De novo identification and quantification of single amino-acid variants in human brain RIM2α is a molecular scaffold for Zona pellucida-induced acrosome reaction High-accuracy mapping of protein binding stability on nucleosomal DNA using a single-molecule

The detection of single amino-acid variants (SAVs) usually depends on single-nucleotide polymorphisms (SNPs) database. Here,we describe a novel method that discovers SAVs at proteome level independent of SNPs data. Using mass spectrometry-based de novo sequencing algorithm, peptide-candidates are identified and compared with theoretical protein database to generate SAVs under pairing strategy, which is followed by database re-searching to control false discovery rate. In human brain tissues, we can confidently identify known and novel protein variants with diverse origins. Combined with DNA/RNA sequencing, we verify SAVs derived from DNA mutations, RNA alternative splicing, and unknown post-transcriptional mechanisms. Furthermore, quantitative analysis in human brain tissues reveals several tissue-specific differential expressions of SAVs. This approach provides a novel access to high-throughput detection of protein variants, which may offer the potential for clinical biomarker discovery and mechanistic research. The acrosome reaction (AR) in mammalian spermatozoa is a prerequisite for successful fertilization, because it leads to the release of hydrolytic enzymes from the acrosomal vesicle along with the exposure ofthe oocyte-recognition protein Izumo on the sperm surface (Bianchi et al., 2014). AR mainly follows the conserved principles of calcium-regulated exocytosis in neurons/ neurosecretory cells, which is reflected by initial vesicle docking/priming steps and calcium-triggered SNARE-mediated membrane fusion (Tsai et al., 2012). However, there are some unique features of AR whose underlying molecular mechanisms are largely unknown: The acrosome is a single, huge vesicle whose exocytosis is realized by multipoint fusions ofthe outer acrosomal and sperm plasma membrane (Tsai et al., 2012). In addition, stimulating sperm with the oocyte's Zona pellucida (ZP) leads to a zipper-like progression of fusion pore formation that starts at the posterior acrosomal region and consistently proceeds in an anterograde direction, whereas the fusion is initiated at random sites following calcium ionophore treatment (Buffone et al., 2009). This directed propagation requires extraordinary temporal and spatial orchestration of individual pore-forming events. Remarkably, individual exocytotic events at neuronal synaptic junctions also require tight coordination, which is accomplished by a protein network termed cytomatrix ofthe active zone (CAZ),consisting ofthe multi-domain CAZ-proteins Munc13, RIM, RIM-BP, Liprin-a, ELKS/ERC, Piccolo/Aczonin, and Bassoon (Sudhof, 2012). Thus, we hypothesized that a CAZ-like network could also coordinate the success of sperm AR. The conformation of nucleosomal DNA is significantly different from that of B-form double stranded DNA (dsDNA) (Richmond and Davey, 2003). In nucleosomal DNA, specific DNA sequences are less flexible and less accessible than in free dsDNA, which might be due to the tight association of histone cores. The allosteric effect via DNA has been documented recently (Kim et al., 2013), suggesting that DNA is not merely a solid rod providing recognition sequences. Previous studies of nucleosomal DNA-protein interactions only demonstrated the key mechanism involved—histone shielding (Li and Wrange, 1993; Hinz et al., 2010; Sahu et al., 2010). How conformational changes in nucleosomal DNA, compared with free dsDNA, affect nucleosomal DNA-protein interactions remains unknown.