OB Fold-containing Protein 1 (OBFC1), a Human Homolog of Yeast Stn1, Associates with TPP1 and Is Implicated in Telomere Length Regulation

The telosome/shelterin, a six-protein complex formed by TRF1, TRF2, RAP1, TIN2, POT1, and TPP1, functions as the core of the telomere interactome, acting as the molecular platform for the assembly of higher order complexes and coordinating cross-talks between various protein subcomplexes. Within the telosome, there are two oligonucleotide- or oligosaccharide-binding (OB) fold-containing proteins, TPP1 and POT1. They can form heterodimers that bind to the telomeric single-stranded DNA, an activity that is central for telomere end capping and telomerase recruitment. Through proteomic analyses, we found that in addition to POT1, TPP1 can associate with another OB fold-containing protein, OBFC1/AAF44. The yeast homolog of OBFC1 is Stn1, which plays a critical role in telomere regulation. We show here that OBFC1/AAF44 can localize to telomeres in human cells and bind to telomeric single-stranded DNA in vitro. Furthermore, overexpression of an OBFC1 mutant resulted in elongated telomeres in human cells, implicating OBFC1/AAF4 in telomere length regulation. Taken together, our studies suggest that OBFC1/AAF44 represents a new player in the telomere interactome for telomere maintenance.Telomeres are specialized linear chromosome end structures, which are regulated and protected by networks of protein complexes (1–4). Telomere length, structure, and integrity are critical for the cells and the organism as a whole. Telomere dysregulation can lead to DNA damage response, cell cycle checkpoint, genome instability, and predisposition to cancer (5–9). Mammalian telomeres are composed of double-stranded (TTAGGG)_n repeats followed by 3′-single-stranded overhangs (10). In addition to the telomerase that directly mediates the addition of telomere repeats to the end of chromosomes (11, 12), a multitude of telomere-specific proteins have been identified that form the telosome/shelterin complex and participate in telomere maintenance (9, 13). The telosome in turn acts as the platform onto which higher order telomere regulatory complexes may be assembled into the telomere interactome (14). The telomere interactome has been proposed to integrate the complex and labyrinthine network of protein signaling pathways involved in DNA damage response, cell cycle checkpoint, and chromosomal end maintenance and protection for telomere homeostasis and genome stability.Of the six telomeric proteins (TRF1, TRF2, RAP1, TIN2, POT1, and TPP1) that make up the telosome, TRF1 and TRF2 have been shown to bind telomeric double-stranded DNA (15, 16), whereas the OB³ fold-containing protein POT1 exhibits high affinities for telomeric ssDNA in vitro (17, 18). Although the OB fold of TPP1 does not show appreciable ssDNA binding activity, heterodimerization of TPP1 and POT1 enhances the POT1 ssDNA binding (17, 18). More importantly, POT1 depends on TPP1 for telomere recruitment, and the POT1-TPP1 heterodimer functions in telomere end protection and telomerase recruitment. Notably, the OB fold of TPP1 is critical for the recruitment of the telomerase (18). Disruption of POT1-TPP1 interaction by dominant negative inhibition, RNA interference, or gene targeting could lead to dysregulation of telomere length as well DNA damage responses at the telomeres (18–21).In budding yeast, the homolog of mammalian POT1, Cdc13, has been shown to interact with two other OB fold-containing proteins, Stn1 and Ten1, to form a Cdc13-Stn1-Ten1 (CST) complex (22, 23). The CST complex participates in both telomere length control and telomere end capping (22, 23). The presence of multiple OB fold-containing proteins from yeast to human suggests a common theme for telomere ssDNA protection (4). Indeed, it has been proposed that the CST complex is structurally analogous to the replication factor A complex and may in fact function as a telomere-specific replication factor A complex (23). Notably, homologs of the CST complex have been found in other species such as Arabidopsis (24), further supporting the notion that multiple OB fold proteins may be involved in evolutionarily conserved mechanisms for telomere end protection and length regulation. It remains to be determined whether the CST complex exists in mammals.Although the circuitry of interactions among telosome components has been well documented and studied, how core telosome subunits such as TPP1 help to coordinate the cross-talks between telomere-specific signaling pathways and other cellular networks remains unclear. To this end, we carried out large scale immunoprecipitations and mass spectrometry analysis of the TPP1 protein complexes in mammalian cells. Through these studies, we identified OB fold-containing protein 1 (OBFC1) as a new TPP1-associated protein. OBFC1 is also known as α-accessory factor AAF44 (36). Sequence alignment analysis indicates that OBFC1 is a homolog of the yeast Stn1 protein (25). Further biochemical and cellular studies demonstrate the association of OBFC1 with TPP1 in live cells. Moreover, we showed that OBFC1 bound to telomeric ssDNA and localized to telomeres in mammalian cells. Dominant expression of an OBFC1 mutant led to telomere length dysregulation, indicating that OBFC1 is a novel telomere-associated OB fold protein functioning in telomere length regulation.     

In-depth Characterization of the Cerebrospinal Fluid (CSF) Proteome Displayed Through the CSF Proteome Resource (CSF-PR)

In this study, the human cerebrospinal fluid (CSF) proteome was mapped using three different strategies prior to Orbitrap LC-MS/MS analysis: SDS-PAGE and mixed mode reversed phase-anion exchange for mapping the global CSF proteome, and hydrazide-based glycopeptide capture for mapping glycopeptides. A maximal protein set of 3081 proteins (28,811 peptide sequences) was identified, of which 520 were identified as glycoproteins from the glycopeptide enrichment strategy, including 1121 glycopeptides and their glycosylation sites. To our knowledge, this is the largest number of identified proteins and glycopeptides reported for CSF, including 417 glycosylation sites not previously reported. From parallel plasma samples, we identified 1050 proteins (9739 peptide sequences). An overlap of 877 proteins was found between the two body fluids, whereas 2204 proteins were identified only in CSF and 173 only in plasma. All mapping results are freely available via the new CSF Proteome Resource (http://probe.uib.no/csf-pr), which can be used to navigate the CSF proteome and help guide the selection of signature peptides in targeted quantitative proteomics.Cerebrospinal fluid (CSF)¹ surrounds and supports the central nervous system (CNS), including the ventricles and subarachnoid space (1). About 80% of the total protein amount in CSF derives from size-dependent filtration of blood across the blood-brain barrier (BBB), and the rest originate from drainage of interstitial fluid from the CNS (2–4). Because CSF is in direct contact with the CNS, it should be a promising source for finding biomarkers for diseases in the CNS (5).Mapping studies characterizing the human CSF proteome and peptidome has previously been carried out using various experimental designs, including both healthy and disease-affected individuals (5–16). A total of 2630 proteins were detected in normal CSF by immunoaffinity depletion of high abundant proteins followed by strong cation exchange fractionation and LC-MS (5), whereas proteome and peptidome analyses of human CSF (collected for diagnostic purposes and turned out normal) by gel separation and trypsin digestion followed by LC-MS analysis have shown 798 proteins and 563 peptide products (derived from 91 precursor proteins) (6). In another publication, Pan et al. combined several proteomics studies in CSF from both normal subjects and subjects with neurological diseases and created a dataset of 2594 identified proteins (16). But in general, the availability and usefulness of published data from proteome mapping experiments is scarce, and the format of the data often makes searching and comparison across datasets difficult. Thus, organizing the data in online databases would greatly benefit the scientific community by making the data more accessible and easier to query. Current online databases containing MS data for CSF include the Sys-BodyFluid, with a total of 1286 CSF proteins from six studies (17). The proteome identifications database (PRIDE) (18) includes 19 studies on human CSF, but none reporting more than 103 identified proteins.Glycosylation is one of the most common post-translational modifications (PTMs), and many known clinical biomarkers as well as therapeutic targets are glycoproteins (19–25). Furthermore, glycosylation plays important roles in cell communication, signaling, aging, and cell adhesion (26, 27). Nevertheless, there are few studies on glycoprotein identification in CSF. One study identified 216 glycoproteins in CSF using both lectin affinity and hydrazide chemistry (8), and another reported 36 N-linked and 44 O-linked glycosylation sites, from 23 and 22 glycoproteins respectively, by enriching for sialic-acid containing glycopeptides (28).Considering the sparse information about the CSF proteome available in public repositories, we have combined several proteomics approaches to create a map of the global CSF proteome, the CSF glycoproteome, and the respective plasma proteome from a pool of 21 (20 for the plasma pool) neurologically healthy individuals. The large amount of data generated through these four datasets (with linked and complementary information) would not easily be accessible through existing repositories. We therefore developed the open access CSF Proteome Resource (CSF-PR, www.probe.uib.no/csf-pr), an online database including the detailed data from the four different proteomics experiments described in this study. CSF-PR will be particularly useful in guiding the selection of appropriate signature peptides for the development of targeted CSF protein assays.     

RNA Interference Screen to Identify Kinases That Suppress Rescue of ΔF508-CFTR

Cystic Fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene encoding the Cystic fibrosis transmembrane conductance regulator (CFTR). ΔF508-CFTR, the most common disease-causing CF mutant, exhibits folding and trafficking defects and is retained in the endoplasmic reticulum, where it is targeted for proteasomal degradation. To identify signaling pathways involved in ΔF508-CFTR rescue, we screened a library of endoribonuclease-prepared short interfering RNAs (esiRNAs) that target ∼750 different kinases and associated signaling proteins. We identified 20 novel suppressors of ΔF508-CFTR maturation, including the FGFR1. These were subsequently validated by measuring channel activity by the YFP halide-sensitive assay following shRNA-mediated knockdown, immunoblotting for the mature (band C) ΔF508-CFTR and measuring the amount of surface ΔF508-CFTR by ELISA. The role of FGFR signaling on ΔF508-CFTR trafficking was further elucidated by knocking down FGFRs and their downstream signaling proteins: Erk1/2, Akt, PLCγ-1, and FRS2. Interestingly, inhibition of FGFR1 with SU5402 administered to intestinal organoids (mini-guts) generated from the ileum of ΔF508-CFTR homozygous mice resulted in a robust ΔF508-CFTR rescue. Moreover, combination of SU5402 and VX-809 treatments in cells led to an additive enhancement of ΔF508-CFTR rescue, suggesting these compounds operate by different mechanisms. Chaperone array analysis on human bronchial epithelial cells harvested from ΔF508/ΔF508-CFTR transplant patients treated with SU5402 identified altered expression of several chaperones, an effect validated by their overexpression or knockdown experiments. We propose that FGFR signaling regulates specific chaperones that control ΔF508-CFTR maturation, and suggest that FGFRs may serve as important targets for therapeutic intervention for the treatment of CF.Cystic fibrosis (CF)¹ is a pleiotropic disease caused by an abnormal ion transport in the secretory epithelia lining the tubular organs of the body such as lungs, intestines, pancreas, liver, and male reproductive tract. In the airways of CF patients, reduced Cl⁻ and bicarbonate secretion caused by lack of functional Cystic fibrosis transmembrane conductance regulator (CFTR) on the apical surface, and hyper-absorption of Na⁺ because of elevated activity of ENaC (1), lead to a dehydration of the airway surface liquid (ASL). This reduces the viscosity of the mucus layer and the deposited layer of thickened mucus creates an environment that promotes bacterial colonization, which eventually leads to chronic infection of the lungs and death (2, 3).CFTR is a transmembrane protein that functions as a cAMP-regulated, ATP-dependent Cl⁻ channel that also allows passage of bicarbonate through its pore (4, 5). It also possesses ATPase activity important for Cl⁻ conductance (6, 7). The CFTR structure is predicted to consist of five domains: two membrane spanning domains (MSD1, MSD2), each composed of six putative transmembrane helices, two nucleotide binding domains (NBD1, NBD2), and a unique regulatory (R) region (8).More than 1900 CFTR mutations have been identified to date (www.genet.sickkids.on.ca/cftr). The most common mutation is a deletion of phenylalanine at position 508 (ΔF508 or ΔF508-CFTR) in NBD1 (9). The ΔF508 mutation causes severe defects in the processing and function of CFTR. The protein exhibits impaired trafficking from the endoplasmic reticulum (ER) to the plasma membrane (PM), impaired intramolecular interactions between NBD1 and the transmembrane domain, and cell surface instability (10–15). Nevertheless, the ΔF508 defect can be corrected, because treating cells expressing ΔF508-CFTR with low temperature or chemical chaperones (e.g. glycerol) can restore some surface expression of the mutant (11, 16).Numerous small molecules that can at least partially correct (or potentiate) the ΔF508-CFTR defect have been identified to date (17–27), and some were already tested in clinical trials (e.g. sildenafil, VX-809/Lumacaftor), or have made it to the clinic (VX-770/Kalydeco/Ivacaftor) (http://www.cff.org/research/DrugDevelopmentPipeline/). However, the need to identify new ΔF508-CFTR correctors remains immense as the most promising corrector, VX-809, has proven ineffective in alleviating lung disease of CF patients when administered alone (27). Thus, our group developed a high-content technology aimed at identifying proteins and small molecules that correct the trafficking and functional defects of ΔF508-CFTR (28). We successfully used this approach to carry out three separate high-content screens: a protein overexpression screen (28), a small-molecule kinase inhibitor screen (29) and a kinome RNA interference (RNAi) screen, described here.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Comprehensive Identification of Proteins from MALDI Imaging

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a powerful tool for the visualization of proteins in tissues and has demonstrated considerable diagnostic and prognostic value. One main challenge is that the molecular identity of such potential biomarkers mostly remains unknown. We introduce a generic method that removes this issue by systematically identifying the proteins embedded in the MALDI matrix using a combination of bottom-up and top-down proteomics. The analyses of ten human tissues lead to the identification of 1400 abundant and soluble proteins constituting the set of proteins detectable by MALDI IMS including >90% of all IMS biomarkers reported in the literature. Top-down analysis of the matrix proteome identified 124 mostly N- and C-terminally fragmented proteins indicating considerable protein processing activity in tissues. All protein identification data from this study as well as the IMS literature has been deposited into MaTisse, a new publically available database, which we anticipate will become a valuable resource for the IMS community.Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS)¹ is an emerging technique that can be described as a multi-color molecular microscope as it allows visualizing the distribution of many molecules as mass to charge (m/z) signals in parallel in situ (1). Originally described some 15 years ago (2) the method has been successfully adapted to different analyte classes including small molecule drugs (3), metabolites (4), lipids (5), proteins (6), and peptides (7) using e.g. formalin fixed paraffin embedded (FFPE) as well as fresh frozen tissue (8). Because the tissue stays intact in the process, MALDI IMS is compatible with histochemistry (9) as well as immunohistochemistry and thus adds an additional dimension of molecular information to classical microscopy based tissue analysis (10). Imaging of proteins is appealing as it conceptually allows determining the localization and abundance of proteoforms (11) that naturally occur in the tissue under investigation including modifications such as phosphorylation, acetylation, or ubiquitination, protease mediated cleavage or truncation (12). Therefore a proteinous m/z species detected by MALDI IMS can be viewed as an in situ molecular probe of a particular biological process. In turn, m/z abundance patterns that discriminate different physiological or pathological conditions might be used as diagnostic or even prognostic markers (13, 14). In recent years, MALDI IMS of proteins has been successfully applied to different cancer types from the brain (15), breast (16, 17), kidney (18), prostate (19), and skin (20). Furthermore, the technique has been applied in the context of colon inflammation (21), embryonic development (22), Alzheimer''s disease (23), and amyotrophic lateral sclerosis (24). With a few notable exceptions (13, 14, 16–18, 20, 24–30), the identity of the proteins constituting the observed characteristic m/z patters has generally remained elusive. This not only precludes the validation of the putative biomarkers by, for example, immunohistochemistry, but also the elucidation of the biological processes that might underlie the observed phenotype.Here, we introduce a straightforward extraction and identification method for proteins embedded in the MALDI matrix layer that represent the molecular species amenable to MALDI IMS. Using a bottom-up proteomics approach including tryptic digestion and liquid chromatography tandem mass spectrometry (LC-MS/MS), we first created an inventory list of proteins derived from this layer, which we term the MALDI matrix proteome. Although the bottom-up approach breaks the link between the identified proteins and the m/z species detected in MALDI IMS, the list of identified proteins serves as the pool of proteins from which all potential biomarkers are most likely derived. Indeed we detected >90% of all human MALDI IMS biomarkers reported in the literature by analyzing just ten human tissues. In addition, the results demonstrate that the same inventory can be used as a focused database for direct top-down sequencing and identification of proteins extracted from the MALDI matrix layer. The proposed method is generic and can be applied to any MALDI IMS study, which is why we believe that one of the major challenges in identifying MALDI IMS biomarkers has now been overcome. In addition, we provide a list of all proteins and peptides identified in the MALDI matrices and tissues studied here as well as a comprehensive list of m/z species identified in the literature dealing with MALDI imaging of humans and rodents. This information has been compiled in MaTisse (http://www.wzw.tum.de/bioanalytik/matisse), a new publically available and searchable database, which we believe will become a valuable tool for the MALDI imaging community.     

Extending the Limits of Quantitative Proteome Profiling with Data-Independent Acquisition and Application to Acetaminophen-Treated Three-Dimensional Liver Microtissues

The data-independent acquisition (DIA) approach has recently been introduced as a novel mass spectrometric method that promises to combine the high content aspect of shotgun proteomics with the reproducibility and precision of selected reaction monitoring. Here, we evaluate, whether SWATH-MS type DIA effectively translates into a better protein profiling as compared with the established shotgun proteomics.We implemented a novel DIA method on the widely used Orbitrap platform and used retention-time-normalized (iRT) spectral libraries for targeted data extraction using Spectronaut. We call this combination hyper reaction monitoring (HRM). Using a controlled sample set, we show that HRM outperformed shotgun proteomics both in the number of consistently identified peptides across multiple measurements and quantification of differentially abundant proteins. The reproducibility of HRM in peptide detection was above 98%, resulting in quasi complete data sets compared with 49% of shotgun proteomics.Utilizing HRM, we profiled acetaminophen (APAP)¹-treated three-dimensional human liver microtissues. An early onset of relevant proteome changes was revealed at subtoxic doses of APAP. Further, we detected and quantified for the first time human NAPQI-protein adducts that might be relevant for the toxicity of APAP. The adducts were identified on four mitochondrial oxidative stress related proteins (GATM, PARK7, PRDX6, and VDAC2) and two other proteins (ANXA2 and FTCD).Our findings imply that DIA should be the preferred method for quantitative protein profiling.Quantitative mass spectrometry is a powerful and widely used approach to identify differentially abundant proteins, e.g. for proteome profiling and biomarker discovery (1). Several tens of thousands of peptides and thousands of proteins can be routinely identified from a single sample injection in shotgun proteomics (2). Shotgun proteomics, however, is limited by low analytical reproducibility. This is due to the complexity of the samples that results in under sampling (supplemental Fig. 1) and to the fact that the acquisition of MS2 spectra is often triggered outside of the elution peak apex. As a result, only 17% of the detectable peptides are typically fragmented, and less than 60% of those are identified. This translates in reliable identification of only 10% of the detectable peptides (3). The overlap of peptide identification across technical replicates is typically 35–60% (4), which results in inconsistent peptide quantification. Alternatively to shotgun proteomics, selected reaction monitoring (SRM) enables quantification of up to 200–300 peptides at very high reproducibility, accuracy, and precision (5–8).Data-independent acquisition (DIA), a novel acquisition type, overcomes the semistochastic nature of shotgun proteomics (9–18). Spectra are acquired according to a predefined schema instead of dependent on the data. Targeted analysis of DIA data was introduced with SWATH-MS (19). For the originally published SWATH-MS, the mass spectrometer cycles through 32 predefined, contiguous, 25 Thomson wide precursor windows, and records high-resolution fragment ion spectra (19). This results in a comprehensive measurement of all detectable precursors of the selected mass range. The main novelty of SWATH-MS was in the analysis of the collected DIA data. Predefined fragment ions are extracted using precompiled spectrum libraries, which results in SRM-like data. Such targeted analyses are now enabled by several publicly available computational tools, in particular Spectronaut², Skyline (20), and OpenSWATH (21). The accuracy of peptide identification is evaluated based on the mProphet method (22).We introduce a novel SWATH-MS-type DIA workflow termed hyper reaction monitoring (HRM) (reviewed in (23)) implemented on a Thermo Scientific Q Exactive platform. It consists of comprehensive DIA acquisition and targeted data analysis with retention-time-normalized spectral libraries (24). Its high accuracy of peptide identification and quantification is due to three aspects. First, we developed a novel, improved DIA method. Second, we reimplemented the mProphet (22) approach in the software Spectronaut (www.spectronaut.org). Third, we developed large, optimized, and retention-time-normalized (iRT) spectral libraries.We compared HRM and state-of-the-art shotgun proteomics in terms of ability to discover differentially abundant proteins. For this purpose, we used a “profiling standard sample set” with 12 non-human proteins spiked at known absolute concentrations into a stable human cell line protein extract. This resulted in quasi complete data sets for HRM and the detection of a larger number of differentially abundant proteins as compared with shotgun proteomics. We utilized HRM to identify changes in the proteome in primary three-dimensional human liver microtissues after APAP exposure (25–27). These primary hepatocytes exhibit active drug metabolism. With a starting material of only 12,000 cells per sample, the abundance of 2,830 proteins was quantified over an APAP concentration range. Six novel NAPQI-cysteine proteins adducts that might be relevant for the toxicity of APAP were found and quantified mainly on mitochondrion-related proteins.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Deletion of the Chloride Transporter Slc26a7 Causes Distal Renal Tubular Acidosis and Impairs Gastric Acid Secretion

SLC26A7 (human)/Slc26a7 (mouse) is a recently identified chloride-base exchanger and/or chloride transporter that is expressed on the basolateral membrane of acid-secreting cells in the renal outer medullary collecting duct (OMCD) and in gastric parietal cells. Here, we show that mice with genetic deletion of Slc26a7 expression develop distal renal tubular acidosis, as manifested by metabolic acidosis and alkaline urine pH. In the kidney, basolateral Cl⁻/HCO₃⁻ exchange activity in acid-secreting intercalated cells in the OMCD was significantly decreased in hypertonic medium (a normal milieu for the medulla) but was reduced only mildly in isotonic medium. Changing from a hypertonic to isotonic medium (relative hypotonicity) decreased the membrane abundance of Slc26a7 in kidney cells in vivo and in vitro. In the stomach, stimulated acid secretion was significantly impaired in isolated gastric mucosa and in the intact organ. We propose that SLC26A7 dysfunction should be investigated as a potential cause of unexplained distal renal tubular acidosis or decreased gastric acid secretion in humans.The collecting duct segment of the distal kidney nephron plays a major role in systemic acid base homeostasis by acid secretion and bicarbonate absorption. The acid secretion occurs via H⁺-ATPase and H-K-ATPase into the lumen and bicarbonate is absorbed via basolateral Cl⁻/HCO₃⁻ exchangers (1–4). The tubules, which are located within the outer medullary region of the kidney collecting duct (OMCD),² have the highest rate of acid secretion among the distal tubule segments and are therefore essential to the maintenance of acid base balance (2).The gastric parietal cell is the site of generation of acid and bicarbonate through the action of cytosolic carbonic anhydrase II (5, 6). The intracellular acid is secreted into the lumen via gastric H-K-ATPase, which works in conjunction with a chloride channel and a K⁺ recycling pathway (7–10). The intracellular bicarbonate is transported to the blood via basolateral Cl⁻/HCO₃⁻ exchangers (11–14).SLC26 (human)/Slc26 (mouse) isoforms are members of a conserved family of anion transporters that display tissue-specific patterns of expression in epithelial cells (15–24). Several SLC26 members can function as chloride/bicarbonate exchangers. These include SLC26A3 (DRA), SLC26A4 (pendrin), SLC26A6 (PAT1 or CFEX), SLC26A7, and SLC26A9 (25–31). SLC26A7 and SLC26A9 can also function as chloride channels (32–34).SLC26A7/Slc26a7 is predominantly expressed in the kidney and stomach (28, 29). In the kidney, Slc26a7 co-localizes with AE1, a well-known Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of (acid-secreting) A-intercalated cells in OMCD cells (29, 35, 36) (supplemental Fig. 1). In the stomach, Slc26a7 co-localizes with AE2, a major Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of acid secreting parietal cells (28). To address the physiological function of Slc26a7 in the intact mouse, we have generated Slc26a7 ko mice. We report here that Slc26a7 ko mice exhibit distal renal tubular acidosis and impaired gastric acidification in the absence of morphological abnormalities in kidney or stomach.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

A Study into the Collision-induced Dissociation (CID) Behavior of Cross-Linked Peptides

Cross-linking/mass spectrometry resolves protein–protein interactions or protein folds by help of distance constraints. Cross-linkers with specific properties such as isotope-labeled or collision-induced dissociation (CID)-cleavable cross-linkers are in frequent use to simplify the identification of cross-linked peptides. Here, we analyzed the mass spectrometric behavior of 910 unique cross-linked peptides in high-resolution MS1 and MS2 from published data and validate the observation by a ninefold larger set from currently unpublished data to explore if detailed understanding of their fragmentation behavior would allow computational delivery of information that otherwise would be obtained via isotope labels or CID cleavage of cross-linkers. Isotope-labeled cross-linkers reveal cross-linked and linear fragments in fragmentation spectra. We show that fragment mass and charge alone provide this information, alleviating the need for isotope-labeling for this purpose. Isotope-labeled cross-linkers also indicate cross-linker-containing, albeit not specifically cross-linked, peptides in MS1. We observed that acquisition can be guided to better than twofold enrich cross-linked peptides with minimal losses based on peptide mass and charge alone. By help of CID-cleavable cross-linkers, individual spectra with only linear fragments can be recorded for each peptide in a cross-link. We show that cross-linked fragments of ordinary cross-linked peptides can be linearized computationally and that a simplified subspectrum can be extracted that is enriched in information on one of the two linked peptides. This allows identifying candidates for this peptide in a simplified database search as we propose in a search strategy here. We conclude that the specific behavior of cross-linked peptides in mass spectrometers can be exploited to relax the requirements on cross-linkers.Cross-linking/mass spectrometry extends the use of mass-spectrometry-based proteomics from identification (1, 2), quantification (3), and characterization of protein complexes (4) into resolving protein structures and protein–protein interactions (5–8). Chemical reagents (cross-linkers) covalently connect amino acid pairs that are within a cross-linker-specific distance range in the native three-dimensional structure of a protein or protein complex. A cross-linking/mass spectrometry experiment is typically conducted in four steps: (1) cross-linking of the target protein or complex, (2) protein digestion (usually with trypsin), (3) LC-MS analysis, and (4) database search. The digested peptide mixture consists of linear and cross-linked peptides, and the latter can be enriched by strong cation exchange (9) or size exclusion chromatography (10). Cross-linked peptides are of high value as they provide direct information on the structure and interactions of proteins.Cross-linked peptides fragment under collision-induced dissociation (CID) conditions primarily into b- and y-ions, as do their linear counterparts. An important difference regarding database searches between linear and cross-linked peptides stems from not knowing which peptides might be cross-linked. Therefore, one has to consider each single peptide and all pairwise combinations of peptides in the database. Having n peptides leads to (n2 + n)/2 possible pairwise combinations. This leads to two major challenges: With increasing size of the database, search time and the risk of identifying false positives increases. One way of circumventing these problems is to use MS2-cleavable cross-linkers (11, 12), at the cost of limited experimental design and choice of cross-linker.In a first database search approach (13), all pairwise combinations of peptides in a database were considered in a concatenated and linearized form. Thereby, all possible single bond fragments are considered in one of the two database entries per peptide pair, and the cross-link can be identified by a normal protein identification algorithm. Already, the second search approach split the peptides for the purpose of their identification (14). Linear fragments were used to retrieve candidate peptides from the database that are then matched based on the known mass of the cross-linked pair and scored as a pair against the spectrum. Isotope-labeled cross-linkers were used to sort the linear and cross-linked fragments apart. Many other search tools and approaches have been developed since (10, 15–19); see (20) for a more detailed list, at least some of which follow the general idea of an open modification search (21–24).As a general concept for open modification search of cross-linked peptides, cross-linked peptides represent two peptides, each with an unknown modification given by the mass of the other peptide and the cross-linker. One identifies both peptides individually and then matches them based on knowing the mass of cross-linked pair (14, 22, 24). Alternatively, one peptide is identified first and, using that peptide and the cross-linker as a modification mass, the second peptide is identified from the database (21, 23). An important element of the open modification search approach is that it essentially converts the quadratic search space of the cross-linked peptides into a linear search space of modified peptides. Still, many peptides and many modification positions have to be considered, especially when working with large databases or when using highly reactive cross-linkers with limited amino acid selectivity (25).We hypothesize that detailed knowledge of the fragmentation behavior of cross-linked peptides might reveal ways to improve the identification of cross-linked peptides. Detailed analyses of the fragmentation behavior of linear peptides exist (26–28), and the analysis of the fragmentation behavior of cross-linked peptides has guided the design of scores (24, 29). Further, cross-link-specific ions have been observed from higher energy collision dissociation (HCD) data (30). Isotope-labeled cross-linkers are used to distinguish cross-linked from linear fragments, generally in low-resolution MS2 of cross-linked peptides (14).We compared the mass spectrometric behavior of cross-linked peptides to that of linear peptides, using 910 high-resolution fragment spectra matched to unique cross-linked peptides from multiple different public datasets at 5% peptide-spectrum match (PSM)¹ false discovery rate (FDR). In addition, we repeated all experiments with a larger sample set that contains 8,301 spectra—also including data from ongoing studies from our lab (Supplemental material S9-S12). This paper presents the mass spectrometric signature of cross-linked peptides that we identified in our analysis and the resulting heuristics that are incorporated into an integrated strategy for the analysis and identification of cross-linked peptides. We present computational strategies that indicate the possibility of alleviating the need for mass-spectrometrically restricted cross-linker choice.