Neutrophils play a pivotal role in the defense against bacterial, viral and fungal infections and are important mediators in the acute inflammatory response. At the same time, neutrophils are also involved in sterile inflammatory responses that are triggered by endogenous ligands. A series of immediate effector functions and the expression of proinflammatory genes enable neutrophils to initiate the immune response against the injurious agent. Among these, interleukin-1b (IL-1b) plays a key role in the orchestration of the inflammatory response. Induction of IL-1b expression leads to production of cytosolic pro-IL-1b, which requires further processing by a proteolytic cleavage event. Caspase-1 was initially identified as the main IL-1b-converting enzyme, and the upstream events leading to caspase-1 activation were identified as so-called inflammasome complexes. Up to now, the inflammasome system has mainly been studied in macrophages, whereas the inflammasome was thought to play a redundant or no role in the cell intrinsic processing of pro-IL-1b in neutrophils. Here, we identify the expression of the components of the NLRP3 inflammasome complex in neutrophils and show that the NLRP3 inflammasome pathway is indeed operational in neutrophils. Our findings establish the NLRP3 inflammasome as a key step in the secretion of matured IL-1b by neutrophils.

We present a concise workflow to enhance the mass spectrometric detection of crosslinked peptides by introducing sequential digestion and the crosslink identification software xiSEARCH. Sequential digestion enhances peptide detection by selective shortening of long tryptic peptides. We demonstrate our simple 12‐fraction protocol for crosslinked multi‐protein complexes and cell lysates, quantitative analysis, and high‐density crosslinking, without requiring specific crosslinker features. This overall approach reveals dynamic protein–protein interaction sites, which are accessible, have fundamental functional relevance and are therefore ideally suited for the development of small molecule inhibitors.

Neutrophil granulocytes are important mediators of innate immunity, but also participate in the pathogenesis of (auto)inflammatory diseases. Neutrophils express a specific set of proteolytic enzymes, the neutrophil serine proteases (NSPs), which are stored in cytoplasmic granules and can be secreted into the extra- and pericellular space upon cellular activation. These NSPs, namely cathepsin G (CG), neutrophil elastase (NE), and proteinase 3 (PR3), have early been implicated in bacterial defense. However, NSPs also regulate the inflammatory response by specifically altering the function of cytokines and chemokines. For instance, PR3 and NE both inactivate the anti-inflammatory mediator progranulin, which may play a role in chronic inflammation. Here, we provide a concise update on NSPs as modulators of inflammation and discuss the biological and pathological significance of this novel function of NSPs. Mounting evidence support an important proinflammatory function for PR3, which may have been underestimated in the past.Electronic supplementary materialThe online version of this article (doi:10.1007/s00109-010-0677-3) contains supplementary material, which is available to authorized users.

We present a concise workflow to enhance the mass spectrometric detection of crosslinked peptides by introducing sequential digestion and the crosslink identification software Xi. Sequential digestion enhances peptide detection by selective shortening of long tryptic peptides. We demonstrate our simple 12-fraction protocol for crosslinked multi-protein complexes and cell lysates, quantitative analysis, and high-density crosslinking, without requiring specific crosslinker features. This overall approach reveals dynamic protein-protein interaction sites, which are accessible, have fundamental functional relevance and are therefore ideally suited for the development of small molecule inhibitors.Crosslinking mass spectrometry (CLMS) has become a standard tool for the topological analysis of multi-protein complexes and has begun delivering high-density information on protein structures, insights into structural changes and the wiring of interaction networks in situ 1 .The technological development currently focuses on enrichment strategies for crosslinked peptides and mass spectrometric data acquisition 2-4 , including newly designed crosslinkers 5 .

BackgroundAnaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma is characterized by the t(2;5) chromosomal translocation, resulting in the expression of a fusion protein formed of nucleophosmin (NPM) and ALK. Recently, we reported the abnormal expression of the transcription factor CCAAT/enhancer binding protein-beta (C/EBPβ) in ALK-positive anaplastic large cell lymphomas, and demonstrated its dependence on NPM-ALK activity. Design and MethodsIn this study, the role of C/EBPβ in proliferation and survival of ALK-positive anaplastic large cell lymphomas was investigated, as well as the mechanism of its expression and activity. Highly effective short hairpin RNA sequences and/or pharmacological inhibitors were used to abrogate the expression or activity of C/EBPβ, signal transducer and activator of transcription 3 (STAT3), AKT, extracellular signal-related kinase 1/2 (ERK1/2) and mammalian target of rapamycin (mTOR). ResultsInterference with C/EBPβ expression resulted in a dramatic decrease in cell proliferation in ALK-positive anaplastic large cell lymphomas, with a mild induction of apoptosis after 6 days. Down-regulation of STAT3 resulted in a marked decrease in C/EBPβ mRNA and protein levels with impairment in cell proliferation and viability, underscoring the important role of these two proteins in ALK-mediated oncogenesis. Additionally, we demonstrated that reduction of ERK1/2 activity led to C/EBPβ Thr 235 dephosphorylation and moderate growth retardation. The AKT/mTOR signaling pathway did not have any influence on C/EBPβ expression or C/EBPβ phosphorylation. ConclusionsThese findings reveal the convergence of STAT3 and ERK1/2 signaling pathways activated by NPM-ALK in mediating the regulation of C/EBPβ expression, a transcription factor central to NPM-ALK transformation.Key words: anaplastic large cell lymphoma (ALCL), C/EBPβ, STAT3, RNA interference, cell proliferation. STAT3 signaling pathway. Haematologica 2010;95:760-767. doi:10.3324/haematol.2009 This is an open-access paper. © F e r r a t a S t o r t i F o u n d a t i o n Citation: Anastasov N, Bonzheim I, Rudelius M, Klier M, Dau T, Angermeier D, Duyster J, Pittaluga S, Fend F, Raffeld M, and Quintanilla-Martinez L. C/EBPβ expression in ALK-positive anaplastic large cell lymphomas is required for cell proliferation and is induced by the C/EBPβ expression in ALK-positive anaplastic large cell lymphomas is required for cell proliferation and is induced by the STAT3 signaling pathway

Cross-linking mass spectrometry has become an important approach for studying protein structures and protein–protein interactions. The amino acid compositions of some protein regions impede the detection of cross-linked residues, although it would yield invaluable information for protein modeling. Here, we report on a sequential-digestion strategy with trypsin and elastase to penetrate regions with a low density of trypsin-cleavage sites. We exploited intrinsic substrate-recognition properties of elastase to specifically target larger tryptic peptides. Our application of this protocol to the TAF4–12 complex allowed us to identify cross-links in previously inaccessible regions.

While AlphaFold2 can predict accurate protein structures from the primary sequence, challenges remain for proteins that undergo conformational changes or for which few homologous sequences are known. Here we introduce AlphaLink, a modified version of the AlphaFold2 algorithm that incorporates experimental distance restraint information into its network architecture. By employing sparse experimental contacts as anchor points, AlphaLink improves on the performance of AlphaFold2 in predicting challenging targets. We confirm this experimentally by using the noncanonical amino acid photo-leucine to obtain information on residue–residue contacts inside cells by crosslinking mass spectrometry. The program can predict distinct conformations of proteins on the basis of the distance restraints provided, demonstrating the value of experimental data in driving protein structure prediction. The noise-tolerant framework for integrating data in protein structure prediction presented here opens a path to accurate characterization of protein structures from in-cell data.

Large artery atherosclerotic stroke (LAS) shows substantial heritability not explained by previous genome-wide association studies. Here, we explore the role of coding variation in LAS by analyzing variants on the HumanExome BeadChip in a total of 3,127 cases and 9,778 controls from Europe, Australia, and South Asia. We report on a nonsynonymous single-nucleotide variant in serpin family A member 1 (SERPINA1) encoding alpha-1 antitrypsin [AAT; p.V213A; P = 5.99E-9, odds ratio (OR) = 1.22] and confirm histone deacetylase 9 (HDAC9) as a major risk gene for LAS with an association in the 3′-UTR (rs2023938; P = 7.76E-7, OR = 1.28). Using quantitative microscale thermophoresis, we show that M1 (A213) exhibits an almost twofold lower dissociation constant with its primary target human neutrophil elastase (NE) in lipoprotein-containing plasma, but not in lipid-free plasma. Hydrogen/deuterium exchange combined with mass spectrometry further revealed a significant difference in the global flexibility of the two variants. The observed stronger interaction with lipoproteins in plasma and reduced global flexibility of the Val-213 variant most likely improve its local availability and reduce the extent of proteolytic inactivation by other proteases in atherosclerotic plaques. Our results indicate that the interplay between AAT, NE, and lipoprotein particles is modulated by the gate region around position 213 in AAT, far away from the unaltered reactive center loop (357-360). Collectively, our findings point to a functionally relevant balance between lipoproteins, proteases, and AAT in atherosclerosis.genetics | ischemic stroke | large artery stroke | antitrypsin | variation S troke is the leading cause of long-term disability and the second most common cause of death worldwide (1, 2). About a quarter of ischemic stroke cases are caused by large artery atherosclerotic stroke (LAS) (3, 4). Atherosclerosis is a chronic inflammatory condition that involves a number of well-characterized steps. Initial stages include the deposition of lipids in vascular endothelial cells, whereas more advanced stages are characterized by fibrotic changes with formation of a fibrotic cap and, eventually, plaque rupture (5). LAS exhibits the highest heritability of all stroke subtypes, with estimates ranging from 40.3 to 66.6% (6, 7). This fact is reflected by recent genome-wide association studies that found common variants for LAS at multiple genomic loci (8-10). The lead SNPs from these regions all reside within intergenic (4, 7, 11) or SignificanceCommon single-amino acid variations of proteins are traditionally regarded as functionally neutral polymorphisms because these substitutions are mostly located outside functionally relevant surfaces. In this study, we present an example of a functionally relevant coding sequence variation, which, as we show here, confers risk for large artery atherosclerotic stroke. The singleresidue variation M1(A213V) in serpin family A member 1 (SERPINA1) [encoding alpha-1 antitrypsin (AAT)] is situated outside the...