Hsp90 picks PIKKs via R2TP and Tel2

Phosphatidylinositol-3 kinase-like kinases (PIKKs) are dependent on Hsp90 for their activation via the R2TP complex and Tel2. In this issue of Structure, Pal and colleagues present the molecular mechanism by which PIKKs are recruited to Hsp90

A hypomorphic allele of ZAP-70 reveals a distinct thymic threshold for autoimmune disease versus autoimmune reactivity

ZAP-70 is critical for T cell receptor (TCR) signaling. Tyrosine to phenylalanine mutations of Y315 and Y319 in ZAP-70 suggest these residues function to recruit downstream effector molecules, but mutagenesis and crystallization studies reveal that these residues also play an important role in autoinhibition ZAP-70. To address the importance of the scaffolding function, we generated a zap70 mutant mouse (YYAA mouse) with Y315 and Y319 both mutated to alanines. These YYAA mice reveal that the scaffolding function is important for normal development and function. Moreover, the YYAA mice have many similarities to a previously identified ZAP-70 mutant mouse, SKG, which harbors a distinct hypomorphic mutation. Both YYAA and SKG mice have impaired T cell development and hyporesponsiveness to TCR stimulation, markedly reduced numbers of thymic T regulatory cells and defective positive and negative selection. YYAA mice, like SKG mice, develop rheumatoid factor antibodies, but fail to develop autoimmune arthritis. Signaling differences that result from ZAP-70 mutations appear to skew the TCR repertoire in ways that differentially influence propensity to autoimmunity versus autoimmune disease susceptibility. By uncoupling the relative contribution from T regulatory cells and TCR repertoire during thymic selection, our data help to identify events that may be important, but alone are insufficient, for the development of autoimmune disease

DNA resection in eukaryotes: deciding how to fix the break

DNA double-strand breaks are repaired by different mechanisms, including homologous recombination and nonhomologous end-joining. DNA-end resection, the first step in recombination, is a key step that contributes to the choice of DSB repair. Resection, an evolutionarily conserved process that generates single-stranded DNA, is linked to checkpoint activation and is critical for survival. Failure to regulate and execute this process results in defective recombination and can contribute to human disease. Here, I review recent findings on the mechanisms of resection in eukaryotes, from yeast to vertebrates, provide insights into the regulatory strategies that control it, and highlight the consequences of both its impairment and its deregulation

Structural basis for phosphorylation-dependent recruitment of tel2 to hsp90 by pih1

Client protein recruitment to the Hsp90 system depends on cochaperones that bind the client and Hsp90 simultaneously and facilitate their interaction. Hsp90 involvement in the assembly of snoRNPs, RNA polymerases, PI3-kinase-like kinases, and chromatin remodeling complexes depends on the TTT (Tel2-Tti1-Tti2), and R2TP complexes-consisting of the AAA-ATPases Rvb1 and Rvb2, Tah1 (Spagh/RPAP3 in metazoa), and Pih1 (Pih1D1 in humans)-that together provide the connection to Hsp90. The biochemistry underlying R2TP function is still poorly understood. Pih1 in particular, at the heart of the complex, has not been described at a structural level, nor have the multiple protein-protein interactions it mediates been characterized. Here we present a structural and biochemical analysis of Hsp90-Tah1-Pih1, Hsp90-Spagh, and Pih1D1-Tel2 complexes that reveal a domain in Pih1D1 specific for binding CK2 phosphorylation sites, and together define the structural basis by which the R2TP complex connects the Hsp90 chaperone system to the TTT complex

The Complete Spectrum of Yeast Chromosome Instability Genes Identifies Candidate CIN Cancer Genes and Functional Roles for ASTRA Complex Components

Chromosome instability (CIN) is observed in most solid tumors and is linked to somatic mutations in genome integrity maintenance genes. The spectrum of mutations that cause CIN is only partly known and it is not possible to predict a priori all pathways whose disruption might lead to CIN. To address this issue, we generated a catalogue of CIN genes and pathways by screening ∼2,000 reduction-of-function alleles for 90% of essential genes in Saccharomyces cerevisiae. Integrating this with published CIN phenotypes for other yeast genes generated a systematic CIN gene dataset comprised of 692 genes. Enriched gene ontology terms defined cellular CIN pathways that, together with sequence orthologs, created a list of human CIN candidate genes, which we cross-referenced to published somatic mutation databases revealing hundreds of mutated CIN candidate genes. Characterization of some poorly characterized CIN genes revealed short telomeres in mutants of the ASTRA/TTT components TTI1 and ASA1. High-throughput phenotypic profiling links ASA1 to TTT (Tel2-Tti1-Tti2) complex function and to TORC1 signaling via Tor1p stability, consistent with the role of TTT in PI3-kinase related kinase biogenesis. The comprehensive CIN gene list presented here in principle comprises all conserved eukaryotic genome integrity pathways. Deriving human CIN candidate genes from the list allows direct cross-referencing with tumor mutational data and thus candidate mutations potentially driving CIN in tumors. Overall, the CIN gene spectrum reveals new chromosome biology and will help us to understand CIN phenotypes in human disease

IGSF4 is a novel TCR ζ-chain–interacting protein that enhances TCR-mediated signaling

The expression of cell surface molecule, IGSF4, on human and mouse T cells associate with the TCR ζ-chain and colocalizes to the c-SMAC to enhance cytokine production and adhesion to APCs

Identity of the elusive IgM Fc receptor (FcμR) in humans

Although Fc receptors (FcRs) for switched immunoglobulin (Ig) isotypes have been extensively characterized, FcR for IgM (FcμR) has defied identification. By retroviral expression and functional cloning, we have identified a complementary DNA (cDNA) encoding a bona fide FcμR in human B-lineage cDNA libraries. FcμR is defined as a transmembrane sialoglycoprotein of ∼60 kD, which contains an extracellular Ig-like domain homologous to two other IgM-binding receptors (polymeric Ig receptor and Fcα/μR) but exhibits an exclusive Fcμ-binding specificity. The cytoplasmic tail of FcμR contains conserved Ser and Tyr residues, but none of the Tyr residues match the immunoreceptor tyrosine-based activation, inhibitory, or switch motifs. Unlike other FcRs, the major cell types expressing FcμR are adaptive immune cells, including B and T lymphocytes. After antigen-receptor ligation or phorbol myristate acetate stimulation, FcμR expression was up-regulated on B cells but was down-modulated on T cells, suggesting differential regulation of FcμR expression during B and T cell activation. Although this receptor was initially designated as Fas apoptotic inhibitory molecule 3, or TOSO, our results indicate that FcμR per se has no inhibitory activity in Fas-mediated apoptosis and that such inhibition is only achieved when anti-Fas antibody of an IgM but not IgG isotype is used for inducing apoptosis

The molecular basis of endolytic activity of a multidomain alginate lyase from Defluviitalea phaphyphila, a representative of a new lyase family, PL39

Alginate is a polymer containing two uronic acid epimers, β-d-mannuronate (M) and α-l-guluronate (G), and is a major component of brown seaweed that is depolymerized by alginate lyases. These enzymes have diverse specificity, cleaving the chain with endo- or exotype activity and with differential selectivity for the sequence of M or G at the cleavage site. Dp0100 is a 201-kDa multi-modular, broad-specificity endotype alginate lyase from the marine thermophile Defluviitalea phaphyphila, which uses brown algae as a carbon source, converting it to ethanol, and bioinformatics analysis suggested that its catalytic domain represents a new polysaccharide lyase family, PLxx. The structure of the Dp0100 catalytic domain, determined at 2.07 Å resolution, revealed that it comprises three regions strongly resembling those of the exotype lyase families PL15 and PL17. The conservation of key catalytic histidine and tyrosine residues belonging to the latter suggest these enzymes share mechanistic similarities. A complex of Dp0100 with a pentasaccharide, M5, showed that the oligosaccharide is located in subsites -2, -1, +1, +2, and +3 in a long, deep canyon open at both ends, explaining the endotype activity of this lyase. This contrasted with the hindered binding sites of the exotype enzymes, which are blocked such that only one sugar moiety can be accommodated at the -1 position in the catalytic site. The biochemical and structural analyses of Dp0100, the first for this new class of endotype alginate lyases, has furthered our understanding of the structure-function and evolutionary relationships within this important class of enzymes