpose FERM, EH, MATH, PHD, PDZ, PROPPIN, PTB and SH2 domains, as well as systems that regulate receptors, GTPases and actin filaments, transfer lipids and assembled bacterial and viral particles. Expert opinion The elucidation of how membranes are recognized has extended the genetic code to the PI code. Novel discoveries include PIP-stop and MET-stop residues to which phosphates and metabolites are attached to block phosphatidylinositol phosphate (PIP) recognition, memteins as functional membrane protein apparatuses, and lipidons as lipid “codons” recognized by membrane readers. At least 5% of the human proteome senses such membrane signals and allows eukaryotic organelles and pathogens to operate and replicate.

Molecular Details of a Coupled Binding and Folding Reaction between the Amyloid Precursor Protein and a Folded Domain.

Intrinsically disordered regions in proteins often function as binding motifs in protein–protein interactions. The mechanistic aspects and molecular details of such coupled binding and folding reactions, which involve formation of multiple noncovalent bonds, have been broadly studied theoretically, but experimental data are scarce. Here, using a combination of protein semisynthesis to incorporate phosphorylated amino acids, backbone amide-to-ester modifications, side chain substitutions, and binding kinetics, we examined the interaction between the intrinsically disordered motif of amyloid precursor protein (APP) and the phosphotyrosine binding (PTB) domain of Mint2. We show that the interaction is regulated by a self-inhibitory segment of the PTB domain previously termed ARM. The helical ARM linker decreases the association rate constant 30-fold through a fast pre-equilibrium between an open and a closed state. Extensive side chain substitutions combined with kinetic experiments demonstrate that the rate-limiting transition state for the binding reaction is governed by native and non-native hydrophobic interactions and hydrogen bonds. Hydrophobic interactions were found to be particularly important during crossing of the transition state barrier. Furthermore, linear free energy relationships show that the overall coupled binding and folding reaction involves cooperative formation of interactions with roughly 30% native contacts formed at the transition state. Our data support an emerging picture of coupled binding and folding reactions following overall chemical principles similar to those of folding of globular protein domains but with greater malleability of ground and transition states.

A Novel Splice Site Variant in the LDLRAP1 Gene Causes Familial Hypercholesterolemia

Background:FH, a hereditary disorder, is caused by pathogenic variants in the LDLR, APOB, and PCSK9 genes. This study has assessed genetic variants in a family, clinically diagnosed with FH. Methods:A family was recruited from MASHAD study in Iran with possible FH based on the Simon Broom criteria. The DNA sample of an affected individual (proband) was analyzed using WES, followed by bioinformatics and segregation analyses. Results:A novel splice site variant (c.345-2A>G) was detected in the LDLRAP1 gene, which was segregated in all affected family members. Moreover, HMGCR rs3846662 g.23092A>G was found to be homozygous (G/G) in the proband, probably leading to reduced response to simvastatin and pravastatin. Conclusion:LDLRAP1 c.345-2A>G could alter the PTB, which acts as an important part of biological pathways related to lipid metabolism.

Describing pre-professional identity in higher education: A case study of exercise science students

Pre-professional identity (PPI) is an understanding of the skills, qualities, conduct, culture and ideology of a students’ intended profession. Understanding PPI is valuable for students and higher-education providers to provide insight into motivation for- and to promote engagement in- learning. Describing PPI is challenging, particularly for evolving health professions. This paper describes a process undertaken to understand PPI, using exercise science (ES), a new and evolving health profession, as a case study. Mixed methods were used to describe three aspects of PPI: 1) student cohort characteristics; 2) personal factors influencing PPI; and 3) perceived career direction. Final year ES students participated in an online survey and a focus group (n=305; 59% male; 75% age range of 20-24 years). Factors contributing to the development of PPI included the desire to help others; interest and experience in sports/exercise. Students had the strongest understanding of the PI domains of affiliation, money and structure and limited understanding of the role of scientist and researcher. Two outcomes were derived from the study findings to advance the theoretical understanding of PPI: 1) a 3-item framework that describes factors specific to students’ PPI; and 2) a worked case study demonstrating how this framework was applied to gain a nuanced understanding of PPI in ES. Our framework can be applied to increase student and higher education providers understanding of PPI and the motivations underpinning student decision-making in higher education.