Phosphorylation of the VAR2CSA extracellular region is associated with enhanced adhesive properties to the placental receptor CSA.

Plasmodium falciparum is the main cause of disease and death from malaria. P. falciparum virulence resides in the ability of infected erythrocytes (IEs) to sequester in various tissues through the interaction between members of the polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesin family to various host receptors. Here, we investigated the effect of phosphorylation of variant surface antigen 2-CSA (VAR2CSA), a member of the PfEMP1 family associated to placental sequestration, on its capacity to adhere to chondroitin sulfate A (CSA) present on the placental syncytium. We showed that phosphatase treatment of IEs impairs cytoadhesion to CSA. MS analysis of recombinant VAR2CSA phosphosites prior to and after phosphatase treatment, as well as of native VAR2CSA expressed on IEs, identified critical phosphoresidues associated with CSA binding. Site-directed mutagenesis on recombinant VAR2CSA of 3 phosphoresidues localised within the CSA-binding region confirmed in vitro their functional importance. Furthermore, using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9), we generated a parasite line in which the phosphoresidue T934 is changed to alanine and showed that this mutation strongly impairs IEs cytoadhesion to CSA. Taken together, these results demonstrate that phosphorylation of the extracellular region of VAR2CSA plays a major role in IEs cytoadhesion to CSA and provide new molecular insights for strategies aiming to reduce the morbidity and mortality of PM.

Drug-induced increase in lysobisphosphatidic acid reduces the cholesterol overload in Niemann-Pick type C cells and mice.

Most cells acquire cholesterol by endocytosis of circulating low-density lipoproteins (LDLs). After cholesteryl ester de-esterification in endosomes, free cholesterol is redistributed to intracellular membranes via unclear mechanisms. Our previous work suggested that the unconventional phospholipid lysobisphosphatidic acid (LBPA) may play a role in modulating the cholesterol flux through endosomes. In this study, we used the Prestwick library of FDA-approved compounds in a high-content, image-based screen of the endosomal lipids, lysobisphosphatidic acid and LDL-derived cholesterol. We report that thioperamide maleate, an inverse agonist of the histamine H3 receptor HRH3, increases highly selectively the levels of lysobisphosphatidic acid, without affecting any endosomal protein or function that we tested. Our data also show that thioperamide significantly reduces the endosome cholesterol overload in fibroblasts from patients with the cholesterol storage disorder Niemann-Pick type C (NPC), as well as in liver of Npc1-/- mice. We conclude that LBPA controls endosomal cholesterol mobilization and export to cellular destinations, perhaps by fluidifying or buffering cholesterol in endosomal membranes, and that thioperamide has repurposing potential for the treatment of NPC.

Lipidomics reveals diurnal lipid oscillations in human skeletal muscle persisting in cellular myotubes cultured in vitro.

Circadian clocks play an important role in lipid homeostasis, with impact on various metabolic diseases. Due to the central role of skeletal muscle in whole-body metabolism, we aimed at studying muscle lipid profiles in a temporal manner. Moreover, it has not been shown whether lipid oscillations in peripheral tissues are driven by diurnal cycles of rest-activity and food intake or are able to persist in vitro in a cell-autonomous manner. To address this, we investigated lipid profiles over 24 h in human skeletal muscle in vivo and in primary human myotubes cultured in vitro. Glycerolipids, glycerophospholipids, and sphingolipids exhibited diurnal oscillations, suggesting a widespread circadian impact on muscle lipid metabolism. Notably, peak levels of lipid accumulation were in phase coherence with core clock gene expression in vivo and in vitro. The percentage of oscillating lipid metabolites was comparable between muscle tissue and cultured myotubes, and temporal lipid profiles correlated with transcript profiles of genes implicated in their biosynthesis. Lipids enriched in the outer leaflet of the plasma membrane oscillated in a highly coordinated manner in vivo and in vitro. Lipid metabolite oscillations were strongly attenuated upon siRNA-mediated clock disruption in human primary myotubes. Taken together, our data suggest an essential role for endogenous cell-autonomous human skeletal muscle oscillators in regulating lipid metabolism independent of external synchronizers, such as physical activity or food intake.

Cyclodextrin triggers MCOLN1-dependent endo-lysosome secretion in Niemann-Pick type C cells.

In specialized cell types, lysosome-related organelles support regulated secretory pathways, whereas in nonspecialized cells, lysosomes can undergo fusion with the plasma membrane in response to a transient rise in cytosolic calcium. Recent evidence also indicates that lysosome secretion can be controlled transcriptionally and promote clearance in lysosome storage diseases. In addition, evidence is also accumulating that low concentrations of cyclodextrins reduce the cholesterol-storage phenotype in cells and animals with the cholesterol storage disease Niemann-Pick type C, via an unknown mechanism. Here, we report that cyclodextrin triggers the secretion of the endo/lysosomal content in nonspecialized cells and that this mechanism is responsible for the decreased cholesterol overload in Niemann-Pick type C cells. We also find that the secretion of the endo/lysosome content occurs via a mechanism dependent on the endosomal calcium channel mucolipin-1, as well as FYCO1, the AP1 adaptor, and its partner Gadkin. We conclude that endo-lysosomes in nonspecialized cells can acquire secretory functions elicited by cyclodextrin and that this pathway is responsible for the decrease in cholesterol storage in Niemann-Pick C cells.

Antibody-based methods for the measurement of α-synuclein concentration in human cerebrospinal fluid - method comparison and round robin study.

α-Synuclein is the major component of Lewy bodies and a candidate biomarker for neurodegenerative diseases in which Lewy bodies are common, including Parkinson's disease and dementia with Lewy bodies. A large body of literature suggests that these disorders are characterized by reduced concentrations of α-synuclein in cerebrospinal fluid (CSF), with overlapping concentrations compared to healthy controls and variability across studies. Several reasons can account for this variability, including technical ones, such as inter-assay and inter-laboratory variation (reproducibility). We compared four immunochemical methods for the quantification of α-synuclein concentration in 50 unique CSF samples. All methods were designed to capture most of the existing α-synuclein forms in CSF ('total' α-synuclein). Each of the four methods showed high analytical precision, excellent correlation between laboratories (R2 0.83-0.99), and good correlation with each other (R2 0.64-0.93), although the slopes of the regression lines were different between the four immunoassays. The use of common reference CSF samples decreased the differences in α-synuclein concentration between detection methods and technologies. Pilot data on an immunoprecipitation mass spectrometry (IP-MS) method is also presented. Our results suggest that the four immunochemical methods and the IP-MS method measure similar forms of α-synuclein and that a common reference material would allow harmonization of results between immunoassays.

Protein Corona: Impact of Lymph Versus Blood in a Complex In Vitro Environment.

In biological environments, the surface of nanoparticles (NPs) are modified by protein corona (PC) that determines their biological behavior. Unfortunately, in vitro tests still give different PC than in vivo tests causing in vitro-in vivo discrepancy; hence, in vitro studies are not indicative for the NPs' behavior in vivo. Here is demonstrated that PC in vitro is strongly influenced by the type of extracellular fluid (ECF), blood or lymph, by their high and low flow conditions and transitions between ECFs, and a combination of these parameters. As a result, this in vitro study approaches fluidic and dynamic variations to which NPs are exposed in vivo: different ECF that NPs encounter first in different injection routes, different transitions in-between ECFs during circulation, and simultaneous change in the exposed flow in these transitions. The most-abundant proteins in PCs are found to be not the most abundant in ECFs, but those having high affinity for binding to the surface of NPs. Moreover, some proteins are differently abundant in PCs at different flows, which indicate force-promoted binding, catch bonds. These results suggest that future in vitro studies should consider more complex incubation conditions to improve the in vitro-in vivo consistency necessary for translational research.

Sexual dimorphism in hepatic lipids is associated with the evolution of metabolic status in mice.

Ectopic lipid accumulation in the liver is implicated in metabolic disease in an age- and sex-dependent manner. The role of hepatic lipids has been well established within the scope of metabolic insults in mice, but has been insufficiently characterized under standard housing conditions, where age-related metabolic alterations are known to occur. We studied a total of 10 male and 10 female mice longitudinally. At 3, 7 and 11 months of age, non-invasive 1 H-magnetic resonance spectroscopy (1 H-MRS) was used to monitor hepatic lipid content (HLC) and fatty acid composition in vivo, and glucose homeostasis was assessed with glucose and insulin challenges. At the end of the study, hepatic lipids were comprehensively characterized by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometric analyses of liver tissue samples. In males, HLC increased from 1.4 ± 0.1% at 3 months to 2.9 ± 0.3% at 7 months (p < 0.01) and 2.7 ± 0.3% at 11 months (p < 0.05), in correlation with fasting insulin levels (p < 0.01, r = 0.51) and parameters from the insulin tolerance test (ITT; p < 0.001, r = -0.69 versus area under the curve; p < 0.01, r = -0.57 versus blood glucose drop at 1 h post-ITT; p < 0.01, r = 0.55 versus blood glucose at 3 h post-ITT). The metabolic performance of females remained the same throughout the study, and HLC was higher than that of males at 3 months (2.7 ± 0.2%, p < 0.01), but comparable at 7 months (2.2 ± 0.2%) and 11 months (2.2 ± 0.1%). Strong sexual dimorphism in bioactive lipid species, including diacylglycerols (higher in males, p < 0.0001), phosphatidylinositols (higher in females, p < 0.001) and omega-3 polyunsaturated fatty acids (higher in females, p < 0.01), was found to be in good correlation with metabolic scores at 11 months. Therefore, in mice housed under standard conditions, sex-specific composition of bioactive lipids is associated with metabolic protection in females, whose metabolic performance was independent of hepatic cytosolic lipid content.

Methods of protein corona isolation for magnetic nanoparticles.

Nanoparticles (NPs) in contact with a biological environment get covered by proteins and some are loosely bound and some are tightly bound. The latter form a hard protein corona (HPC) which is known to determine their biological behavior. Therefore, in order to study the biological behaviour of NPs one needs to start from the HPC. However, established methods and standards of HPC isolation are still not known. This is especially a challenge in the case of magnetic NPs which form a major branch of nanomedicine. Therefore, we developed a novel HPC isolation method, a multi-step centrifugation method (MSCM), for single-domain magnetic NPs. The MSCM was applied to iron oxide NPs in interaction with human blood and lymph serum with different dilutions in triplicate. The analysis of the composition of the obtained HPCs showed the reproducibility of the MSCM. This new method was also compared with the existing magnetic separation method (MagSep) and a study of the obtained HPC allowed us to establish the validity limits of MagSep and MSCM on only superparamagnetic NPs and on any single-domain magnetic NPs, respectively. Surprisingly, the HPCs obtained by these two isolation methods were quite different, up to 50%, suggesting that only these proteins, which are found in the HPCs of both isolation methods, are in fact real HPCs.

Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics.

The cellular abundance of transcription factors (TFs) is an important determinant of their regulatory activities. Deriving TF copy numbers is therefore crucial to understanding how these proteins control gene expression. We describe a sensitive selected reaction monitoring-based mass spectrometry assay that allowed us to determine the copy numbers of up to ten proteins simultaneously. We applied this approach to profile the absolute levels of key TFs, including PPARγ and RXRα, during terminal differentiation of mouse 3T3-L1 pre-adipocytes. Our analyses revealed that individual TF abundance differs dramatically (from ∼250 to >300,000 copies per nucleus) and that their dynamic range during differentiation can vary up to fivefold. We also formulated a DNA binding model for PPARγ based on TF copy number, binding energetics and local chromatin state. This model explains the increase in PPARγ binding sites during the final differentiation stage that occurs despite a concurrent saturation in PPARγ copy number.

Quantitative mass spectrometry reveals plasticity of metabolic networks in Mycobacterium smegmatis.

Mycobacterium tuberculosis has a remarkable ability to persist within the human host as a clinically inapparent or chronically active infection. Fatty acids are thought to be an important carbon source used by the bacteria during long term infection. Catabolism of fatty acids requires reprogramming of metabolic networks, and enzymes central to this reprogramming have been targeted for drug discovery. Mycobacterium smegmatis, a nonpathogenic relative of M. tuberculosis, is often used as a model system because of the similarity of basic cellular processes in these two species. Here, we take a quantitative proteomics-based approach to achieve a global view of how the M. smegmatis metabolic network adjusts to utilization of fatty acids as a carbon source. Two-dimensional liquid chromatography and mass spectrometry of isotopically labeled proteins identified a total of 3,067 proteins with high confidence. This number corresponds to 44% of the predicted M. smegmatis proteome and includes most of the predicted metabolic enzymes. Compared with glucose-grown cells, 162 proteins showed differential abundance in acetate- or propionate-grown cells. Among these, acetate-grown cells showed a higher abundance of proteins that could constitute a functional glycerate pathway. Gene inactivation experiments confirmed that both the glyoxylate shunt and the glycerate pathway are operational in M. smegmatis. In addition to proteins with annotated functions, we demonstrate carbon source-dependent differential abundance of proteins that have not been functionally characterized. These proteins might play as-yet-unidentified roles in mycobacterial carbon metabolism. This study reveals several novel features of carbon assimilation in M. smegmatis, which suggests significant functional plasticity of metabolic networks in this organism.

Amyloid-beta oligomerization is associated with the generation of a typical peptide fragment fingerprint.

Amyloid-beta (Aß) peptide oligomerization plays a central role in the pathogenesis of Alzheimer's disease (AD), and Aß oligomers are collectively considered an appealing therapeutic target for the treatment of AD. However, the molecular mechanisms leading to the pathologic accumulation of oligomers are unclear, and the exact structural composition of oligomers is being debated. Using targeted and quantitative mass spectrometry, we reveal site-specific Aß autocleavage during the early phase of aggregation, producing a typical Aß fragment signature and that truncated Aß peptides can form stable oligomeric complexes with full-length Aß peptide. We show that the use of novel anti-Aß antibodies raised against these truncated Aß isoforms allows for monitoring and targeting the accumulation of truncated Aß fragments. Antibody-enabled screening of transgenic models of AD as well as human postmortem brain tissue and cerebrospinal fluid revealed that aggregation-associated Aß cleavage is a highly relevant clinical feature of AD.

A mammalian transcription factor-specific peptide repository for targeted proteomics.

Site-specific transcription factors (TFs) play an essential role in mammalian development and function as they are vital for the majority of cellular processes. Despite their biological importance, TF proteomic data is scarce in the literature, likely due to difficulties in detecting peptides as the abundance of TFs in cells tends to be low. In recent years, significant improvements in MS-based technologies in terms of sensitivity and specificity have increased the interest in developing quantitative methodologies specifically targeting relatively lowly abundant proteins such as TFs in mammalian models. Such efforts would be greatly aided by the availability of TF peptide-specific information as such data would not only enable improvements in speed and accuracy of protein identifications, but also ameliorate cross-comparisons of quantitative proteomics data and allow for a more efficient development of targeted proteomics assays. However, to date, no comprehensive TF proteotypic peptide database has been developed. To address this evident lack of TF peptide data in public repositories, we are generating a comprehensive, experimentally derived TF proteotypic peptide spectral library dataset based on in vitro protein expression. Our library currently contains peptide information for 89 TFs and this number is set to increase in the near future. All MS data have been deposited in the ProteomeXchange with identifier PXD001212 (http://proteomecentral.proteomexchange.org/dataset/PXD001212).

Identification of new Presenilin-1 phosphosites: implication for γ-secretase activity and Aß production.

An important pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid-beta (Aß) peptides in the brain parenchyma, leading to neuronal death and impaired learning and memory. The protease γ-secretase is responsible for the intramembrane proteolysis of the amyloid-ß precursor protein (APP), which leads to the production of the toxic Aß peptides. Thus, an attractive therapeutic strategy to treat AD is the modulation of the γ-secretase activity, to reduce Aß42 production. Because phosphorylation of proteins is a post-translational modification known to modulate the activity of many different enzymes, we used electrospray (LC-MS/MS) mass spectrometry to identify new phosphosites on highly purified human γ-secretase. We identified 11 new single or double phosphosites in two well-defined domains of Presenilin-1 (PS1), the catalytic subunit of the γ-secretase complex. Next, mutagenesis and biochemical approaches were used to investigate the role of each phosphosite in the maturation and activity of γ-secretase. Together, our results suggest that the newly identified phosphorylation sites in PS1 do not modulate γ-secretase activity and the production of the Alzheimer's Aß peptides. Individual PS1 phosphosites shall probably not be considered therapeutic targets for reducing cerebral Aß plaque formation in AD. In this study, we identified 11 new phosphosites in Presenilin-1 (PS1), the catalytic subunit of the Alzheimer's γ-secretase complex. By combining a mutagenesis approach with cell-based and cell-free γ-secretase assays, we demonstrate that the new phosphosites do not modulate the maturation and activity of γ-secretase. Individual PS1 phosphosites shall thus not be considered therapeutic targets for reducing cerebral Aß plaque formation in Alzheimer's Disease. Aß, amyloid beta.

Alpha-synuclein post-translational modifications as potential biomarkers for Parkinson disease and other synucleinopathies.

The development of novel therapies against neurodegenerative disorders requires the ability to detect their early, presymptomatic manifestations in order to enable treatment before irreversible cellular damage occurs. Precocious signs indicative of neurodegeneration include characteristic changes in certain protein levels, which can be used as diagnostic biomarkers when they can be detected in fluids such as blood plasma or cerebrospinal fluid. In the case of synucleinopathies, cerebrospinal alpha-synuclein (α-syn) has attracted great interest as a potential biomarker; however, there is ongoing debate regarding the association between cerebrospinal α-syn levels and neurodegeneration in Parkinson disease and synucleinopathies. Post-translational modifications (PTMs) have emerged as important determinants of α-syn's physiological and pathological functions. Several PTMs are enriched within Lewy bodies and exist at higher levels in α-synucleinopathy brains, suggesting that certain modified forms of α-syn might be more relevant biomarkers than the total α-syn levels. However, the quantification of PTMs in bodily fluids poses several challenges. This review describes the limitations of current immunoassay-based α-syn quantification methods and highlights how these limitations can be overcome using novel mass-spectrometry-based assays. In addition, we describe how advances in chemical synthesis, which have enabled the preparation of α-syn proteins that are site-specifically modified at single or multiple residues, can facilitate the development of more accurate assays for detecting and quantifying α-syn PTMs in health and disease.

Involvement of Plasmodium falciparum protein kinase CK2 in the chromatin assembly pathway.

BACKGROUND: Protein kinase CK2 is a pleiotropic serine/threonine protein kinase with hundreds of reported substrates, and plays an important role in a number of cellular processes. The cellular functions of Plasmodium falciparum CK2 (PfCK2) are unknown. The parasite's genome encodes one catalytic subunit, PfCK2α, which we have previously shown to be essential for completion of the asexual erythrocytic cycle, and two putative regulatory subunits, PfCK2ß1 and PfCK2ß2. RESULTS: We now show that the genes encoding both regulatory PfCK2 subunits (PfCK2ß1 and PfCK2ß2) cannot be disrupted. Using immunofluorescence and electron microscopy, we examined the intra-erythrocytic stages of transgenic parasite lines expressing hemagglutinin (HA)-tagged catalytic and regulatory subunits (HA-CK2α, HA-PfCK2ß1 or HA-PfCK2ß2), and localized all three subunits to both cytoplasmic and nuclear compartments of the parasite. The same transgenic parasite lines were used to purify PfCK2ß1- and PfCK2ß2-containing complexes, which were analyzed by mass spectrometry. The recovered proteins were unevenly distributed between various pathways, with a large proportion of components of the chromatin assembly pathway being present in both PfCK2ß1 and PfCK2ß2 precipitates, implicating PfCK2 in chromatin dynamics. We also found that chromatin-related substrates such as nucleosome assembly proteins (Naps), histones, and two members of the Alba family are phosphorylated by PfCK2α in vitro. CONCLUSIONS: Our reverse-genetics data show that each of the two regulatory PfCK2 subunits is required for completion of the asexual erythrocytic cycle. Our interactome study points to an implication of PfCK2 in many cellular pathways, with chromatin dynamics being identified as a major process regulated by PfCK2. This study paves the way for a kinome-wide interactomics-based approach to elucidate protein kinase function in malaria parasites.

Tissue transglutaminase-mediated glutamine deamidation of beta-amyloid peptide increases peptide solubility, whereas enzymatic cross-linking and peptide fragmentation may serve as molecular triggers for rapid peptide aggregation.

Tissue transglutaminase (TGase) has been implicated in a number of cellular processes and disease states, where the enzymatic actions of TGase may serve in both, cell survival and apoptosis. To date, the precise functional properties of TGase in cell survival or cell death mechanisms still remain elusive. TGase-mediated cross-linking has been reported to account for the formation of insoluble lesions in conformational diseases. We report here that TGase induces intramolecular cross-linking of ß-amyloid peptide (Aß), resulting in structural changes of monomeric Aß. Using high resolution mass spectrometry (MS) of cross-linked Aß peptides, we observed a shift in mass, which is, presumably associated with the loss of NH3 due to enzymatic transamidation activity and hence intramolecular peptide cross-linking. We have observed that a large population of Aß monomers contained an 0.984 Da increase in mass at a glutamine residue, indicating that glutamine 15 serves as an indispensable substrate in TGase-mediated deamidation to glutamate 15. We provide strong analytical evidence on TGase-mediated Aß peptide dimerization, through covalent intermolecular cross-linking and hence the formation of Aß1-40 dimers. Our in depth analyses indicate that TGase-induced post-translational modifications of Aß peptide may serve as an important seed for aggregation.

Activation of a PAK-MEK signalling pathway in malaria parasite-infected erythrocytes.

Merozoites of malaria parasites invade red blood cells (RBCs), where they multiply by schizogony, undergoing development through ring, trophozoite and schizont stages that are responsible for malaria pathogenesis. Here, we report that a protein kinase-mediated signalling pathway involving host RBC PAK1 and MEK1, which do not have orthologues in the Plasmodium kinome, is selectively stimulated in Plasmodium falciparum-infected (versus uninfected) RBCs, as determined by the use of phospho-specific antibodies directed against the activated forms of these enzymes. Pharmacological interference with host MEK and PAK function using highly specific allosteric inhibitors in their known cellular IC50 ranges results in parasite death. Furthermore, MEK inhibitors have parasiticidal effects in vitro on hepatocyte and erythrocyte stages of the rodent malaria parasite Plasmodium berghei, indicating conservation of this subversive strategy in malaria parasites. These findings have profound implications for the development of novel strategies for antimalarial chemotherapy.

Phosphorylation at S87 is enhanced in synucleinopathies, inhibits alpha-synuclein oligomerization, and influences synuclein-membrane interactions.

Increasing evidence suggests that phosphorylation may play an important role in the oligomerization, fibrillogenesis, Lewy body (LB) formation, and neurotoxicity of alpha-synuclein (alpha-syn) in Parkinson disease. Herein we demonstrate that alpha-syn is phosphorylated at S87 in vivo and within LBs. The levels of S87-P are increased in brains of transgenic (TG) models of synucleinopathies and human brains from Alzheimer disease (AD), LB disease (LBD), and multiple system atrophy (MSA) patients. Using antibodies against phosphorylated alpha-syn (S129-P and S87-P), a significant amount of immunoreactivity was detected in the membrane in the LBD, MSA, and AD cases but not in normal controls. In brain homogenates from diseased human brains and TG animals, the majority of S87-P alpha-syn was detected in the membrane fractions. A battery of biophysical methods were used to dissect the effect of S87 phosphorylation on the structure, aggregation, and membrane-binding properties of monomeric alpha-syn. These studies demonstrated that phosphorylation at S87 expands the structure of alpha-syn, increases its conformational flexibility, and blocks its fibrillization in vitro. Furthermore, phosphorylation at S87, but not S129, results in significant reduction of alpha-syn binding to membranes. Together, our findings provide novel mechanistic insight into the role of phosphorylation at S87 and S129 in the pathogenesis of synucleinopathies and potential roles of phosphorylation in alpha-syn normal biology.

Human proteinase 3 resistance to inhibition extends to alpha-2 macroglobulin.

Polymorphonuclear neutrophils contain at least four serine endopeptidases, namely neutrophil elastase (NE), proteinase 3 (PR3), cathepsin G (CatG), and NSP4, which contribute to the regulation of infection and of inflammatory processes. In physiological conditions, endogenous inhibitors including α2-macroglobulin (α2-M), serpins [α1-proteinase inhibitor (α1-PI)], monocyte neutrophil elastase inhibitor (MNEI), α1-antichymotrypsin, and locally produced chelonianins (elafin, SLPI) control excessive proteolytic activity of neutrophilic serine proteinases. In contrast to human NE (hNE), hPR3 is weakly inhibited by α1-PI and MNEI but not by SLPI. α2-M is a large spectrum inhibitor that traps a variety of proteinases in response to cleavage(s) in its bait region. We report here that α2-M was more rapidly processed by hNE than hPR3 or hCatG. This was confirmed by the observation that the association between α2-M and hPR3 is governed by a kass in the ≤ 105  m-1 ·s-1 range. Since α2-M-trapped proteinases retain peptidase activity, we first predicted the putative cleavage sites within the α2-M bait region (residues 690-728) using kinetic and molecular modeling approaches. We then identified by mass spectrum analysis the cleavage sites of hPR3 in a synthetic peptide spanning the 39-residue bait region of α2-M (39pep-α2-M). Since the 39pep-α2-M peptide and the corresponding bait area in the whole protein do not contain sequences with a high probability of specific cleavage by hPR3 and were indeed only slowly cleaved by hPR3, it can be concluded that α2-M is a poor inhibitor of hPR3. The resistance of hPR3 to inhibition by endogenous inhibitors explains at least in part its role in tissue injury during chronic inflammatory diseases and its well-recognized function of major target autoantigen in granulomatosis with polyangiitis.

Analysis of the S. pombe Meiotic Proteome Reveals a Switch from Anabolic to Catabolic Processes and Extensive Post-transcriptional Regulation.

Meiotic progression in S. pombe is regulated by stage-specific gene expression and translation, changes in RNA stability, expression of anti-sense transcripts, and targeted proteolysis of regulatory proteins. We have used SILAC labeling to examine the relative levels of proteins in diploid S. pombe cells during meiosis. Among the 3,268 proteins quantified at all time points, the levels of 880 proteins changed at least 2-fold; the majority of proteins showed stepwise increases or decreases during the meiotic divisions, while some changed transiently. Overall, we observed reductions in proteins involved in anabolism and increases in proteins involved in catabolism. We also observed increases in the levels of proteins of the ESCRT-III complex and revealed a role for ESCRT-III components in chromosome segregation and spore formation. Correlation with studies of meiotic gene expression and ribosome occupancy reveals that many of the changes in steady-state protein levels are post-transcriptional.