C2CD6 regulates targeting and organization of the CatSper calcium channel complex in sperm flagella

The CatSper cation channel is essential for sperm capacitation and male fertility. The multi-subunit CatSper complexes form highly organized calcium signaling nanodomains on flagellar membranes. Here we report identification of an uncharacterized protein C2CD6 as a novel subunit of the CatSper complex. C2CD6 contains a calcium-dependent membrane targeting C2 domain. C2CD6 associates with the CatSper calcium-selective core forming subunits. Deficiency of C2CD6 depletes the CatSper nanodomains from the flagellum and results in male sterility. C2CD6-deficient sperm are defective in hyperactivation and fail to fertilize oocytes both in vitro and in vivo. CatSper currents are present but at a significantly lower level in C2CD6-deficient sperm. Transient treatments with either Ca2+ ionophore, starvation, or a combination of both restore the fertilization capacity of C2CD6-deficient sperm. C2CD6 interacts with EFCAB9, a pH-dependent calcium sensor in the CatSper complex. We postulate that C2CD6 facilitates incorporation of the CatSper complex into the flagellar plasma membrane and may function as a calcium sensor. The identification of C2CD6 may enable the long-sought reconstitution of the CatSper ion channel complex in a heterologous system for male contraceptive development.

Validated Determination of NRG1 Ig-like Domain Structure by Mass Spectrometry Coupled with Computational Modeling

High resolution hydroxyl radical protein footprinting (HR-HRPF) is a mass spectrometry-based method that measures the solvent exposure of multiple amino acids in a single experiment, offering constraints for experimentally-informed computational modeling. HR-HRPF-based modeling has previously been used to accurately model the structure of proteins of known structure, but the technique has never been used to determine the structure of a protein of unknown structure leaving questions of unintentional bias and applicability to unknown structures unresolved. Here, we present the use of HR-HRPF-based modeling to determine the structure of the Ig-like domain of NRG1, a protein with no close homolog of known structure. Independent determination of the protein structure by both HR-HRPF-based modeling and heteronuclear NMR was carried out, with results compared only after both processes were complete. The HR-HRPF-based model was highly similar to the lowest energy NMR model, with a backbone RMSD of 1.6 Å. To our knowledge, this is the first use of HR-HRPF-based modeling to determine a previously uncharacterized protein structure.

A novel sequence-based predictor for identifying and characterizing thermophilic proteins using estimated propensity scores of dipeptides

Owing to their ability to maintain a thermodynamically stable fold at extremely high temperatures, thermophilic proteins (TTPs) play a critical role in basic research and a variety of applications in the food industry. As a result, the development of computation models for rapidly and accurately identifying novel TTPs from a large number of uncharacterized protein sequences is desirable. In spite of existing computational models that have already been developed for characterizing thermophilic proteins, their performance and interpretability remain unsatisfactory. We present a novel sequence-based thermophilic protein predictor, termed SCMTPP, for improving model predictability and interpretability. First, an up-to-date and high-quality dataset consisting of 1853 TPPs and 3233 non-TPPs was compiled from published literature. Second, the SCMTPP predictor was created by combining the scoring card method (SCM) with estimated propensity scores of g-gap dipeptides. Benchmarking experiments revealed that SCMTPP had a cross-validation accuracy of 0.883, which was comparable to that of a support vector machine-based predictor (0.906–0.910) and 2–17% higher than that of commonly used machine learning models. Furthermore, SCMTPP outperformed the state-of-the-art approach (ThermoPred) on the independent test dataset, with accuracy and MCC of 0.865 and 0.731, respectively. Finally, the SCMTPP-derived propensity scores were used to elucidate the critical physicochemical properties for protein thermostability enhancement. In terms of interpretability and generalizability, comparative results showed that SCMTPP was effective for identifying and characterizing TPPs. We had implemented the proposed predictor as a user-friendly online web server at http://pmlabstack.pythonanywhere.com/SCMTPP in order to allow easy access to the model. SCMTPP is expected to be a powerful tool for facilitating community-wide efforts to identify TPPs on a large scale and guiding experimental characterization of TPPs.

The Heat Shock Protein 60 and Pap1 Participate in the Sporothrix schenckii-Host Interaction

Sporothrixschenckii is one of the etiological agents of sporotrichosis, a worldwide-distributed subcutaneous mycosis. Its cell wall contains a glycoconjugate composed of rhamnose, mannose, glucuronic acid, and proteins, named peptidorhamnomannan, which harbors important Sporothrix-specific immunogenic epitopes. Although the peptidorhamnomannan carbohydrate moiety has been extensively studied, thus far, little is known about the protein core. Here, using LC-MS/MS, we analyzed the S.schenckii peptidorhamnomannan peptide fraction and generated mass signals of 325 proteins, most of them likely to be moonlighting proteins. Among the identified proteins, chaperonin GroEL/Hsp60 and the uncharacterized protein Pap1 were selected for further analysis. Both proteins were heterologously expressed in bacteria, and they showed adhesive properties to the extracellular matrix proteins laminin, elastin, fibrinogen, and fibronectin, although Pap1 also was bound to type-I and type-II collagen. The inoculation of concentrations higher than 40 μg of these proteins, separately, increased immune effectors in the hemolymph of Galleriamellonella larvae and protected animals from an S.schenckii lethal challenge. These observations were confirmed when yeast-like cells, pre-incubated with anti-rHsp60 or anti-rPap1 antibodies were used to inoculate larvae. The animals inoculated with pretreated cells showed increased survival rates when compared to the control groups. In conclusion, we report that Hsp60 and Pap1 are part of the cell wall peptidorhamnomannan, can bind extracellular matrix components, and contribute to the S.schenckii virulence. To our knowledge, this is the first report about moonlighting protein in the S.schenckii cell wall with an important role during the pathogen–host interaction.

An Uncharacterized Protein from the Metagenome with no Obvious Homology to known Lipases Shows Excellent Alkaline Lipase Properties and Potential Applications in the Detergent Industry

A novel lipase, Lip486, which has no obvious homology with known lipases, was discovered using functional metagenomics technology. Phylogenetic tree analysis suggested that the enzyme belongs to a new subfamily called lipolytic enzyme family II. To explore the enzymatic properties, lip486 was expressed heterologously and efficiently in Escherichia coli. The recombinant enzyme displayed the highest activity on the substrate p-nitrophenyl caprate with a carbon chain length of 10, and its optimum temperature and pH were 53 °C and 8.0, respectively. The recombinant Lip486 showed good activity and stability in strong alkaline and medium-low -temperature environments. The results of compatibility and soaking tests showed that the enzyme had good compatibility with 4 kinds of commercial detergents, and an appropriate soaking time could further improve the enzyme activity. Oil stain removal test results for a cotton cloth indicated that the washing performance of commercial laundry detergent supplemented with Lip486 was further improved. In addition, as one of the smallest lipases found to date, Lip486 also has the advantages of high yield, good stability and easy molecular modification. These characteristics reflect the good application prospects for Lip486 in the detergent and other industries in the future.

Systematic analysis of membrane contact sites in Saccharomyces cerevisiae uncovers modulators of cellular lipid distribution

Actively maintained close appositions, or contact sites, between organelle membranes, enable the efficient transfer of biomolecules between the various cellular compartments. Several such sites have been described together with their tethering machinery. Despite these advances we are still far from a comprehensive understanding of the function and regulation of most contact sites. To systematically characterize the proteome of contact sites and support the discovery of new tethers and functional molecules, we established a high throughput screening approach in Saccharomyces cerevisiae based on co-localization imaging. We imaged split fluorescence reporters for six different contact sites, two of which have never been studied before, on the background of 1165 strains expressing a mCherry-tagged yeast protein that have a cellular punctate distribution (a hallmark of contact sites). By scoring both co-localization events and effects on reporter size and abundance, we discovered over 100 new potential contact site residents and effectors in yeast. Focusing on several of the newly identified residents, we identified one set of hits as previously unrecognized homologs to Vps13 and Atg2. These proteins share their lipid transport domain, thus expanding this family of lipid transporters. Analysis of another candidate, Ypr097w, which we now call Lec1 (Lipid-droplet Ergosterol Cortex 1), revealed that this previously uncharacterized protein dynamically shifts between lipid droplets and the cell cortex, and plays a role in regulation of ergosterol distribution in the cell.

Transcriptome analysis of conditionally immortalized atrial myocytes: identification of a novel atrium-enriched protein involved in sarcomere assembly and maintenance

Background Heart development relies on the tight spatiotemporal control of cardiac gene expression. Genes involved in these processes have been identified using mainly (transgenic) animals models and pluripotent stem cell-derived cardiomyocytes (CMs). Recently, the repertoire of cardiomyocyte differentiation models has been expanded with iAM-1, a monoclonal cell line of conditionally immortalized neonatal rat atrial myocytes (NRAMs) which allows toggling between proliferative and differentiated (i.e. excitable and contractile) phenotypes in a synchronized and homogenous manner. Purpose To identify and characterize (lowly expressed) genes with an as-of-yet uncharacterized role in cardiomyocyte differentiation, dedifferentiation and proliferation by exploiting the unique properties of conditionally immortalized NRAMs (iAMs). Methods and results RNA sequencing was performed during a full cycle of iAM-1 differentiation and subsequent dedifferentiation, identifying ±13,000 transcripts, of which the dynamic expressional changes during cardiomyogenic differentiation in most cases opposed those during dedifferentiation. Among the genes whose expression increased during differentiation and decreased during dedifferentiation were many genes with a known (lineage-specific) role in cardiac muscle formation, thereby confirming the relevance of iAMs as cardiomyogenic differentiation model. Filtering for cardiomyocyte-enriched low abundancy transcripts, resulted in the identification of an uncharacterized protein, which is highly conserved among Nephrozoa and up- and downregulated during cardiomyocyte differentiation and dedifferentiation, respectively. In neonatal and adult rats, this protein is muscle-specific, highly atrium-enriched and localized around the C-zone of cardiac sarcomeres. Lentiviral shRNA-mediated knockdown resulted in loss of sarcomeric organization in both NRAMs and iAMs. Neither knockdown nor overexpression of this protein affected the electrophysiological properties of differentiated iAM monolayers. Conclusions iAM-1 cells offer a relevant model to identify and characterize novel (low abundancy) genes involved in cardiomyocyte (de)differentiation as exemplified by the identification a novel uncharacterized protein that is muscle-specific, highly atrium-enriched, localized around the C-zone of cardiac sarcomeres and plays a specific role in atrial sarcomerigenesis. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Netherlands Organisation for Health Research and Development (ZonMw) Leiden Regenerative Medicine Platform Holding project with number (LRMPH) Figure 1. (A) Experimental setup. At the indicated timepoints iAM-1 cells were fixed for immunostaining and RNA extraction for transcriptome analysis. (B) Immunochemical staining of iAM-1 cells for the proliferation marker Ki-67 and the Z-line marker sarcomeric α-actinin. (C & D) Immunohistological double stainings of longitudinal sections of neonatal rat hearts for the uncharacterized protein (GOI 1) and the sarcomeric protein cardiac troponin I (TNNI3). LA, left atrium; RA, right atrium; LV, left ventricle; RV, right ventricle. Scale bar, 250 μm.

Cryptococcus neoformans releases proteins during intracellular residence that affect the outcome of the fungal-macrophage interaction

Cryptococcus neoformans is a facultative intracellular pathogen that can replicate and disseminate in mammalian macrophages. In this study, we analyzed fungal proteins identified in murine macrophage-like cells after infection with C. neoformans. To accomplish this, we developed a protocol to identify proteins released from cryptococcal cells inside macrophage-like cells; we identified 127 proteins of fungal origin in infected macrophage-like cells. Among the proteins identified was urease, a known virulence factor, and others such as transaldolase and phospholipase D, which have catalytic activities that could contribute to virulence. This method provides a straightforward methodology to study host-pathogen interactions. We chose to study further Yor1, a relatively uncharacterized protein belonging to the large family of ATP binding cassette transporter (ABC transporters). These transporters belong to a large and ancient protein family found in all extant phyla. While ABC transporters have an enormous diversity of functions across varied species, in pathogenic fungi they are better studied as drug efflux pumps. Analysis of C. neoformans yor1Δ strains revealed defects in non-lytic exocytosis and capsule size, when compared to wild-type strains. We detected no difference in growth rates, cell body size and vesicle secretion. Our results indicate that C. neoformans releases a large suite of proteins during macrophage infection, some of which can modulate fungal virulence and are likely to affect the fungal-macrophage interaction.

A poplar short-chain dehydrogenase reductase plays a potential key role in biphenyl detoxification

Polychlorinated biphenyls (PCBs) are persistent organic pollutants with severe effects on human health and the biosphere. Plant-based remediation offers many benefits over conventional PCB remediation, but its development has been hampered by our poor understanding of biphenyl metabolism in eukaryotes, among other factors. We report here a major PCB-responsive protein in poplar, a plant model system capable of PCB uptake and translocation. We provide structural and functional evidence that this uncharacterized protein, termed SDR57C, belongs to the heterogeneous short-chain dehydrogenase reductase (SDR) superfamily. Despite sequence divergence, structural modeling hinted at structural and functional similarities between SDR57C and BphB, a central component of the Bph pathway for biphenyl/PCB degradation in aerobic bacteria. By combining gas chromatography/mass spectrometry (GC/MS) profiling with a functional complementation scheme, we found that poplar SDR57C can replace BphB activity in the upper Bph pathway of Pseudomonas furukawaii KF707 and therefore catalyze the oxidation of 2,3-dihydro-2,3-dihydroxybiphenyl (2,3-DHDB) to 2,3-dihydroxybiphenyl (2,3-DHB). Consistent with this biochemical activity, we propose a mechanism of action based on prior quantum studies, general properties of SDR enzymes, and the modeled docking of 2,3-DHDB to the SDR57C-NAD+ complex. The putative detoxifying capacity of SDR57C was substantiated through reverse genetics in Arabidopsis thaliana. Phenotypic characterization of the SDR lines underscored an inducible plant pathway with the potential to catabolize toxic biphenyl derivatives. Partial similarities with aerobic bacterial degradation notwithstanding, real-time messenger RNA quantification indicates the occurrence of plant-specific enzymes and features. Our results may help explain differences in degradative abilities among plant genotypes and also provide elements to improve them.

High-Resolution Proteomics of Aedes Aegypti Salivary Glands Infected With DENV2, ZIKV and CHIKV Reveal Virus-Specific and Broad Antiviral Factors

Arboviruses such as dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses infect close to half a billion people per year, and are transmitted through Aedes aegypti bites. Infection-induced changes in mosquito salivary glands (SG) influence transmission by inducing immunity, which restricts virus replication, and by altering saliva composition, which influences skin infection. Here, we profiled SG responses to DENV2, ZIKV and CHIKV infections by using high-resolution quantitative proteomics. We identified 218 proteins related to immunity, blood-feeding or cellular machinery. We observed that 58, 27 and 29 proteins were regulated by DENV2, ZIKV and CHIKV infections, respectively. While the regulation patterns were mostly virus-specific, we determined the function of four uncharacterized proteins that were upregulated by all three viruses. We revealed the anti-ZIKV function of gamma-interferon responsive lysosomal thiol-like (GILT-like), the anti-CHIKV function of adenosine deaminase (ADA), the pro-ZIKV function of salivary gland surface protein 1 (SGS1) and the antiviral function against all three viruses of an uncharacterized protein we called salivary gland broad-spectrum antiviral protein (SGBAP). The comprehensive description of SG responses to three global pathogenic viruses and the identification of new restriction factors improves our understanding of the molecular mechanisms influencing transmission.