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Dysregulation of proteolytic enzymes has huge impact on epidermal homeostasis, which can result in severe pathological conditions such as fibrosis or Netherton syndrome. The metalloprotease meprin β was found to be upregulated in hyperproliferative skin diseases. AP-1 transcription factor complex has been reported to induce Mep1b expression. Since AP-1 and its subunit fos-related antigen 2 (fra-2) are associated with the onset and progression of psoriasis, we wanted to investigate if this could partially be attributed to increased meprin β activity. Here, we demonstrate that fra-2 transgenic mice show increased meprin β expression and proteolytic activity in the epidermis. To avoid influence by other fra-2 regulated genes, we additionally generated a mouse model that enabled tamoxifen-inducible expression of meprin β under the Krt5-promotor to mimic the pathological condition. Interestingly, induced meprin β expression in the epidermis resulted in hyperkeratosis, hair loss and mottled pigmentation of the skin. Employing N-terminomics revealed syndecan-1 as a substrate of meprin β in skin. Shedding of syndecan-1 at the cell surface caused delayed calcium-induced differentiation and impaired adhesion of keratinocytes, which was blocked by the meprin β inhibitor fetuin-B.

The identification of proteins below approximately 70-100 amino acids in bottom-up proteomics is still a challenging task due to the limited number of peptides generated by proteolytic digestion. This includes the short open reading frame-encoded peptides (SEPs), which are a subset of the small proteins that were not previously annotated or that are alternatively encoded. Here, we systematically investigated the use of multiple proteases (trypsin, chymotrypsin, LysC, LysargiNase, and GluC) in GeLC-MS/MS analysis to improve the sequence coverage and the number of identified peptides for small proteins, with a focus on SEPs, in the archaeon Methanosarcina mazei. Combining the data of all proteases, we identified 63 small proteins and additional 28 SEPs with at least two unique peptides, while only 55 small proteins and 22 SEP could be identified using trypsin only. For 27 small proteins and 12 SEPs, a complete sequence coverage was achieved. Moreover, for five SEPs, incorrectly predicted translation start points or potential in vivo proteolytic processing were identified, confirming the data of a previous top-down proteomics study of this organism. The results show clearly that a multi-protease approach allows to improve the identification and molecular characterization of small proteins and SEPs. LC-MS data: ProteomeXchange PXD023921.

Bacterial small RNAs (sRNAs) are well known to modulate gene expression by base pairing with trans‐encoded transcripts and are typically non‐coding. However, several sRNAs have been reported to also contain an open reading frame and thus are considered dual‐function RNAs. In this study, we discovered a dual‐function RNA from Vibrio cholerae, called VcdRP, harboring a 29 amino acid small protein (VcdP), as well as a base‐pairing sequence. Using a forward genetic screen, we identified VcdRP as a repressor of cholera toxin production and link this phenotype to the inhibition of carbon transport by the base‐pairing segment of the regulator. By contrast, we demonstrate that the VcdP small protein acts downstream of carbon transport by binding to citrate synthase (GltA), the first enzyme of the citric acid cycle. Interaction of VcdP with GltA results in increased enzyme activity and together VcdR and VcdP reroute carbon metabolism. We further show that transcription of vcdRP is repressed by CRP allowing us to provide a model in which VcdRP employs two different molecular mechanisms to synchronize central metabolism in V. cholerae., A screen for repressors of cholera toxin production identifies VcdPR as the first V. cholerae RNA that acts both as a non‐coding expression modulator and by encoding a small protein.

Meprin β is a membrane-bound metalloprotease involved in extracellular matrix assembly and inflammatory processes in health and disease. A disintegrin and metalloproteinase (ADAM)10 and ADAM17 are physiologic relevant sheddases of inactive promeprin β, which influences its substrate repertoire and subsequent biologic functions. Proteomic analysis also revealed several ADAMs as putative meprin β substrates. Here, we demonstrate specific N-terminal processing of ADAM9, 10, and 17 by meprin β and identify cleavage sites within their prodomains. Because ADAM prodomains can act as specific inhibitors, we postulate a role for meprin β in the regulation of ADAM activities. Indeed, prodomain cleavage by meprin β caused increased ADAM protease activities, as observed by peptide-based cleavage assays and demonstrated by increased ectodomain shedding activity. Direct interaction of meprin β and ADAM proteases could be shown by immunofluorescence microscopy and immunoprecipitation experiments. As demonstrated by a bacterial activator of meprin β and additional measurement of TNF-α shedding on bone marrow–derived macrophages, meprin β/ADAM protease interactions likely influence inflammatory conditions. Thus, we identified a novel proteolytic pathway of meprin β with ADAM proteases to control protease activities at the cell surface as part of the protease web.—Wichert, R., Scharfenberg, F., Colmorgen, C., Koudelka, T., Schwarz, J., Wetzel, S., Potempa, B., Potempa, J., Bartsch, J. W., Sagi, I., Tholey, A., Saftig, P., Rose-John, S., Becker-Pauly, C. Meprin β induces activities of A disintegrin and metalloproteinases 9, 10, and 17 by specific prodomain cleavage.

Detyrosination is a major post-translational modification of microtubules (MT), which has significant impact on MT function in cell division, differentiation, growth, migration, polarity, and intracellular trafficking. Detyrosination of α-tubulin occurs via the recently identified complex of vasohibin 1/2 (vash1/2) and small vasohibin binding protein (SVBP). However, there is still remaining detyrosinating activity in the absence of vash1/2/SVBP, and little is known about the regulation of detyrosination. Using cellular and cell-free assays we showed that the calcium-dependent proteases calpains 1 and 2 regulate MT detyrosination. We identified new calpain cleavage sites in the N-terminal disordered region of vash1 using in vitro proteolysis followed by mass spectrometry. However, this cleavage did not affect the detyrosination activity of vasohibin. In conclusion, the regulation of MT detyrosination by calpains occurs via another yet unknown tubulin carboxypeptidase. Importantly, calpains’ calcium dependency could allow a fine regulation of MT detyrosination. Thus, identifying the calpain-regulated pathway of MT detyrosination can be of major importance for several basic and clinical research and should be focused on in future studies.Summary StatementThe conventional calpains 1 and 2 play an important role in the regulation of microtubule detyrosination in a vasohibin independent way. Thus, they possibly control another still unknown tubulin carboxypeptidase.

Though proteases were long regarded as nonspecific degradative enzymes, over time, it was recognized that they also hydrolyse peptide bonds very specifically with a limited substrate pool. This irreversible posttranslational modification modulates the fate and activity of many proteins, making proteolytic processing a master switch in the regulation of e.g., the immune system, apoptosis and cancer progression. N- and C-terminomics, the identification of protein termini, has become indispensable in elucidating protease substrates and therefore protease function. Further, terminomics has the potential to identify yet unknown proteoforms, e.g. formed by alternative splicing or the recently discovered alternative ORFs. Different strategies and workflows have been developed that achieve higher sensitivity, a greater depth of coverage or higher throughput. In this review, we summarize recent developments in both N- and C-terminomics and include the potential of top-down proteomics which inherently delivers information on both ends of analytes in a single analysis.

Murine T cells express the GPI-anchored ADP-ribosyltransferase 2.2 (ARTC2.2) on the cell surface. In response to T cell activation or extracellular NAD+ or ATP-mediated gating of the P2X7 ion channel ARTC2.2 is shed from the cell surface as a soluble enzyme. Shedding alters the target specificity of ARTC2.2 from cell surface proteins to secreted proteins. Here we demonstrate that shed ARTC2.2 potently ADP-ribosylates IFN-γ in addition to other cytokines. Using mass spectrometry, we identify arginine 128 as the target site of ADP-ribosylation. This residue has been implicated to play a key role in binding of IFN-γ to the interferon receptor 1 (IFNR1). Indeed, binding of IFN-γ to IFNR1 blocks ADP-ribosylation of IFN-γ. Moreover, ADP-ribosylation of IFN-γ inhibits the capacity of IFN-γ to induce STAT1 phosphorylation in macrophages and upregulation of the proteasomal subunit ß5i and the proteasomal activator PA28-α in podocytes. Our results show that ADP-ribosylation inhibits the signaling functions of IFN-γ and point to a new regulatory mechanism for controlling signaling by IFN-γ.

Proteomics 21, 2000246 (1-5) (2021). doi:10.1002/pmic.202000246, The genome of coronaviruses, including SARS‐CoV‐2, encodes for two proteases, a papain like (PL$^{pro}$) protease and the so‐called main protease (M$^{pro}$), a chymotrypsin‐like cysteine protease, also named 3CL$^{pro}$ or non‐structural protein 5 (nsp5). Mpro is activated by autoproteolysis and is the main protease responsible for cutting the viral polyprotein into functional units. Aside from this, it is described that M$^{pro}$ proteases are also capable of processing host proteins, including those involved in the host innate immune response. To identify substrates of the three main proteases from SARS‐CoV, SARS‐CoV‐2, and hCoV‐NL63 coronviruses, an LC‐MS based N‐terminomics in vitro analysis is performed using recombinantly expressed proteases and lung epithelial and endothelial cell lysates as substrate pools. For SARS‐CoV‐2 M$^{pro}$, 445 cleavage events from more than 300 proteins are identified, while 151 and 331 M$^{pro}$ derived cleavage events are identified for SARS‐CoV and hCoV‐NL63, respectively. These data enable to better understand the cleavage site specificity of the viral proteases and will help to identify novel substrates in vivo. All data are available via ProteomeXchange with identifier PXD021406., Published by Wiley VCH, Weinheim

Myocardial inflammation has recently been recognized as a distinct feature of cardiac hypertrophy and heart failure. HectD3, a HECT domain containing E3 ubiquitin ligase has previously been investigated in the host defense against infections as well as neuroinflammation; its cardiac function however is still unknown. Here we show that HectD3 simultaneously attenuates Calcineurin-NFAT driven cardiomyocyte hypertrophy and the pro-inflammatory actions of LPS/interferon-γ via its cardiac substrates SUMO2 and Stat1, respectively. AAV9-mediated overexpression of HectD3 in mice in vivo not only reduced cardiac SUMO2/Stat1 levels and pathological hypertrophy but also largely abolished macrophage infiltration and fibrosis induced by pressure overload. Taken together, we describe a novel cardioprotective mechanism involving the ubiquitin ligase HectD3, which links anti-hypertrophic and anti-inflammatory effects via dual regulation of SUMO2 and Stat1. In a broader perspective, these findings support the notion that cardiomyocyte growth and inflammation are more intertwined than previously anticipated., Rangrez et al. show that overexpression of the HECT domain E3 ubiquitin protein ligase 3 (HectD3) reduces cardiac hypertrophy while reducing macrophage infiltration in mice. This study provides a cardioprotective mechanism, where HectD3 targets SUMO2 and Stat1 to exert its anti-hypertrophic and anti-inflammatory effects.

Summary A 5,000-year-old Yersinia pestis genome (RV 2039) is reconstructed from a hunter-fisher-gatherer (5300–5050 cal BP) buried at Riņņukalns, Latvia. RV 2039 is the first in a series of ancient strains that evolved shortly after the split of Y. pestis from its antecessor Y. pseudotuberculosis ∼7,000 years ago. The genomic and phylogenetic characteristics of RV 2039 are consistent with the hypothesis that this very early Y. pestis form was most likely less transmissible and maybe even less virulent than later strains. Our data do not support the scenario of a prehistoric pneumonic plague pandemic, as suggested previously for the Neolithic decline. The geographical and temporal distribution of the few prehistoric Y. pestis cases reported so far is more in agreement with single zoonotic events.

The genome of the halophilic archaeon Haloferax volcanii encodes more than 40 one-domain zinc finger µ-proteins. Only one of these, HVO_2753, contains four C(P)XCG motifs, suggesting the presence of two zinc binding pockets (ZBPs). Homologs of HVO_2753 are widespread in many euryarchaeota. An in frame deletion mutant of HVO_2753 grew indistinguishably from the wild-type in several media, but had a severe defect in swarming and in biofilm formation. For further analyses, the protein was produced homologously as well as heterologously in Escherichia coli. HVO_2753 was stable and folded in low salt, in contrast to many other haloarchaeal proteins. Only haloarchaeal HVO_2753 homologs carry a very hydrophilic N terminus, and NMR analysis showed that this region is very flexible and not part of the core structure. Surprisingly, both NMR analysis and a fluorimetric assay revealed that HVO_2753 binds only one zinc ion, despite the presence of two ZBPs. Notably, the analysis of cysteine to alanine mutant proteins by NMR as well by in vivo complementation revealed that all four C(P)XCG motifs are essential for folding and function. The NMR solution structure of the major conformation of HVO_2753 was solved. Unexpectedly, it was revealed that ZBP1 was comprised of C(P)XCG motifs 1 and 3, and ZBP2 was comprised of C(P)XCG motifs 2 and 4. There are several indications that ZBP2 is occupied by zinc, in contrast to ZBP1. To our knowledge, this study represents the first in-depth analysis of a zinc finger µ-protein in all three domains of life.

Proteolytic processing of membrane proteins by A disintegrin and metalloprotease-17 (ADAM17) is a key regulatory step in many physiological and pathophysiological processes. This so-called shedding is essential for development, regeneration and immune defense. An uncontrolled ADAM17 activity promotes cancer development, chronic inflammation and autoimmune diseases. Consequently, the ADAM17 activity is tightly regulated. As a final trigger for the shedding event a phosphatidylserine (PS) flip to the outer leaflet of the cell membrane was recently described. PS interacts with the extracellular part of ADAM17, which results in the shedding event by shifting the catalytic domain towards the membrane close to the cleavage sites within ADAM17 substrates. Our data indicate that the intrinsic proteolytic activity of the catalytic domain is prerequisite for the shedding activity and constantly present. However, the accessibility for substrate cleavage sites is controlled on several levels. In this report, we demonstrate that the positioning of the catalytic domain towards the cleavage sites is a crucial part of the shedding process. This finding contributes to the understanding of the complex and multilayered regulation of ADAM17 at the cell surface.

The identification and quantification of molecules involved in bacterial communication are major prerequisites for the understanding of interspecies interactions at the molecular level. We developed a procedure allowing the determination of 2-heptyl-4(1H)-quinolone (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) and the virulence factor pyocyanin (PYO) formed by the Gram-negative bacterium Pseudomonas aeruginosa. The method is based on dispersive liquid-liquid microextraction from small supernatant volumes (below 10 μL) followed by quantitative matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The use of ionic liquid matrix led to a lowered limit of detection for pyocyanin and, due to suppression of matrix background signals, easy to interpret mass spectra compared to crystalline matrices. Using an isotope-labeled pyocyanin standard synthesized in small-scale synthesis, quantitative analysis spanning approximately one order of magnitude (0.5 to 250 fmol) was feasible. The method was successfully applied to the detection of the signaling molecules PQS and HHQ in cultures of P. aeruginosa strains isolated from sputum of cystic fibrosis patients and allowed a highly sensitive quantification of PYO from these cultures. Hence, the developed method bears the potential to be used for screening purposes in clinical settings and will help to decipher the molecular basis of bacterial communication. Graphical abstract Ionic liquid matrices for the detection and quantification of the toxin pyocyanin and other signaling molecules from P. aeruginosa by MALDI MS.

Infectious agents such as lipopolysaccharides (LPS) challenge the functional properties of the alveolar‐capillary barrier (ACB) in the lung. In this study, we analyse the site‐specific effects of LPS on the ACB and reveal the effects on the individual cell types and the ACB as a functional unit. Monocultures of H441 epithelial cells and co‐cultures of H441 with endothelial cells cultured on Transwells® were treated with LPS from the apical or basolateral compartment. Barrier properties were analysed by the transepithelial electrical resistance (TEER), by transport assays, and immunostaining and assessment of tight junctional molecules at protein level. Furthermore, pro‐inflammatory cytokines and immune‐modulatory molecules were evaluated by ELISA and semiquantitative real‐time PCR. Liquid chromatography–mass spectrometry‐based proteomics (LS‐MS) was used to identify proteins and effector molecules secreted by endothelial cells in response to LPS. In co‐cultures treated with LPS from the basolateral compartment, we noticed a significant reduction of TEER, increased permeability and induction of pro‐inflammatory cytokines. Conversely, apical treatment did not affect the barrier. No changes were noticed in H441 monoculture upon LPS treatment. However, LPS resulted in an increased expression of pro‐inflammatory cytokines such as IL‐6 in OEC and in turn induced the reduction of TEER and an increase in SP‐A expression in H441 monoculture, and H441/OEC co‐cultures after LPS treatment from basolateral compartment. LS‐MS‐based proteomics revealed factors associated with LPS‐mediated lung injury such as ICAM‐1, VCAM‐1, Angiopoietin 2, complement factors and cathepsin S, emphasizing the role of epithelial–endothelial crosstalk in the ACB in ALI/ARDS.

Mass spectrometry based proteomics is an indispensable tool for almost all research areas relevant for the understanding of proteolytic processing, ranging from the identification of substrates, products and cleavage sites up to the analysis of structural features influencing protease activity. The majority of methods for these studies are based on bottom-up proteomics performing analysis at peptide level. As this approach is characterized by a number of pitfalls, e.g. loss of molecular information, there is an ongoing effort to establish top-down proteomics, performing separation and MS analysis both at intact protein level. We briefly introduce major approaches of bottom-up proteomics used in the field of protease research and highlight the shortcomings of these methods. We then discuss the present state-of-the-art of top-down proteomics. Together with the discussion of known challenges we show the potential of this approach and present a number of successful applications of top-down proteomics in protease research. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.

The shedding of ectodomains is a crucial mechanism in many physiological and pathological events. A disintegrin and metalloprotease-17 (ADAM17) is a key sheddase involved in essential processes, such as development, regeneration, and immune defense. ADAM17 exists in two conformations which differ in their disulfide connection in the membrane-proximal domain (MPD). Protein-disulfide isomerases (PDIs) on the cell surface convert the open MPD into a rigid closed form, which corresponds to inactive ADAM17. ADAM17 is expressed in its open activatable form in the endoplasmic reticulum (ER) and consequently must be protected against ER-resident PDI activity. Here, we show that the chaperone 78-kDa glucose-regulated protein (GRP78) protects the MPD against PDI-dependent disulfide-bond isomerization by binding to this domain and, thereby, preventing ADAM17 inhibition.

// Tillmann Bedau 1 , Neele Schumacher 1 , Florian Peters 1 , Johannes Prox 1 , Philipp Arnold 2 , Tomas Koudelka 3 , Ole Helm 4 , Frederike Schmidt 1 , Bjorn Rabe 1 , Marlene Jentzsch 5 , Philip Rosenstiel 5 , Susanne Sebens 4 , Andreas Tholey 3 , Stefan Rose-John 1 and Christoph Becker-Pauly 1 1 Unit for Degradomics of the Protease Web, Institute of Biochemistry, University of Kiel, 24118 Kiel, Germany 2 Anatomical Institute, University of Kiel, 24118 Kiel, Germany 3 Institute of Experimental Medicine, University of Kiel, 24118 Kiel, Germany 4 Institute for Experimental Tumor Research, University of Kiel, 24118 Kiel, Germany 5 Institute of Clinical Molecular Biology, University of Kiel, 24118 Kiel, Germany Correspondence to: Christoph Becker-Pauly, email: cbeckerpauly@biochem.uni-kiel.de Keywords: CD99, meprin β, proteolytic shedding, lung cancer, inflammation Received: January 18, 2017 Accepted: June 17, 2017 Published: July 04, 2017 ABSTRACT Transendothelial cell migration (TEM) is crucial for inflammation and metastasis. The adhesion molecule CD99 was shown to be important for correct immune cell extravasation and is highly expressed on certain cancer cells. Recently, we demonstrated that ectodomain shedding of CD99 by the metalloprotease meprin β promotes TEM in vitro . In this study, we employed an acute inflammation model (air pouch/carrageenan) and found significantly less infiltrated cells in meprin β knock-out animals validating the previously observed pro-inflammatory activity. To further analyze the impact of meprin β on CD99 shedding with regard to cell adhesion and proliferation we characterized two lung cancer associated CD99 variants (D92H, D92Y), carrying point mutations at the main cleavage site. Interestingly, ectodomain shedding of these variants by meprin β was still detectable. However the cleavage site shifted to adjacent positions. Nevertheless, expression of CD99 variants D92H and D92Y revealed partial misfolding and proteasomal degradation. A previously observed influence of CD99 on Src activation and increased proliferation could not be confirmed in this study, independent of wild-type CD99 or the variants D92H and D92Y. However, we identified meprin β as a potent inducer of Src phosphorylation. Importantly, we found significantly increased cell migration when expressing the cancer-associated CD99 variant D92H compared to the wild-type protein.

Inflammasomes are cytosolic protein complexes, which orchestrate the maturation of active IL-1 beta by proteolytic cleavage via caspase-1. Although many principles of inflammasome activation have been described, mechanisms that limit inflammasome-dependent immune responses remain poorly defined. Here, we show that the thiol-specific peroxidase peroxiredoxin-4 (Prdx4) directly regulates IL-1 beta generation by interfering with caspase-1 activity. We demonstrate that caspase-1 and Prdx4 form a redox-sensitive regulatory complex via caspase-1 cysteine 397 that leads to caspase-1 sequestration and inactivation. Mice lacking Prdx4 show an increased susceptibility to LPS-induced septic shock. This effect was phenocopied in mice carrying a conditional deletion of Prdx4 in the myeloid lineage (Prdx4-Delta LysMCre). Strikingly, we demonstrate that Prdx4 co-localizes with inflammasome components in extracellular vesicles (EVs) from inflammasome-activated macrophages. Purified EVs are able to transmit a robust IL-1 beta-dependent inflammatory response in vitro and also in recipient mice in vivo. Loss of Prdx4 boosts the pro-inflammatory potential of EVs. These findings identify Prdx4 as a critical regulator of inflammasome activity and provide new insights into remote cell-to-cell communication function of inflammasomes via macrophage-derived EVs.

Objective: In this study, using a Yeast two-hybrid screen, we identified the HECT domain-containing E3 ubiquitin ligase 3 (HECTD3) as one of the novel cardiac binding partners of SUMO2. Present study thus focusses on deciphering the cardiac function of HECTD3. Methods and Results: HECTD3-SUMO2 interaction was further validated by co-immunoprecipitation analysis. Interesting, adenovirus-mediated overexpression of HECTD3 in neonatal rat ventricular cardiomyocytes (NRVCMs) resulted in downregulation of endogenous as well as overexpressed SUMO2. Proteasome inhibitor MG132 treatment however inhibited HECTD3-mediated SUMO2 downregulation, suggesting SUMO2 as a likely substrate of HECTD3 in ubiquitin-proteasome system dependent protein degradation. To get further insights into cardiac substrates of HECTD3, we performed mass-spectrometry analysis of NRVCM protein lysate. Interestingly, several proteins from interferon signaling, including signal transducer and activator of transcription-1 (STAT1), were significantly downregulated by HECTD3 overexpression. Functional dissection of HECTD3-SUMO2-STAT1 interaction revealed attenuation of cellular hypertrophy and LPS mediated activation of inflammatory cascade in NRVCMs. Importantly, in vitro findings were consistent in vivo , where, AAV-9 mediated overexpression of HECTD3 substantially reduced cardiac hypertrophy and fibrosis, inhibited activation of STAT1-mediated downstream signaling, and attenuated infiltration of inflammatory cells to the heart after transverse-aortic constriction or Angiotensin II treatment. Conclusions: In conclusion, we describe here a novel cardioprotective mechanism involving the ubiquitin ligase HECTD3, which exerts anti-hypertrophic and anti-inflammatory effects via dual regulation of SUMO2 and its sumoylation target STAT1. This “dual pathway” inhibition may be exploited for the prevention and/or therapy of cardiac hypertrophy and heart failure.

The intramembrane proteases SPPL2a/b control pro-atherogenic signaling of membrane-bound proteolytic fragments derived from the oxLDL receptor LOX-1. In mice deficient for these proteases, plaque development and fibrosis is enhanced. This highlights SPPL2a/b as crucial players of a novel athero-protective mechanism, which is conserved in humans., The lectin-like oxidized LDL receptor 1 (LOX-1) is a key player in the development of atherosclerosis. LOX-1 promotes endothelial activation and dysfunction by mediating uptake of oxidized LDL and inducing pro-atherogenic signaling. However, little is known about modulators of LOX-1–mediated responses. Here, we show that the function of LOX-1 is controlled proteolytically. Ectodomain shedding by the metalloprotease ADAM10 and lysosomal degradation generate membrane-bound N-terminal fragments (NTFs), which we identified as novel substrates of the intramembrane proteases signal peptide peptidase–like 2a and b (SPPL2a/b). SPPL2a/b control cellular LOX-1 NTF levels which, following self-association via their transmembrane domain, can activate MAP kinases in a ligand-independent manner. This leads to an up-regulation of several pro-atherogenic and pro-fibrotic targets including ICAM-1 and the connective tissue growth factor CTGF. Consequently, SPPL2a/b-deficient mice, which accumulate LOX-1 NTFs, develop larger and more advanced atherosclerotic plaques than controls. This identifies intramembrane proteolysis by SPPL2a/b as a novel atheroprotective mechanism via negative regulation of LOX-1 signaling., Graphical Abstract

In the search for potential mechanisms underlying the remarkable resistance of healthy skin against infection by soil bacteria like Pseudomonas (P.) aeruginosa we identified fragments of the intrinsically disordered protein hornerin as potent microbicidal agents in the stratum corneum. We found that, independent of the amino acid (AA)-sequence, any tested linear cationic peptide containing a high percentage of disorder-promoting AA and a low percentage of order-promoting AA is a potent microbicidal antimicrobial. We further show that the antimicrobial activity of these cationic intrinsically disordered antimicrobial peptides (CIDAMPs) depends on the peptide chain length, its net charge, lipidation and environmental conditions. The ubiquitous presence of latent CIDAMP sources in nature suggests a common and yet overlooked adapted innate disinfection system of body surfaces. The simple structure and virtually any imaginable sequence or composition of disorder-promoting AA allow the generation of a plethora of CIDAMPs. These are potential novel microbicidal anti-infectives for various bacterial pathogens, including P. aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA) and fungal pathogens like Candida albicans and Cryptococcus neoformans.

Purpose Amyloidosis is a disease group caused by pathological aggregation and deposition of peptides in diverse tissue sites. Recently, matrix-assisted laser desorption/ionization mass spectrometry imaging coupled with ion mobility separation (MALDI-IMS MSI) was introduced as a novel tool to identify and classify amyloidosis using single sections from formalin-fixed and paraffin-embedded cardiac biopsies. Here, we tested the hypothesis that MALDI-IMS MSI can be applied to lung and gastrointestinal specimens. Experimental design Forty six lung and 65 gastrointestinal biopsy and resection specimens with different types of amyloid were subjected to MALDI-IMS MSI. Ninety three specimens included tissue areas without amyloid as internal negative controls. Nine cases without amyloid served as additional negative controls. Results Utilizing a peptide filter method and 21 known amyloid specific tryptic peptides we confirmed the applicability of a universal peptide signature with a sensitivity of 100% and a specificity of 100% for the detection of amyloid deposits in the lung and gastrointestinal tract. Additionally, the frequencies of individual m/z-values of the 21 tryptic marker peptides showed organ- and tissue-type specific differences. Conclusions and clinical relevance MALDI-IMS MSI adds a valuable analytical approach to diagnose and classify amyloid and the detection frequency of individual tryptic peptides is organ-/tissue-type specific.

Background We previously showed that the protein pattern of lenses removed in cataract surgery differs between patients with Parkinson's disease and age-matched controls. In this study, we identified the protein reduced in abundance in the 34- to 37-kDa gel band. Methods During cataract surgery (phacoemulsification), we collected the rinsing fluid and lens particles from the eyes of PD patients and controls. Residual lens fragments in the supernatant of 3 PD patients and aged-matched controls were studied for protein profiles using liquid chromatography-mass spectrometry and Western blots. Results We identified glyceraldehyde-3-phosphate dehydrogenase by mass spectrometry as the protein reduced in abundance and verified this finding in Western blots. Conclusions Glyceraldehyde-3-phosphate dehydrogenase has been implicated in PD development. The reduction of glyceraldehyde-3-phosphate dehydrogenase in the lenses of PD patients may be a new biomarker for PD and might also indicate an important role for this protein in PD development. © 2016 International Parkinson and Movement Disorder Society

Disintegrin and metalloproteinases (ADAMs) 10 and 17 can release the extracellular part of a variety of membrane-bound proteins via ectodomain shedding important for many biological functions. So far, substrate identification focused exclusively on membrane-anchored ADAM10 and ADAM17. However, besides known shedding of ADAM10, we identified ADAM8 as a protease capable of releasing the ADAM17 ectodomain. Therefore, we investigated whether the soluble ectodomains of ADAM10/17 (sADAM10/17) exhibit an altered substrate spectrum compared to their membrane-bound counterparts. A mass spectrometry-based N-terminomics approach identified 134 protein cleavage events in total and 45 common substrates for sADAM10/17 within the secretome of murine cardiomyocytes. Analysis of these cleavage sites confirmed previously identified amino acid preferences. Further in vitro studies verified fibronectin, cystatin C, sN-cadherin, PCPE-1 as well as sAPP as direct substrates of sADAM10 and/or sADAM17. Overall, we present the first degradome study for sADAM10/17, thereby introducing a new mode of proteolytic activity within the protease web.

Cationic intrinsically disordered antimicrobial peptides (CIDAMPs) belong to a novel class of epithelial peptide antibiotics with microbicidal activity against various pathogens, including Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans. Here we show that treatment of distinct bacteria with different hornerin (HRNR)-derived CIDAMPs cause formation of unique cytoplasmic protein aggregates, suggesting a common intracellular mode of action. We further found that, unlike most amphipathic antimicrobial peptides, HRNR traverses bacterial membranes energy-dependently and accumulates within the cytoplasm. Strikingly, certain structurally different, HRNR-based CIDAMPs were found to bind to an identical panel of distinct bacterial ribosomal proteins, thereby manifesting features of several known classes of antibiotics. This may cause the formation of aberrant proteins and toxic protein aggregates in HRNR-treated pathogens which eventually may induce its death. Our study reveals evidence that structurally distinct CIDAMPs of an abundant body surface protein simultaneously target multiple sites of the bacterial protein synthesis machinery.

The hepatitis B virus (HBV) is one of the most widespread human pathogens known today, yet its origin and evolutionary history are still unclear and controversial. Here, we report the analysis of three ancient HBV genomes recovered from human skeletons found at three different archaeological sites in Germany. We reconstructed two Neolithic and one medieval HBV genomes byde novoassembly from shotgun DNA sequencing data. Additionally, we observed HBV-specific peptides using paleo-proteomics. Our results show that HBV circulates in the European population for at least 7000 years. The Neolithic HBV genomes show a high genomic similarity to each other. In a phylogenetic network, they do not group with any human-associated HBV genome and are most closely related to those infecting African non-human primates. These ancient virus forms appear to represent distinct lineages that have no close relatives today and went possibly extinct. Our results reveal the great potential of ancient DNA from human skeletons in order to study the long-time evolution of blood borne viruses.

ARTC2.2 is a toxin-related, GPI-anchored ADP-ribosyltransferase expressed by murine T cells. In response to NAD+ released from damaged cells during inflammation, ARTC2.2 ADP-ribosylates and thereby gates the P2X7 ion channel. This induces ectodomain shedding of metalloprotease-sensitive cell surface proteins. In this study, we show that ARTC2.2 itself is a target for P2X7-triggered ectodomain shedding. We identify the metalloprotease cleavage site 3 aa upstream of the predicted GPI anchor attachment site of ARTC2.2. Intravenous injection of NAD+ increased the level of enzymatically active ARTC2.2 in serum, indicating that this mechanism is operative also under inflammatory conditions in vivo. Radio–ADP-ribosylation assays reveal that shedding refocuses the target specificity of ARTC2.2 from membrane proteins to secretory proteins. Our results uncover nucleotide-induced membrane-proximal proteolysis as a regulatory mechanism to control the substrate specificity of ARTC2.2.

Amyloids are pathological intra- and extracellular fibrillar aggregates of polypeptides with a cross-β-sheet structure and characteristic tinctorial properties. The amyloid deposits commonly enclose several non-fibrillar components of the extracellular matrix. Their potential to regulate the formation and aggregation process of amyloid fibrils is still poorly understood. For a better understanding of the role of the extracellular matrix in amyloidosis, it is essential to gain deeper insights into the composition of amyloid deposits. Here, we utilized matrix-assisted laser desorption and ionization mass spectrometry imaging to identify extracellular matrix compounds in amyloid deposits. Using this technique, we identified and determined the spatial distribution of vitronectin within AApoAI-, ALλ-, ATTR- and AIns amyloid deposits and, using immunohistochemistry, validated the spatial overlap of vitronectin with amyloids in 175 cases with diverse types of amyloid in several different tissues.

The nematode Caenorhabditis elegans interacts with a variety of bacteria as it feeds on microbes, and a number of these both associate and persist within the worm's intestine. Host-microbe interactions in C. elegans have been analyzed primarily at the transcriptome level with the host response often been monitored after challenge with pathogens. We assessed the proteome of C. elegans after growth on bacteria capable of colonizing its gut, via a comparative analysis of the nematode exposed to two naturally associated Ochrobactrum spp. (MYb71, MYb237) versus C. elegans grown on Escherichia coli OP50. A total of 4677 C. elegans proteins were identified, 3941 quantified. Significant alterations in protein abundances were observed for 122 proteins, 48 higher and 74 lower in abundance. We observed an increase in abundance of proteins potentially regulated via host signaling pathways, in addition to proteins involved in processing of foreign entities (e.g., lipase, proteases, glutathione metabolism). Decreased in abundance were proteins involved in both degradation and biosynthesis of amino acids, and enzymes associated with the degradation of peptidoglycan (lysozymes). The protein level differences between C. elegans grown on native microbiome members compared to the laboratory food bacterium may help to identify molecular processes involved in host-microbe interactions.

The largest numbers of commensal bacteria reside within our intestinal tract, with an increasing density from mouth to anus. Recently, a new estimate for the total number of bacteria (3.8×1013) in the 70 kg ‘reference man’ was reported.1 For human cells, the same authors revised past estimates to 3.0×1013 cells, out of which approximately 90% belong to the haematopoietic lineage. Hence, the widely cited 10:1 ratio of bacteria versus human cells received an update, showing that the number of bacteria in the body is actually of the same order as the number of human cells, and that the cumulative bacterial mass is about 200 g. Still, this large number of bacteria highlights their importance in maintaining health and metabolism. Different parts of the intestinal tract have different functions, tissue structure varies accordingly and gradients exist for several physicochemical parameters such as nutrients, pH or oxygen levels.2 Consequently, microbiota composition varies along the gut, but also between luminal and mucosa-attached communities of the same intestinal segment, and even along the crypt-villus axis in the epithelium. Thus, host–microbiota interactions are likely regionally specific and the local crosstalk determines intestinal function and physiology. Probably each human individual carries its own ‘microbial fingerprint’ (especially when considering genomic variation within the bacterial species’ populations), which is why forensic scientists started to exploit the use of this non-human organ.3 Recent large-scale analyses of population-based cohorts with >1000 samples validated that body mass index, age at sampling and gender are important covariates that need to be included in microbiome association analyses.4 In sum, Falony et al identified 18 covariates, including stool consistency, dietary factors and blood traits, cumulatively explaining 7.7% of the total variation in the gut microbiota, leaving 92.3% of the interindividual microbial variation unexplained. In a …

A novel archaeal lytic virus targeting species of the genus Methanosarcina was isolated using Methanosarcina mazei strain Gö1 as the host. Due to its spherical morphology, the virus was designated Met hanosarcina s pherical v irus (MetSV). Molecular analysis demonstrated that MetSV contains double-stranded linear DNA with a genome size of 10,567 bp containing 22 open reading frames (ORFs), all oriented in the same direction. Functions were predicted for some of these ORFs, i.e., such as DNA polymerase, ATPase, and DNA-binding protein as well as envelope (structural) protein. MetSV-derived spacers in CRISPR loci were detected in several published Methanosarcina draft genomes using bioinformatic tools, revealing a potential protospacer-adjacent motif (PAM) motif (TTA/T). Transcription and expression of several predicted viral ORFs were validated by reverse transcription-PCR (RT-PCR), PAGE analysis, and liquid chromatography-mass spectrometry (LC-MS)-based proteomics. Analysis of core lipids by atmospheric pressure chemical ionization (APCI) mass spectrometry showed that MetSV and Methanosarcina mazei both contain archaeol and glycerol dialkyl glycerol tetraether without a cyclopentane moiety (GDGT-0). The MetSV host range is limited to Methanosarcina strains growing as single cells ( M. mazei , Methanosarcina barkeri and Methanosarcina soligelidi ). In contrast, strains growing as sarcina-like aggregates were apparently protected from infection. Heterogeneity related to morphology phases in M. mazei cultures allowed acquisition of resistance to MetSV after challenge by growing cultures as sarcina-like aggregates. CRISPR/Cas-mediated resistance was excluded since neither of the two CRISPR arrays showed MetSV-derived spacer acquisition. Based on these findings, we propose that changing the morphology from single cells to sarcina-like aggregates upon rearrangement of the envelope structure prevents infection and subsequent lysis by MetSV. IMPORTANCE Methanoarchaea are among the most abundant organisms on the planet since they are present in high numbers in major anaerobic environments. They convert various carbon sources, e.g., acetate, methylamines, or methanol, to methane and carbon dioxide; thus, they have a significant impact on the emission of major greenhouse gases. Today, very little is known about viruses specifically infecting methanoarchaea that most probably impact the abundance of methanoarchaea in microbial consortia. Here, we characterize the first identified Methanosarcina -infecting virus (MetSV) and show a mechanism for acquiring resistance against MetSV. Based on our results, we propose that growth as sarcina-like aggregates prevents infection and subsequent lysis. These findings allow new insights into the virus-host relationship in methanogenic community structures, their dynamics, and their phase heterogeneity. Moreover, the availability of a specific virus provides new possibilities to deepen our knowledge of the defense mechanisms of potential hosts and offers tools for genetic manipulation.

The protease web is a synonym for highly regulated molecular networks comprising enzymes, substrates, inhibitors, and other regulatory proteins. Latest high-throughput methods provided huge data sets, revealing an amazing complexity of proteolytic systems important for health and disease. Based on our previous studies, we discuss major problems and questions that have to be solved to gain precise insight into the regulation of the protease web and its impact on pathophysiological conditions. The goal is a combination of different proteomic approaches that help to investigate specific protease function at a glance. Exemplarily, the characterization of the metalloproteases meprin α and meprin β by proteomic identification of cleavage sites and terminal amine isotopic labeling of substrates demonstrates the power of MS-based techniques. Meprins are rather orphan proteases and could not be assigned to precise biological functions until recently. Proteomics helped to identify meprin α and meprin β being important for collagen assembly and deposition in skin, which makes them potential therapeutic targets in fibrotic conditions. Additionally, identification of the cleavage site specificity provides the basis for the development of activity-based probes and small compound inhibitors, important for the regulation of meprin activity and subsequent treatment of associated diseases.

Quantitative mass spectrometry is a powerful tool for the determination of enzyme activities as it does not require labeled substrates and simultaneously allows for the identification of reaction products. However, major restrictions are the limited number of samples which can be measured in parallel due to the need for isotope labeled internal standards. Here we describe the use of metal labeling of peptides for the setup of multiplexed enzyme activity assays. After proteolytic reaction, using the protease trypsin, remaining substrates and peptide products formed in the reaction were labeled with metal chelators complexing rare earth metal ions. Labeled peptides were quantified with high accuracy and over a wide dynamic range (at least 2 orders of magnitude) using MALDI MS in case of simple peptide mixtures or by LC-MALDI MS for complex substrate mixtures and used for the monitoring of time-dependent product formation and substrate consumption. Due to multiplexing capabilities and accuracy, the presented approach will be useful for the determination of enzyme activities with a wide range of biochemical and biotechnological applications.

The determination of a protease's cleavage site specificity is one of the major goals in degradomics. In the last years, the use of proteome-derived peptide libraries and liquid chromatography-mass spectrometry (LC-MS) in a proteomic identification of protease cleavage sites (PICS) experiment became popular for that purpose.In this chapter, we offer a step-by-step protocol for the execution of a quantitative proteomic identification of protease cleavage sites (Q-PICS) experiment, which enables the relative quantification of proteolytic events by isobaric labeling, e.g., with tandem mass tags (TMT). In this way, the cleavage site specificity and activity of a protease can be compared under different reaction conditions (e.g., buffer, pH, temperature, inhibitor). Multiplexing can further be used to analyze replicate experiments in parallel, decreasing instrument times and work effort significantly, or to perform internal controls.

Signaling of the cytokine interleukin-6 (IL-6) via its soluble IL-6 receptor (sIL-6R) is responsible for the proinflammatory properties of IL-6 and constitutes an attractive therapeutic target, but how the sIL-6R is generated in vivo remains largely unclear. Here, we use liquid chromatography–mass spectrometry to identify an sIL-6R form in human serum that originates from proteolytic cleavage, map its cleavage site between Pro-355 and Val-356, and determine the occupancy of all O- and N-glycosylation sites of the human sIL-6R. The metalloprotease a disintegrin and metalloproteinase 17 (ADAM17) uses this cleavage site in vitro, and mutation of Val-356 is sufficient to completely abrogate IL-6R proteolysis. N- and O-glycosylation were dispensable for signaling of the IL-6R, but proteolysis was orchestrated by an N- and O-glycosylated sequon near the cleavage site and an N-glycan exosite in domain D1. Proteolysis of an IL-6R completely devoid of glycans is significantly impaired. Thus, glycosylation is an important regulator for sIL-6R generation., Author Summary Interleukin-6 (IL-6) is a cytokine secreted by our body upon infection or trauma to stimulate the immune system response. IL-6 is partially responsible for fever and triggers inflammation in many diseases. It activates its target cells via the membrane-bound IL-6 receptor (IL-6R), and soluble forms of this receptor (sIL-6R) are present in high amounts in the serum of healthy individuals and mediate the inflammatory response in all cells of the human body. However, it remains unclear how the soluble form of this cytokine is generated in humans. In this study, we isolate sIL-6R from human serum and show that the majority is produced via cleavage of the membrane-bound IL-6R by a protease. We identify the exact cleavage site and find that it is identical to a cleavage site used by the metalloprotease ADAM17. We further show that glycosylation, a post-transcriptional modification, is dispensable for the transport and biological function of IL-6R and map the occupancy of all O- and N-glycosylation sites. However, we find that only a single N-glycan is critically involved in the regulation of proteolysis by ADAM17 and conclude that glycosylation is an important regulator for sIL-6R generation.

The adhesion molecule CD99 is essential for the transendothelial migration of leukocytes. In this study, we used biochemical and cellular assays to show that CD99 undergoes ectodomain shedding by the metalloprotease meprin β and subsequent intramembrane proteolysis by γ-secretase. The cleavage site in CD99 was identified by mass spectrometry within an acidic region highly conserved through different vertebrate species. This finding fits perfectly to the unique cleavage specificity of meprin β with a strong preference for aspartate residues and suggests coevolution of protease and substrate. We hypothesized that limited CD99 cleavage by meprin β would alter cellular transendothelial migration (TEM) behavior in tissue remodeling processes, such as inflammation and cancer. Indeed, meprin β induced cell migration of Lewis lung carcinoma cells in an in vitro TEM assay. Accordingly, deficiency of meprin β in Mep1b-/- mice resulted in significantly increased CD99 protein levels in the lung. Therefore, meprin β could serve as a therapeutic target, given that in a proof-of-concept approach we showed accumulation of CD99 protein in lungs of meprin β inhibitor-treated mice.-Bedau, T., Peters, F., Prox, J., Arnold, P., Schmidt, F., Finkernagel, M., Kollmann, S., Wichert, R., Otte, A., Ohler, A., Stirnberg, M., Lucius, R., Koudelka, T., Tholey, A., Biasin, V., Pietrzik, C. U., Kwapiszewska, G., Becker-Pauly, C. Ectodomain shedding of CD99 within highly conserved regions is mediated by the metalloprotease meprin β and promotes transendothelial cell migration.

Background: A growing number of cardiac muscle diseases are characterized by depositions of misfolded proteins, including cardiac amyloidosis and desmin-releated cardiomyopathy (DRM). The continued presence and chronic accumulation of misfolded or unfolded proteins can lead to aggregation and/or the formation of soluble peptides that are proteotoxic. This in turn leads to compromised protein quality control and precipitates a downward spiral of the cell’s ability to maintain homeostasis and may eventually result in cell death. We recently identified massive protein aggregates in the hearts of transgenic mice overexpressing the intercalated disc (ID) protein myozap (Myozap-tg). We now sought to investigate the precise composition of these aggregates and the role of Myozap in other proteinopathies such as DRM. Methods and Results: We employed multi-dimensional proteomics, transcriptomics, confocal microscopy, and molecular biology approaches to decipher the underlying causes and consequences of protein aggregate formation in Myozap-tg mice. Transcriptome profiling of these mice revealed striking upregulation of autophagy, protein synthesis, and pro-inflammatory pathways, whereas protein degradation pathways were down-regulated. Surprisingly, proteomics analyses revealed Desmin and α-crystallin B (CryAB) as the major constituents of the aggregates, which was further validated by confocal microscopy. Moreover, we identified the presence of toxic preamyloid oligomers in Myozap-tg mouse hearts, a hallmark in many protein aggregation-based diseases including DRM. Most interestingly, we also observed co-localization of Myozap with protein aggregates observed in both transgenic mouse hearts overexpressing mutant Desmin (D7) and mutant CryAB (R120G), as well as in human DRM patients. Conclusion: The present study implies a new role for Myozap, which was previously reported to affect cardiac SRF signaling: (1) Myozap accumulates in various forms of experimental and human protein aggregation cardiomyopathy, suggesting involvement in protein homoestasis. (2) The fact that Myozap is now the third ID protein (after desmin and CryAB) to cause cardiac proteinopathy points to a general role of the ID in its molecular pathogenesis.

The identification of protein C-termini in complex proteomes is challenging due to the poor ionization efficiency of the carboxyl group. Amidating the negatively charged C-termini with ethanolamine (EA) has been suggested to improve the detection of C-terminal peptides and allows for a directed depletion of internal peptides after proteolysis using carboxyl reactive polymers. In the present study, the derivatization with N,N-dimethylethylenediamine (DMEDA) and (4-aminobutyl)guanidine (AG) leading to a positively charged C-terminus was investigated. C-terminal charge-reversed peptides showed improved coverage of b- and y-ion series in the MS/MS spectra compared to their noncharged counterparts. DMEDA-derivatized peptides resulted in many peptides with charge states of 3+, which benefited from ETD fragmentation. This makes the charge-reversal strategy particularly useful for the analysis of protein C-termini, which may also be post-translationally modified. The labeling strategy and the indirect enrichment of C-termini worked with similar efficiency for both DMEDA and EA, and their applicability was demonstrated on an E. coli proteome. Utilizing two proteases and different MS/MS activation mechanisms allowed for the identification of400 C-termini, encompassing both canonical and truncated C-termini.

Stromal factors play a critical role in the development of the mammary gland. Using a three dimensional-coculture model we demonstrate a significant role for stromal fibroblasts in the regulation of normal mammary epithelial morphogenesis and the control of tumor growth. Both soluble factors secreted by fibroblasts and fibroblast-derived modifications of the matrix compliance contribute to the regulation of epithelial cell morphogenesis. Readjustment of matrix tension by fibroblasts can even induce a phenotypic reversion of breast carcinoma cells. These data offer a basis to develop new strategies for the normalization of the tumor stroma as an innovative target in cancer therapy.

Knowledge of cleavage site specificity and activity are major prerequisites for understanding protease function. On the basis of a recently presented approach for proteomic identification of cleavage sites (PICS) in proteome-derived peptide libraries, we developed an isobaric labeling quantitative LC-MALDI-TOF/TOF MS/MS approach (Q-PICS) for simultaneous determination of cleavage site specificity and robust relative quantification of proteolytic events. For GluC-protease, 737 cleavage sites were identified in a yeast proteome-derived peptide library; 94.0% showed the typical GluC specificity for peptide bonds at glutamyl and aspartyl residues. The six-plex tandem mass tagging strategy allowed for three simultaneous replicates in a single run, guaranteeing high confidence and robust statistics for quantitative measurements. Using the quantitative capacity of Q-PICS, we performed a comparison of cleavage site specificity of GluC in two different buffer systems. The results support earlier findings describing that apparent difference between the buffer systems are probably caused by the inhibitory effect of bicarbonate on the overall GluC activity and that the preference for Glu-X bonds compared to Asp-X bonds is independent of the buffer system used.

Protease-catalyzed hydrolysis of peptide bonds is one of the most pivotal post-translational modifications fulfilling manifold functions in the regulation of cellular processes. Therefore, dysregulation of proteolytic reactions plays a central role in many pathophysiological events. For this reason, understanding the molecular mechanisms in proteolytic reactions, in particular the knowledge of proteases involved in complex processes, expression levels and activity of protease and knowledge of the targeted substrates are an indispensable prerequisite for targeted drug development. The present review focuses on mass spectrometry-based proteomic methods for the analysis of protease cleavage sites, including the identification of the hydrolyzed bonds as well as of the surrounding sequence. Peptide- and protein-centric approaches and bioinformatic tools for experimental data interpretation will be presented and the major advantages and drawbacks of the different approaches will be addressed. The recent applications of these approaches for the analysis of biological function of different protease classes and potential future directions will be discussed.

Relative protein abundances of Escherichia coli MG1655 growing exponentially on minimal medium with acetate or glucose as the sole carbon source were investigated in a quantitative shotgun proteome analysis with TMT6-plex isobaric tags. Peptides were separated by high resolution high/low pH 2D-LC, using an optimized fraction pooling scheme followed by mass spectrometric analysis. Quantitative data were acquired for 2099 proteins covering 49% of the predicted E. coli proteins, showing system-wide effects of growth conditions. In total, 507 proteins showed a fold change of at least 1.5 and 205 proteins changed by more than twofold. Significant differences in abundance were observed for most of the proteins in the central carbon metabolism and in proteins relevant for amino acid and protein synthesis, processing of environmental information and scavenging of a variety of alternate carbon sources. Periplasmic-binding proteins were also more abundant on acetate, especially proteins involved in scavenging extracellular resources such as sugars. All MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (dataset identifier PXD003863).

The whey protein β-lactoglobulin has been proposed as a transporter for covalent bound bioactive compounds in order to enhance their stability and reduce their sensory perception. The garlic derived compounds allicin and diallyl disulfide were bound covalently to the native and heat denatured protein. The binding site and the influence of the modification on the digestibility were determined by mass spectrometric analysis of the modified β-lactoglobulin. Further, the conformation of the modified protein was assessed by circular dichroism and dynamic light scattering. The free thiol group of Cys(121) turned out to be the major binding site. After proteolysis with trypsin at pH 7 but not with pepsin at pH 2, a limited transfer to other cysteinyl residues was observed. The covalently bound ligands did not mask any proteolytic cleavage sites of pepsin, trypsin or chymotrypsin. The modified β-lactoglobulin showed a native like conformation, besides a moderate loosening of protein folding. The covalent binding of organosulfur compounds to β-lactoglobulin provides a bioactive ingredient without impairing the digestibility and functional properties of the protein.

ADAM17, a prominent member of the ‘Disintegrin and Metalloproteinase' (ADAM) family, controls vital cellular functions through cleavage of transmembrane substrates. Here we present evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. PS exposure is tightly coupled to substrate shedding provoked by diverse ADAM17 activators. PS dependency is demonstrated in the following: (a) in Raji cells undergoing apoptosis; (b) in mutant PSA-3 cells with manipulatable PS content; and (c) in Scott syndrome lymphocytes genetically defunct in their capacity to externalize PS in response to intracellular Ca2+ elevation. Soluble phosphorylserine but not phosphorylcholine inhibits substrate cleavage. The isolated membrane proximal domain (MPD) of ADAM17 binds to PS but not to phosphatidylcholine liposomes. A cationic PS-binding motif is identified in this domain, replacement of which abrogates liposome-binding and renders the protease incapable of cleaving its substrates in cells. We speculate that surface-exposed PS directs the protease to its targets where it then executes its shedding function., ADAM17 is a member of the ‘Disintegrin and Metalloproteinase' family of proteases, that cleaves transmembrane substrates from the surfaces of cells. Here the authors show that surface exposure of phosphatidylserine is required for ADAM17 sheddase activity, possibly by directing the protease to its substrates.

Amyloidosis is a group of diseases caused by extracellular accumulation of fibrillar polypeptide aggregates. So far, diagnosis is performed by Congo red staining of tissue sections in combination with polarization microscopy. Subsequent identification of the causative protein by immunohistochemistry harbors some difficulties regarding sensitivity and specificity. Mass spectrometry based approaches have been demonstrated to constitute a reliable method to supplement typing of amyloidosis, but still depend on Congo red staining. In the present study, we used matrix-assisted laser desorption/ionization mass spectrometry imaging coupled with ion mobility separation (MALDI-IMS MSI) to investigate amyloid deposits in formalin-fixed and paraffin-embedded tissue samples. Utilizing a novel peptide filter method, we found a universal peptide signature for amyloidoses. Furthermore, differences in the peptide composition of ALλ and ATTR amyloid were revealed and used to build a reliable classification model. Integrating the peptide filter in MALDI-IMS MSI analysis, we developed a bioinformatics workflow facilitating the identification and classification of amyloidosis in a less time and sample-consuming experimental setup. Our findings demonstrate also the feasibility to investigate the amyloid's protein composition, thus paving the way to establish classification models for the diverse types of amyloidoses and to shed further light on the complex process of amyloidogenesis.