Human WIPF1 blocking peptide

Nck is a ubiquitously expressed, primarily cytosolic adapter protein consisting of one SH2 domain and three SH3 domains. It links receptor and nonreceptor tyrosine kinases to actin cytoskeleton reorganizing proteins. In T lymphocytes, Nck is a crucial component of signaling pathways for T cell activation and effector function. It recruits actin remodeling proteins to T cell receptor (TCR)-associated activation clusters and thereby initiates changes in cell polarity and morphology. Moreover, Nck is crucial for the TCR-induced mobilization of secretory vesicles to the cytotoxic immunological synapse. To identify the interactome of Nck in human T cells, we performed a systematic screen for interaction partners in untreated or pervanadate-treated cells. We used GST fusion proteins containing full length Nck, the combined SH3 domains or the individual SH3 and SH2 domains to precipitate putative Nck interactors from cellular lysates. Protein bands were excised from gels, processed by tryptic in-gel digestion and analyzed by mass spectrometry. Using this approach, we confirmed previously established interactions (e.g., with Slp76, CD3(8), WASP, and WIPF1) and identified several novel putative Nck-binding proteins. We subsequently verified the SH2 domain binding to the actin-binding protein HIP55 and to FYB/ADAP, and the SH3-mediated binding to the nuclear proteins SFPQ/NONO. Using laser scanning microscopy, we provide new evidence for a nuclear localization of Nck in human T cells. Our data highlight the fundamental role of Nck in the TCR-to-cytoskeleton crosstalk and point to yet unknown nuclear functions of Nck also in T lymphocytes.

Inherited bone marrow failure (IBMF) syndromes are rare blood disorders characterized by hematopoietic cell dysfunction and predisposition to hematologic malignancies. Despite advances in the understanding of molecular pathogenesis of these heterogeneous diseases, genetic variant interpretation, genotype-phenotype correlation, and outcome prognostication remain difficult. As new IBMF and other myelodysplastic syndrome (MDS) predisposition genes continue to be discovered (frequently in small kindred studies), there is an increasing need for a systematic framework to evaluate penetrance and prevalence of mutations in genes associated with IBMF phenotypes. To address this need, we analyzed population-based genomic data from >125 000 individuals in the Genome Aggregation Database for loss-of-function (LoF) variants in 100 genes associated with IBMF. LoF variants in genes associated with IBMF/MDS were present in 0.426% of individuals. Heterozygous LoF variants in genes in which haploinsufficiency is associated with IBMF/MDS were identified in 0.422% of the population; homozygous LoF variants associated with autosomal recessive IBMF/MDS diseases were identified in only.004% of the cohort. Using age distribution of LoF variants and 2 measures of mutational constraint, LOEUF ("loss-of-function observed/expected upper bound fraction") and pLI ("probability of being loss-of-function intolerance"), we evaluated the pathogenicity, tolerance, and age-related penetrance of LoF mutations in specific genes associated with IBMF syndromes. This analysis led to insights into rare IBMF diseases, including syndromes associated with DHX34, MDM4, RAD51, SRP54, and WIPF1. Our results provide an important population-based framework for the interpretation of LoF variant pathogenicity in rare and emerging IBMF syndromes.