Exome sequencing and functional analyses revealed CETN1 variants leads to impaired cell division and male fertility.

Human spermatogenesis requires an orchestrated expression of numerous genes in various germ cell subtypes. Therefore, the genetic landscape of male infertility is highly complex. Known genetic factors alone account for at least 15% of male infertility. However, ~40% of infertile men remain undiagnosed and are classified as idiopathic infertile men. We performed exome sequencing in 47 idiopathic infertile men (discovery cohort), followed by replication study (40 variants in 33 genes) in 844 infertile men and 709 controls using Sequenom MassARRAY® based genotyping. We report 17 variants in twelve genes that comprise both previously reported (DNAH8, DNAH17, FISP2 and SPEF2) and novel candidate genes (BRDT, CETN1, CATSPERD, GMCL1, SPATA6, TSSK4, TSKS and ZNF318) for male infertility. The latter have a strong biological nexus to human spermatogenesis and their respective mouse knockouts are concordant with human phenotypes. One candidate gene CETN1, identified in this study, was sequenced in another independent cohort of 840 infertile and 689 fertile men. Further, CETN1 variants were functionally characterized using biophysical and cell biology approaches. We demonstrate that CETN1 variant- p.Met72Thr leads to multipolar cells, fragmented nuclei during mitosis leading to cell death and show significantly perturbed ciliary disassembly dynamics. Whereas CETN1-5' UTR variant; rs367716858 leads to loss of a methylation site and increased reporter gene expression in vitro. We report a total of eight novel candidate genes identified by exome sequencing, which may have diagnostic relevance and can contribute to improved diagnostic workup and clinical management of male infertility.

Secretagogin is a Ca2+-dependent stress-responsive chaperone that may also play a role in aggregation-based proteinopathies.

Secretagogin (SCGN) is a three-domain hexa-EF-hand Ca2+-binding protein that plays a regulatory role in the release of several hormones. SCGN is expressed largely in pancreatic ß-cells, certain parts of the brain, and also in neuroendocrine tissues. The expression of SCGN is altered in several diseases, such as diabetes, cancers, and neurodegenerative disorders; however, the precise associations that closely link SCGN expression to such pathophysiologies are not known. In this work, we report that SCGN is an early responder to cellular stress, and SCGN expression is temporally upregulated by oxidative stress and heat shock. We show the overexpression of SCGN efficiently prevents cells from heat shock and oxidative damage. We further demonstrate that in the presence of Ca2+, SCGN efficiently prevents the aggregation of a broad range of model proteins in vitro. Small-angle X-ray scattering (BioSAXS) studies further reveal that Ca2+ induces the conversion of a closed compact apo-SCGN conformation into an open extended holo-SCGN conformation via multistate intermediates, consistent with the augmentation of chaperone activity of SCGN. Furthermore, isothermal titration calorimetry establishes that Ca2+ enables SCGN to bind α-synuclein and insulin, two target proteins of SCGN. Altogether, our data not only demonstrate that SCGN is a Ca2+-dependent generic molecular chaperone involved in protein homeostasis with broad substrate specificity but also elucidate the origin of its altered expression in several cancers. We describe a plausible mechanism of how perturbations in Ca2+ homeostasis and/or deregulated SCGN expression would hasten the process of protein misfolding, which is a feature of many aggregation-based proteinopathies.

Interface interactions between ßγ-crystallin domain and Ig-like domain render Ca2+ -binding site inoperative in abundant perithecial protein of Neurospora crassa.

We describe a set of proteins in which a ßγ-crystallin domain pairs with an Ig-like domain, and which are confined to microbes, like bacteria, slime molds and fungi. DdCAD-1 (Ca2+ -dependent cell adhesion molecule-1) and abundant perithecial protein (APP) represent this class of molecules. Using the crystal structure of APP-NTD (N-terminal domain of APP), we describe its mode of Ca2+ binding and provide a generalized theme for correct identification of the Ca2+ -binding site within this class of molecules. As a common feature, one of the two Ca2+ -binding sites is non-functional in the ßγ-crystallin domains of these proteins. While APP-NTD binds Ca2+ with a micromolar affinity which is comparable to DdCAD-1, APP surprisingly does not bind Ca2+ . Crystal structures of APP and Ca2+ -bound APP-NTD reveal that the interface interactions in APP render its Ca2+ -binding site inoperative. Thus, heterodomain association provides a novel mode of Ca2+ -binding regulation in APP. Breaking the interface interactions (mutating Asp30Ala, Leu132Ala and Ile135Ala) or separation from the Ig-like domain removes the constraints upon the required conformational transition and enables the ßγ-crystallin domain to bind Ca2+ . In mechanistic detail, our work demonstrates an interdomain interface adapted to distinct functional niches in APP and its homolog DdCAD-1.

Abundant Perithecial Protein (APP) from Neurospora is a primitive functional analog of ocular crystallins.

The eye arose during the Cambrian explosion from pre-existing proteins that would have been recruited for the formation of the specialized components of this organ, such as the transparent lens. Proteins suitable for the role of lens crystallins would need to possess unusual physical properties and the study of such earliest analogs of ocular crystallins would add to our understanding of the nature of recruitment of proteins as lens/corneal crystallins. We show that the Abundant Perithecial Protein (APP) of the fungi Neurospora and Sordaria fulfils the criteria for an early crystallin analog. The perithecia in these fungal species are phototropic, and APP accumulates at a high concentration in the neck of the pitcher-shaped perithecium. Spores are formed at the base of the perithecium, and light contributes to their maturation. The hydrodynamic properties of APP appear to exclude dimer formation or aggregation at high protein concentrations. APP is also deficient in Ca2+ binding, a property seen in its close homolog, the calcium-binding cell adhesion molecule (DdCAD-1) from Dictyostelium discoidum. Comparable to crystallins, APP occurs in high concentrations and seems to have dispensed with Ca2+ binding in exchange for greater stability. These crystallin-like attributes of APP lead us to demonstrate that it is a primitive form of ocular crystallins.

Strategizing for the purification of a multiple Big domain-containing protein in native conformation is worth it!

The reliability and accuracy of conformational or functional studies of any novel multidomain protein rely on the quality of protein. The bottleneck in structural studies with the complete Big_2 domain containing proteins like LigA, LigB or MpIBP is usually their large molecular size owing to their multidomain (>10-12 domains) architectures. Interestingly, a soil bacterium Paenarthrobacter aurescens TC1, harbours a gene that encodes a protein comprising of four predicted Big_2 domains. We report here the expression and purification of this novel, multiple Big_2 domains containing protein, Arig of P. aurescens TC1. During overexpression, recombinant Arig formed inclusion bodies and hence was purified by on-column refolding. The refolded Arig revealed a ß-sheet conformation and a well-resolved near-UV CD spectra but did not exhibit a well-dispersed 2D [1H-15N]-HSQC NMR spectrum, as expected for a well-folded ß-sheet native conformation. We, therefore, further optimized Arig overexpression in the soluble fraction by including osmolytes. CD spectroscopic and 2D [1H-15N]-HSQC analyses consolidate that Arig purified alternatively has a well-folded native conformation. While we describe different strategies for purification of Arig, we also present the spectral properties of this novel all-ß-sheet protein.

1H, 13C and 15N NMR assignments of a bacterial immunoglobulin-like domain (group 2) of a protein of a bacterium Paenarthrobacter aurescens TC1.

The bacterial immunoglobulin-like (Big) domain is one of the prevalent domain types, which facilitates cell-cell adhesion by assembling into multi-domain architectures. We selected a four Big_2 domain protein (named 'Arig') from a Gram positive, Paenarthrobacter aurescens TC1 (known earlier as Arthrobacter aurescens TC1). In an attempt to characterize structural and ligand-binding features of individual Big_2 domains, we have cloned, overexpressed, isolated and purified the second Big_2 domain of Arig along with a few of its adjacent Big_2 domain residues (residue 143 to 269) referred to as 'Arig2'. The 13C/15N-doubly-labeled His-tagged Arig2 (133 residues long) showed an ordered conformation as revealed by the well dispersed 2D [15N-1H]-HSQC spectrum. Subsequently, a suite of heteronuclear 3D NMR experiments has enabled almost complete 1H, 13C and 15N NMR resonance assignments of Arig2.