Bcl-xL acts as an inhibitor of IP3R channels, thereby antagonizing Ca2+-driven apoptosis.

Anti-apoptotic Bcl-2-family members not only act at mitochondria but also at the endoplasmic reticulum, where they impact Ca2+ dynamics by controlling IP3 receptor (IP3R) function. Current models propose distinct roles for Bcl-2 vs. Bcl-xL, with Bcl-2 inhibiting IP3Rs and preventing pro-apoptotic Ca2+ release and Bcl-xL sensitizing IP3Rs to low [IP3] and promoting pro-survival Ca2+ oscillations. We here demonstrate that Bcl-xL too inhibits IP3R-mediated Ca2+ release by interacting with the same IP3R regions as Bcl-2. Via in silico superposition, we previously found that the residue K87 of Bcl-xL spatially resembled K17 of Bcl-2, a residue critical for Bcl-2's IP3R-inhibitory properties. Mutagenesis of K87 in Bcl-xL impaired its binding to IP3R and abrogated Bcl-xL's inhibitory effect on IP3Rs. Single-channel recordings demonstrate that purified Bcl-xL, but not Bcl-xLK87D, suppressed IP3R single-channel openings stimulated by sub-maximal and threshold [IP3]. Moreover, we demonstrate that Bcl-xL-mediated inhibition of IP3Rs contributes to its anti-apoptotic properties against Ca2+-driven apoptosis. Staurosporine (STS) elicits long-lasting Ca2+ elevations in wild-type but not in IP3R-knockout HeLa cells, sensitizing the former to STS treatment. Overexpression of Bcl-xL in wild-type HeLa cells suppressed STS-induced Ca2+ signals and cell death, while Bcl-xLK87D was much less effective in doing so. In the absence of IP3Rs, Bcl-xL and Bcl-xLK87D were equally effective in suppressing STS-induced cell death. Finally, we demonstrate that endogenous Bcl-xL also suppress IP3R activity in MDA-MB-231 breast cancer cells, whereby Bcl-xL knockdown augmented IP3R-mediated Ca2+ release and increased the sensitivity towards STS, without altering the ER Ca2+ content. Hence, this study challenges the current paradigm of divergent functions for Bcl-2 and Bcl-xL in Ca2+-signaling modulation and reveals that, similarly to Bcl-2, Bcl-xL inhibits IP3R-mediated Ca2+ release and IP3R-driven cell death. Our work further underpins that IP3R inhibition is an integral part of Bcl-xL's anti-apoptotic function.

Scop3P: A Comprehensive Resource of Human Phosphosites within Their Full Context.

Protein phosphorylation is a key post-translational modification in many biological processes and is associated to human diseases such as cancer and metabolic disorders. The accurate identification, annotation, and functional analysis of phosphosites are therefore crucial to understand their various roles. Phosphosites are mainly analyzed through phosphoproteomics, which has led to increasing amounts of publicly available phosphoproteomics data. Several resources have been built around the resulting phosphosite information, but these are usually restricted to the protein sequence and basic site metadata. What is often missing from these resources, however, is context, including protein structure mapping, experimental provenance information, and biophysical predictions. We therefore developed Scop3P: a comprehensive database of human phosphosites within their full context. Scop3P integrates sequences (UniProtKB/Swiss-Prot), structures (PDB), and uniformly reprocessed phosphoproteomics data (PRIDE) to annotate all known human phosphosites. Furthermore, these sites are put into biophysical context by annotating each phosphoprotein with per-residue structural propensity, solvent accessibility, disordered probability, and early folding information. Scop3P, available at https://iomics.ugent.be/scop3p, presents a unique resource for visualization and analysis of phosphosites and for understanding of phosphosite structure-function relationships.

The CEP5 Peptide Promotes Abiotic Stress Tolerance, As Revealed by Quantitative Proteomics, and Attenuates the AUX/IAA Equilibrium in Arabidopsis.

Peptides derived from non-functional precursors play important roles in various developmental processes, but also in (a)biotic stress signaling. Our (phospho)proteome-wide analyses of C-TERMINALLY ENCODED PEPTIDE 5 (CEP5)-mediated changes revealed an impact on abiotic stress-related processes. Drought has a dramatic impact on plant growth, development and reproduction, and the plant hormone auxin plays a role in drought responses. Our genetic, physiological, biochemical, and pharmacological results demonstrated that CEP5-mediated signaling is relevant for osmotic and drought stress tolerance in Arabidopsis, and that CEP5 specifically counteracts auxin effects. Specifically, we found that CEP5 signaling stabilizes AUX/IAA transcriptional repressors, suggesting the existence of a novel peptide-dependent control mechanism that tunes auxin signaling. These observations align with the recently described role of AUX/IAAs in stress tolerance and provide a novel role for CEP5 in osmotic and drought stress tolerance.

COSS: A Fast and User-Friendly Tool for Spectral Library Searching.

Spectral similarity searching to identify peptide-derived MS/MS spectra is a promising technique, and different spectrum similarity search tools have therefore been developed. Each of these tools, however, comes with some limitations, mainly because of low processing speed and issues with handling large databases. Furthermore, the number of spectral data formats supported is typically limited, which also creates a threshold to adoption. We have therefore developed COSS (CompOmics Spectral Searching), a new and user-friendly spectral library search tool supporting two scoring functions. COSS also includes decoy spectra generation for result validation. We have benchmarked COSS on three different spectral libraries and compared the results with established spectral searching tools and a sequence database search tool. Our comparison showed that COSS more reliably identifies spectra, is capable of handling large data sets and libraries, and is an easy to use tool that can run on low computer specifications. COSS binaries and source code can be freely downloaded from https://github.com/compomics/COSS.

Bcl-2 and IP3 compete for the ligand-binding domain of IP3Rs modulating Ca2+ signaling output.

Bcl-2 proteins have emerged as critical regulators of intracellular Ca2+ dynamics by directly targeting and inhibiting the IP3 receptor (IP3R), a major intracellular Ca2+-release channel. Here, we demonstrate that such inhibition occurs under conditions of basal, but not high IP3R activity, since overexpressed and purified Bcl-2 (or its BH4 domain) can inhibit IP3R function provoked by low concentration of agonist or IP3, while fails to attenuate against high concentration of agonist or IP3. Surprisingly, Bcl-2 remained capable of inhibiting IP3R1 channels lacking the residues encompassing the previously identified Bcl-2-binding site (a.a. 1380-1408) located in the ARM2 domain, part of the modulatory region. Using a plethora of computational, biochemical and biophysical methods, we demonstrate that Bcl-2 and more particularly its BH4 domain bind to the ligand-binding domain (LBD) of IP3R1. In line with this finding, the interaction between the LBD and Bcl-2 (or its BH4 domain) was sensitive to IP3 and adenophostin A, ligands of the IP3R. Vice versa, the BH4 domain of Bcl-2 counteracted the binding of IP3 to the LBD. Collectively, our work reveals a novel mechanism by which Bcl-2 influences IP3R activity at the level of the LBD. This allows for exquisite modulation of Bcl-2's inhibitory properties on IP3Rs that is tunable to the level of IP3 signaling in cells.

The study of degradation mechanisms of glyco-engineered plant produced anti-rabies monoclonal antibodies E559 and 62-71-3.

Rabies is an ancient and neglected zoonotic disease caused by the rabies virus, a neurotropic RNA virus that belongs to the Rhabdoviridae family, genus Lyssavirus. It remains an important public health problem as there are cost and health concerns imposed by the current human post exposure prophylaxis therapy. The use of monoclonal antibodies (mAbs) is therefore an attractive alternative. Rabies mostly affects people that reside in resource-limited areas where there are occasional failures in the cold-chain. These environmental changes may upset the stability of the mAbs. This study focused on mAbs 62-71-3 and E559; their structures, responses to freeze/thaw (F/T) and exposure to reactive oxygen species were therefore studied with the aid of a wide range of biophysical and in silico techniques in order to elucidate their stability and identify aggregation prone regions. E559 was found to be less stable than 62-71-3. The complementarity determining regions (CDR) contributed the most to its instability, more specifically: peptides 99EIWD102 and 92ATSPYT97 found in CDR3, Trp33 found in CDR1 and the oxidised Met34. The constant region "158SWNSGALTGHTFPAVL175" was also flagged by the special aggregation propensity (SAP) tool and F/T experiments to be highly prone to aggregation. The E559 peptides "4LQESGSVL11 from the heavy chain and 4LTQSPSSL11 from the light chain, were also highly affected by F/T. These residues may serve as good candidates for mutation, in the aim to bring forward more stable therapeutic antibodies, thus paving a way to a more safe and efficacious antibody-based cocktail treatment against rabies.

Cross-linked peptide identification: A computational forest of algorithms.

Chemical cross-linking analyzed by mass spectrometry (XL-MS) has become an important tool in unravelling protein structure, dynamics, and complex formation. Because the analysis of cross-linked proteins with mass spectrometry results in specific computational challenges, many computational tools have been developed to identify cross-linked peptides from mass spectra and subsequently interpret the identified cross-links within their structural context. In this review, we will provide an overview of the different tools that are currently available to tackle the computational part of an XL-MS experiment. First, we give an introduction on the computational challenges encountered when processing data from a cross-linking experiment. We then discuss available tools to identify peptides that are linked by intact or MS-cleavable cross-linkers, and we provide an overview of tools to interpret cross-linked peptides in the context of protein structure. Finally, we give an outlook on data management and dissemination challenges and opportunities for cross-linking experiments.

The mutational landscape of MYCN, Lin28b and ALKF1174L driven murine neuroblastoma mimics human disease.

Genetically engineered mouse models have proven to be essential tools for unraveling fundamental aspects of cancer biology and for testing novel therapeutic strategies. To optimally serve these goals, it is essential that the mouse model faithfully recapitulates the human disease. Recently, novel mouse models for neuroblastoma have been developed. Here, we report on the further genomic characterization through exome sequencing and DNA copy number analysis of four of the currently available murine neuroblastoma model systems (ALK, Th-MYCN, Dbh-MYCN and Lin28b). The murine tumors revealed a low number of genomic alterations - in keeping with human neuroblastoma - and a positive correlation of the number of genetic lesions with the time to onset of tumor formation was observed. Gene copy number alterations are the hallmark of both murine and human disease and frequently affect syntenic genomic regions. Despite low mutational load, the genes mutated in murine disease were found to be enriched for genes mutated in human disease. Taken together, our study further supports the validity of the tested mouse models for mechanistic and preclinical studies of human neuroblastoma.

A double point mutation at residues Ile14 and Val15 of Bcl-2 uncovers a role for the BH4 domain in both protein stability and function.

B-cell lymphoma 2 (Bcl-2) protein is the archetype apoptosis suppressor protein. The N-terminal Bcl-2-homology 4 (BH4) domain of Bcl-2 is required for the antiapoptotic function of this protein at the mitochondria and endoplasmic reticulum (ER). The involvement of the BH4 domain in Bcl-2's antiapoptotic functions has been proposed based on Gly-based substitutions of the Ile14/Val15 amino acids, two hydrophobic residues located in the center of Bcl-2's BH4 domain. Following this strategy, we recently showed that a BH4-domain-derived peptide in which Ile14 and Val15 have been replaced by Gly residues, was unable to dampen proapoptotic Ca2+ -release events from the ER. Here, we investigated the impact of these mutations on the overall structure, stability, and function of full-length Bcl-2 as a regulator of Ca2+ signaling and cell death. Our results indicate that full-length Bcl-2 Ile14Gly/Val15Gly, in contrast to wild-type Bcl-2, (a) displayed severely reduced structural stability and a shortened protein half-life; (b) failed to interact with Bcl-2-associated X protein (BAX), to inhibit the inositol 1,4,5-trisphosphate receptor (IP3 R) and to protect against Ca2+ -mediated apoptosis. We conclude that the hydrophobic face of Bcl-2's BH4 domain (Ile14, Val15) is an important structural regulatory element by affecting protein stability and turnover, thereby likely reducing Bcl-2's ability to modulate the function of its targets, like IP3 R and BAX. Therefore, Bcl-2 structure/function studies require pre-emptive and reliable determination of protein stability upon introduction of point mutations at the level of the BH4 domain.

An extra dimension in protein tagging by quantifying universal proteotypic peptides using targeted proteomics.

The use of protein tagging to facilitate detailed characterization of target proteins has not only revolutionized cell biology, but also enabled biochemical analysis through efficient recovery of the protein complexes wherein the tagged proteins reside. The endogenous use of these tags for detailed protein characterization is widespread in lower organisms that allow for efficient homologous recombination. With the recent advances in genome engineering, tagging of endogenous proteins is now within reach for most experimental systems, including mammalian cell lines cultures. In this work, we describe the selection of peptides with ideal mass spectrometry characteristics for use in quantification of tagged proteins using targeted proteomics. We mined the proteome of the hyperthermophile Pyrococcus furiosus to obtain two peptides that are unique in the proteomes of all known model organisms (proteotypic) and allow sensitive quantification of target proteins in a complex background. By combining these 'Proteotypic peptides for Quantification by SRM' (PQS peptides) with epitope tags, we demonstrate their use in co-immunoprecipitation experiments upon transfection of protein pairs, or after introduction of these tags in the endogenous proteins through genome engineering. Endogenous protein tagging for absolute quantification provides a powerful extra dimension to protein analysis, allowing the detailed characterization of endogenous proteins.

Resolution of protein structure by mass spectrometry.

Typically, mass spectrometry is used to identify the peptides present in a complex peptide mixture and subsequently the precursor proteins. As such, mass spectrometry focuses mainly on the primary structure, the (modified) amino acid sequence of peptides and proteins. In contrast, the three-dimensional structure of a protein is typically determined with protein X-ray crystallography or NMR. Despite the close relationship between these two aspects of protein studies (sequence and structure), mass spectrometry and structure determination are not frequently combined. Nevertheless, this combination of approaches, dubbed conformational proteomics, can offer insight into the function, working mechanism, and conformational status of a protein. In this review, we will discuss the developments at the intersection of mass spectrometry-based proteomics and protein structure determination and start from a brief overview of the classic approaches to identify protein structure along with their advantages and disadvantages. We will subsequently discuss the ability of mass spectrometry to overcome some of the hurdles of these classic methods. Finally, we will provide an outlook on the interplay of mass spectrometry and protein structure determination, and highlight several recent experiments in which mass spectrometry was successfully used to either aid or complement structure elucidation. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 35:653-665, 2016.

Limited Proteolysis Combined with Stable Isotope Labeling Reveals Conformational Changes in Protein (Pseudo)kinases upon Binding Small Molecules.

Likely due to conformational rearrangements, small molecule inhibitors may stabilize the active conformation of protein kinases and paradoxically promote tumorigenesis. We combined limited proteolysis with stable isotope labeling MS to monitor protein conformational changes upon binding of small molecules. Applying this method to the human serine/threonine kinase B-Raf, frequently mutated in cancer, we found that binding of ATP or its nonhydrolyzable analogue AMP-PNP, but not ADP, stabilized the structure of both B-Raf(WT) and B-Raf(V600E). The ATP-competitive type I B-Raf inhibitor vemurafenib and the type II inhibitor sorafenib stabilized the kinase domain (KD) but had distinct effects on the Ras-binding domain. Stabilization of the B-Raf(WT) KD was confirmed by hydrogen/deuterium exchange MS and molecular dynamics simulations. Our results are further supported by cellular assays in which we assessed cell viability and phosphorylation profiles in cells expressing B-Raf(WT) or B-Raf(V600E) in response to vemurafenib or sorafenib. Our data indicate that an overall stabilization of the B-Raf structure by specific inhibitors activates MAPK signaling and increases cell survival, helping to explain clinical treatment failure. We also applied our method to monitor conformational changes upon nucleotide binding of the pseudokinase KSR1, which holds high potential for inhibition in human diseases.

Ryanodine receptors are targeted by anti-apoptotic Bcl-XL involving its BH4 domain and Lys87 from its BH3 domain.

Anti-apoptotic B-cell lymphoma 2 (Bcl-2) family members target several intracellular Ca(2+)-transport systems. Bcl-2, via its N-terminal Bcl-2 homology (BH) 4 domain, inhibits both inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), while Bcl-XL, likely independently of its BH4 domain, sensitizes IP3Rs. It remains elusive whether Bcl-XL can also target and modulate RyRs. Here, Bcl-XL co-immunoprecipitated with RyR3 expressed in HEK293 cells. Mammalian protein-protein interaction trap (MAPPIT) and surface plasmon resonance (SPR) showed that Bcl-XL bound to the central domain of RyR3 via its BH4 domain, although to a lesser extent compared to the BH4 domain of Bcl-2. Consistent with the ability of the BH4 domain of Bcl-XL to bind to RyRs, loading the BH4-Bcl-XL peptide into RyR3-overexpressing HEK293 cells or in rat hippocampal neurons suppressed RyR-mediated Ca(2+) release. In silico superposition of the 3D-structures of Bcl-2 and Bcl-XL indicated that Lys87 of the BH3 domain of Bcl-XL could be important for interacting with RyRs. In contrast to Bcl-XL, the Bcl-XL(K87D) mutant displayed lower binding affinity for RyR3 and a reduced inhibition of RyR-mediated Ca(2+) release. These data suggest that Bcl-XL binds to RyR channels via its BH4 domain, but also its BH3 domain, more specific Lys87, contributes to the interaction.

Scop3D: three-dimensional visualization of sequence conservation.

The integration of a protein's structure with its known sequence variation provides insight on how that protein evolves, for instance in terms of (changing) function or immunogenicity. Yet, collating the corresponding sequence variants into a multiple sequence alignment, calculating each position's conservation, and mapping this information back onto a relevant structure is not straightforward. We therefore built the Sequence Conservation on Protein 3D structure (scop3D) tool to perform these tasks automatically. The output consists of two modified PDB files in which the B-values for each position are replaced by the percentage sequence conservation, or the information entropy for each position, respectively. Furthermore, text files with absolute and relative amino acid occurrences for each position are also provided, along with snapshots of the protein from six distinct directions in space. The visualization provided by scop3D can for instance be used as an aid in vaccine development or to identify antigenic hotspots, which we here demonstrate based on an analysis of the fusion proteins of human respiratory syncytial virus and mumps virus.

Structural investigation of B-Raf paradox breaker and inducer inhibitors.

The V600E missense mutation in B-Raf kinase leads to an anomalous regulation of the MAPK pathway, uncontrolled cell proliferation, and initiation of tumorigenesis. While the ATP-competitive B-Raf inhibitors block the MAPK pathway in B-Raf mutant cells, they induce conformational changes to wild-type B-Raf kinase domain leading to heterodimerization with C-Raf causing a paradoxical hyperactivation of MAPK pathway. A new class of inhibitors (paradox breakers) has been developed that inhibit B-Raf(V600E) activity without agonistically affecting the MAPK pathway in wild-type B-Raf cells. In this study, we explore the structural, conformational, and cellular effects on the B-Raf kinase domain upon binding of paradox breakers and inducers. Our results indicate that a subtle structural difference between paradox inducers and breakers leads to significant conformational differences when complexed with B-Raf. This study provides a novel insight into the activation of B-Raf by ATP-competitive inhibitors and can aid in the design of more potent and selective inhibitors without agonistic function.

Getting intimate with trypsin, the leading protease in proteomics.

Nowadays, mass spectrometry-based proteomics is carried out primarily in a bottom-up fashion, with peptides obtained after proteolytic digest of a whole proteome lysate as the primary analytes instead of the proteins themselves. This experimental setup crucially relies on a protease to digest an abundant and complex protein mixture into a far more complex peptide mixture. Full knowledge of the working mechanism and specificity of the used proteases is therefore crucial, both for the digestion step itself as well as for the downstream identification and quantification of the (fragmentation) mass spectra acquired for the peptides in the mixture. Targeted protein analysis through selected reaction monitoring, a relative newcomer in the specific field of mass spectrometry-based proteomics, even requires a priori understanding of protease behavior for the proteins of interest. Because of the rapidly increasing popularity of proteomics as an analytical tool in the life sciences, there is now a renewed demand for detailed knowledge on trypsin, the workhorse protease in proteomics. This review addresses this need and provides an overview on the structure and working mechanism of trypsin, followed by a critical analysis of its cleavage behavior, typically simply accepted to occur exclusively yet consistently after Arg and Lys, unless they are followed by a Pro. In this context, shortcomings in our ability to understand and predict the behavior of trypsin will be highlighted, along with the downstream implications. Furthermore, an analysis is carried out on the inherent shortcomings of trypsin with regard to whole proteome analysis, and alternative approaches will be presented that can alleviate these issues. Finally, some reflections on the future of trypsin as the workhorse protease in mass spectrometry-based proteomics will be provided.