Protease Inhibitor Cocktail Research Articles

A Novel PD-L1-targeting Antagonistic DNA Aptamer With Antitumor Effects.

The PD-1/PD-L1 axis is a major pathway involved in tumor immune evasion. Here, we report the novel PD-L1 antagonizing DNA aptamer (aptPD-L1) and demonstrate an integrated pipeline that expedites therapeutic aptamer development. Aptamer can exert antibody-mimic functions and is advantageous over antibody for its chemically synthetic nature, low immunogenicity, and efficient tissue penetration. Our results showed that aptPD-L1 blocked the binding between human PD-1 and PD-L1. Experiments using murine models showed that aptPD-L1 promoted in vitro lymphocyte proliferation and suppressed in vivo tumor growth without the induction of observable liver or renal toxicity. Analyses on the aptPD-L1-treated tumors further revealed elevated levels of infiltrating CD4+ and CD8+ T cells, intratumoral IL-2, TNF-α, interferon (IFN)-γ and the C-X-C motif chemokines, CXCL9 and CXCL10. The CD8+ T cells in the aptPD-L1-treated tumors had higher CXCR3 expression level compared to the random-sequence oligonucleotides-treated ones. Besides, the length and density of CD31+ intratumoral microvessels were significantly decreased in the aptPD-L1 treatment group. Collectively, our data suggested that aptPD-L1 helps T cell function restoration and modifies tumor microenvironment. These chemokines might orchestrate together to attract more T cells into the tumor tissues to form the positive amplification loop against tumor growth, indicating the translational potential of aptPD-L1 in cancer immunotherapy.

Heterogeneous Expression of OLFM4 in Neutrophils and Plasma

Introduction: Olfactomedin 4 (OLFM4) is a glycoprotein predominantly expressed in myeloid cells and in gastrointestinal tissues. OLFM4 is stored in specific granules of human neutrophils where it defines a subset of neutrophils ranging from 5-40% OLFM4 positive neutrophils. OLFM4 has been proposed to inhibit cathepsin C, a cysteine protease essential for activation of serine proteases. Elevated levels of OLFM4 has been seen primary myelofibrosis (PMF) patients, these patients furthermore display a significantly elevated serum OLFM4.Methods: We developed monoclonal antibodies against OLFM4 and established an Enzyme Linked Immunosorbent Assay for OLFM4 for further investigation of OLFM4 in myeloid cells.Results: We observed two populations of individuals with respect to OLFM4 levels in plasma, the majority with OLFM4 in plasma between 0 and 0.1 μg/mL, mean 0.028 μg/mL while approximately 10% had OLFM4 between 4 and 60 μg/mL, mean 15 μg/mL. The levels were constant over time.The level did not relate to the size of the OLFM4 positive neutrophil subset detected in peripheral blood. We studied the biosynthesis of OLFM4 in isolated bone marrow cells from an individual with high plasma OLFM4 and an individual with low plasma OLFM4. The levels of OLFM4 mRNA were comparable and the amounts of OLFM4 synthesized and retained in cells were similar between the two individuals. We next determined whether OLFM4 might be produced by bone marrow cells and released into bone marrow plasma. Corresponding levels of OLFM4 determined in bone marrow plasma and blood plasma from two persons with high levels of OLFM4 showed lower levels in bone marrow plasma than in blood plasma, arguing against bone marrow as the direct source of OLFM4 in plasma.To estimate the amount of OLFM4 generated daily during myelopoiesis, we quantitated the amount of OLFM4 in neutrophils from 3 sets of buffy coat neutrophils, each pooled from 4 healthy donors. The amount of OLFM4 was 1.2 μg/107 neutrophils. As the production of neutrophils is about 1 x 109 cells/kg body weight/day, this would indicate production of 10 mg OLFM4/day in an adult.To rule out the liver as a production site, mRNA was determined by Affymetrix gene array in liver biopsies from 42 patients evaluated for liver steatosis. OLFM4 mRNA levels were uniformly at the border of detection in all (data not shown).OLFM4 is secreted from PMA stimulated neutrophils in parallel with other specific granule proteins. When purified OLFM4 was added to medium from neutrophils induced to degranulate with PMA, the ability to detect OLFM4 was rapidly lost, indicating that OLFM4 is highly sensitive to proteolysis. Adding a cocktail of protease inhibitors to the material secreted from PMA activated neutrophils preserved OLFM4.As we find that OLFM4 is highly sensitive to proteolysis we suggest that the differences in OLFM4 levels in plasma may be related to individual differences in the susceptibility of OLFM4 to escape degradation when neutrophils decease as part of their normal life cycle. Production of 10 mg OLFM4/day would support a plasma level of 3-4 µg/ml plasma depending of the half-life of OLFM4 in plasma. This hypothesis is not easily tested, but if proven correct, might open for novel insight into the fate of neutrophils after exiting circulation, an issue that is still a matter of debate. DisclosuresNo relevant conflicts of interest to declare.

Factors Affecting Inducible Expression of Outer Membrane Protein A (OmpA) of Shigella dysenteriae Type-1 in Lactococcus lactis Using Nisin Inducible Controlled Expression (NICE).

Potential use of Lactococcus lactis (L. lactis) as a heterologous protein expression host as well as for delivery of multiple therapeutic proteins has been investigated extensively using Nisin Inducible Controlled Expression (NICE) system. Optimum inducible expression of heterologous protein by NICE system in L. lactis depends on multiple factors. To study the unexplored role of factors affecting heterologous protein expression in L. lactis using NICE, the present study outlines the optimization of various key parameters such as inducer concentration, host's proteases and precipitating agent using Outer membrane protein A (OmpA). For efficient expression and secretion of OmpA, pSEC:OmpA vector was successfully constructed. To circumvent the troubles encountered during detection of expressed OmpA, the precipitating agent was switched from TCA to methanol. Nevertheless, detection was achieved accompanied by degraded protein products. Speculating the accountability of observed degradation at higher inducer concentration, different nisin concentrations were evaluated. Lower nisin concentrations were found desirable for optimum expression of OmpA. Consistently observed degradation was eliminated by incorporation of protease inhibitor cocktail which inhibits intracellular proteases and expression in VEL1153 (NZ9000 ΔhtrA) strain which inhibits extracellular protease leading to optimum expression of OmpA. Versatility and complexity of NICE system in L. lactis requires fine-tuning of target protein specific parameters for optimum expression.

Yersinia enterocolitica-mediated degradation of neutrophil extracellular traps (NETs)

Neutrophil extracellular trap (NET) formation is described as a tool of the innate host defence to fight against invading pathogens. Fibre-like DNA structures associated with proteins such as histones, cell-specific enzymes and antimicrobial peptides are released, thereby entrapping invading pathogens. It has been reported that several bacteria are able to degrade NETs by nucleases and thus evade the NET-mediated entrapment. Here we studied the ability of three different Yersinia serotypes to induce and degrade NETs. We found that the common Yersinia enterocolitica serotypes O:3, O:8 and O:9 were able to induce NETs in human blood-derived neutrophils during the first hour of co-incubation. At later time points, the NET amount was reduced, suggesting that degradation of NETs has occurred. This was confirmed by NET degradation assays with phorbol-myristate-acetate-pre-stimulated neutrophils. In addition, we found that the Yersinia supernatants were able to degrade purified plasmid DNA. The absence of Ca(2+) and Mg(2+) ions, but not that of a protease inhibitor cocktail, completely abolished NET degradation. We therefore postulate that Y. enterocolitica produces Ca(2+)/Mg(2+)-dependent NET-degrading nucleases as shown for some Gram-positive pathogens.

Abstract 108: C1q-TNF Related Protein-9, a Novel Cardioprotective Cardiokine, Requires Proteolytic Cleavage to Generate a Biologically Active Globular Domain Isoform

The discovery of the cardiac secretomes, known as “cardiokines”, significantly enhanced appreciation of the local microenvironment’s influence upon disease development. We previously reported cardiomyocytes produce cardioprotective adiponectin (APN). However, cardiac APN levels are several orders of magnitude below adipocytes, thereby making APN an unlikely physiologically relevant cardiokine. C1qTNF-related proteins (CTRP1-15) is a newly descovered family of APN paralogs. However, whether any of the CTRP members may function as a “true” cardiokine remains unknown. Here we demonstrated that several CTRPs (CTRP1,4,7,9,13) were expressed in the heart at levels significantly greater than APN. Most notably, the mRNA level of CTRP9 exceeded APN by >100-fold in cardiac tissue. Adult cardiac cells were isolated and cultured in vitro. Conditioned medium was collected 1-8 hours after culture and CTRP9 protein levels were determined by ELISA. A significant increase of CTRP9 in conditioned medium was detected as early as 1 hour in culture, steadily accumulating thereafter. These results indicate CTRP9 is secreted by cardiac cell at levels that may function as a cardiokine. Addional experiemnt unexpectedly demonstarted that CTRP9 circulates in the plasma primarily in the globular domain isoform (gCTRP9), opposing from APN which circulates as full length multimers. Recombinant full length CTRP9 (fCTRP9) was cleaved when incubated with cardiac tissue extracts, generating gCTRP9, a process inhibited by protease inhibitor cocktail. To gain more insight into the biological significance of gCTRP9 production, isolated adult cardiomyocytes were treated with gCTRP9 or fCTRP9. gCTRP9 rapidly activates cardiac survival kinases, including AMPK, Akt, and eNOS. However, fCTRP9-mediated kinase activation is much less potent, and significantly delayed. Kinase activation by fCTRP9, but not gCTRP9, is inhibited by protease inhibitor cocktail. These results demonstrate for the first time CTRP9 is a novel cardiokine undergoing proteolytic cleavage to generate cardioprotective isoform, gCTRP9. Enhancing cardiac CTRP9 production and/or its proteolytic post-translational modification are of therapeutic potential, attenuating cardiac injury.

Mast cell degranulation inhibits motor patterns of human ileum and sigmoid colon in vitro: relevance for postoperative ileus.

Local release of mast cell proteases during gastrointestinal surgery is associated with the inhibition of motility and postoperative ileus (POI). We determined whether activation of intramuscular mast cell affects the motor patterns of the human ileum and colon and whether proteases are involved. Motor response of ileal and colonic circular muscle strips was measured in organ bath. Mast cell degranulation was induced by compound 48/80 (c48/80; 25-675 μg/mL). Motor response was quantified as tone, rhythmic phasic contractions (RPCs) and contractions to electric field stimulation (EFS; 40 Hz), and bethanechol-evoked contractions. Ketotifen (10(-6) mol/L) and a protease inhibitor cocktail (P8340) were used to evaluate the role of mast cell mediators. (a) c48/80 impaired the spontaneous and the electrically evoked motor response in small bowel and colonic strips (sigmoid colon EC50 : 460.0 μg/mL for RPCs and 8.9 μg/mL for electrically evoked contraction amplitudes) and bethanechol-evoked contractions. (b) Preincubation with ketotifen (10(-6) mol/L, 1 h) prevented the impairment of RPCs and EFS-evoked contractions in the sigmoid colon and ileum but not in the right colon. (c) Preincubation with P8340 also prevented the impairment of contractions in the sigmoid colon but not in the ileum or the right colon. Mast cell degranulation by c48/80 inhibits the spontaneous and the nerve-mediated motor response in the human ileum and colon. The effect is partially mediated by mast cell proteases and could be relevant in the pathophysiology of POI.

Protein oxidation affects proteolysis in a meat model system

The effect of hydrogen peroxide-induced protein oxidation and pH (4.8 and 5.2) on meat proteolysis was investigated in a meat model system for dry fermented sausages. In oxidised samples, increased protein carbonyl contents and decreased thiol concentrations were found. The initial concentration of protein carbonyls was significantly lower in oxidised samples at pH4.8 than in ones at pH5.2, but after ten days comparable levels were reached. The inhibition of proteolysis by the addition of a protease inhibitor cocktail did not influence protein oxidation. Yet, proteolysis was negatively affected by low pH values as well as by oxidation, resulting in a reduced release of amino acids during ripening.

Effects of different media supplements on the production of an active recombinant plant peroxidase in a Pichia pastoris Δoch1 strain

Recombinant protein production in microorganisms is one of the most studied areas of research in biotechnology today. In this respect the yeast Pichia pastoris is an important microbial production host due to its capability of secreting the target protein and performing posttranslational modifications. In a recent study, we described the development of a robust bioprocess for a glyco-engineered recombinant P. pastoris strain where the native α-1,6-mannosyltransfrease OCH1 was knocked out (Δoch1 strain). This strain produced the glycosylated enzyme horseradish peroxidase (HRP) with more homogeneous and shorter surface glycans than the respective benchmark strain. However, the recombinant Δoch1 strain was physiologically impaired and thus hard to cultivate. We faced cell cluster formation, cell lysis and consequent intensive foam formation. Thus, we investigated the effects of the 3 process parameters temperature, pH and dissolved oxygen concentration on (1) cell physiology, (2) cell morphology, (3) cell lysis, (4) productivity and (5) product purity in a multivariate manner. However, not only process parameters might influence these characteristics, but also media supplements might have an impact. Here, we describe the effects of different heme-precursors as well as of a protease-inhibitor cocktail on the production of active HRP in therecombinant P. pastoris Δoch1strain.

Enzyme Activities of Muscle Tissue Lysates can be Unexpectedly Affected by Protease Inhibitor Cocktails

Previous studies in our lab have shown that the protective use of certain protease inhibitor cocktails with skeletal muscle tissue lysates can decrease protein expression on PAGE (polyacrylamide gel electrophoresis). The purpose of the present study was to determine whether inclusion of cocktails in two different rat skeletal muscle (biceps femoris and rectus abdominis) tissue lysates could affect enzyme activities of endogenous non-proteases. After lysates were incubated with cocktails at 37°C for one hour, they were run on non-denaturing polyacrylamide gel electrophoresis (native-PAGE) and assayed for enzyme activities. Results indicate that acetyl-cholinesterase (AChE) activities of lysates incubated with cocktails were significantly diminished; in contrast, malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) activities of lysates incubated with cocktails were unaffected. When muscle lysates were challenged with high trypsin concentrations (0.14%), enzyme activities of MDH (mitochondrial isozyme) and LDH were inhibited; however, the inclusion of certain cocktails rescued activities. Enzyme activities in cardiac and small intestinal muscle tissue lysates treated with cocktails appear to show a similar pattern. In conclusion, it is suggested that the inclusion of cocktails with tissue lysates should be carefully evaluated when measuring tissue enzyme activity. (Support from MSMC Research Funding and Pace Scholarly Research)

C1q-TNF-related protein-9, a novel cardioprotetcive cardiokine, requires proteolytic cleavage to generate a biologically active globular domain isoform.

Prevalence and severity of postmyocardial infarction heart failure continually escalate in type 2 diabetes via incompletely understood mechanisms. The discovery of the cardiac secretomes, collectively known as "cardiokines", has significantly enhanced appreciation of the local microenvironment's influence on disease development. Recent studies demonstrated that C1q-TNF-related protein-9 (CTRP9), a newly discovered adiponectin (APN) paralog, is highly expressed in the heart. However, its relationship with APN (concerning diabetic cardiovascular injury in particular) remains unknown. Plasma CTRP9 levels are elevated in APN knockout and reduced in diabetic mice. In contrast to APN, which circulates as full-length multimers, CTRP9 circulates in the plasma primarily in the globular domain isoform (gCTRP9). Recombinant full-length CTRP9 (fCTRP9) was cleaved when incubated with cardiac tissue extracts, generating gCTRP9, a process inhibited by protease inhibitor cocktail. gCTRP9 rapidly activates cardiac survival kinases, including AMPK, Akt, and endothelial NOS. However, fCTRP9-mediated kinase activation is much less potent and significantly delayed. Kinase activation by fCTRP9, but not gCTRP9, is inhibited by protease inhibitor cocktail. These results demonstrate for the first time that the novel cardiokine CTRP9 undergoes proteolytic cleavage to generate gCTRP9, the dominant circulatory and actively cardioprotective isoform. Enhancing cardiac CTRP9 production and/or its proteolytic posttranslational modification are of therapeutic potential, attenuating diabetic cardiac injury.

Functional Significance of Point Mutations in Stress Chaperone Mortalin and Their Relevance to Parkinson Disease

Mortalin/mtHsp70/Grp75 (mot-2), a heat shock protein 70 family member, is an essential chaperone, enriched in cancers, and has been shown to possess pro-proliferative and anti-apoptosis functions. An allelic form of mouse mortalin (mot-1) that differs by two amino acids, M618V and G624R, in the C terminus substrate-binding domain has been reported. Furthermore, genome sequencing of mortalin from Parkinson disease patients identified two missense mutants, R126W and P509S. In the present study, we investigated the significance of these mutations in survival, proliferation, and oxidative stress tolerance in human cells. Using mot-1 and mot-2 recombinant proteins and specific antibodies, we performed screening to find their binding proteins and then identified ribosomal protein L-7 (RPL-7) and elongation factor-1 α (EF-1α), which differentially bind to mot-1 and mot-2, respectively. We demonstrate that mot-1, R126W, or P509S mutant (i) lacks mot-2 functions involved in carcinogenesis, such as p53 inactivation and hTERT/hnRNP-K (heterogeneous nuclear ribonucleoprotein K) activation; (ii) causes increased level of endogenous oxidative stress; (iii) results in decreased tolerance of cells to exogenous oxidative stress; and (iv) shows differential binding and impact on the RPL-7 and EF-1α proteins. These factors may mediate the transformation of longevity/pro-proliferative function of mot-2 to the premature aging/anti-proliferative effect of mutants, and hence may have significance in cellular aging, Parkinson disease pathology, and prognosis.

Plasma Protease-Mediated Metabolism of Carfilzomib and Its Implications on Clinical Effects and Side Effects

Introduction: Similar to most other anti-cancer drugs, carfilzomib is administered at a defined dose per m2. Inspired by preclinical data showing that extrahepatic peptidases play an important role in the metabolism of carfilzomib, using samples from a prospective clinical trial, we conducted an investigation focusing on concentrations of albumin in peripheral blood and its effects on plasma protease-mediated metabolism of carfilzomib.Methods: Baseline laboratory values, pharmacokinetics data, and response data were collected from the clinical trial. Minimal residual disease was determined by multi-parametric flow cytometry utilizing, 8-color flow panel and analyzes ≥ 3 x 106 events (sensitivity 1 x 10-5). To study the rate of metabolism in plasma, apheresis samples were obtained from the NIH blood bank, lyophilized albumin was added to plasma with and without a non-specific protease inhibitor cocktail (1X). Carfilzomib concentrations were obtained at several time points over the course of 24 hours by mass spectrometry via previously published methods.Results: Patients with baseline serum albumin >4.0 g/dL had a near 4-fold greater clearance (1576 L/h vs 401.1 L/h; n=9 vs n=34) that was further increased in patients with normal white blood cell count. Consistent with the increase in clearance, 80% of patients (29/33) with albumin <4.0 g/dL became minimum residual disease (MRD) negative whereas only 44.4% of patients (4/9) with albumin >4.0 g/dL had a similar outcome. Therefore, individuals with baseline albumin >4.0 g/dL are at significantly greater risk for not responding to the combination of carfilzomib, lenalidomide, and dexamethasone [OR (95%CI) = 7.3 (1.4-36.7); P=0.0049]. Median progression-free survival was also longer in patients with albumin <4.0 g/dL vs. albumin >4.0 g/dL (unreached vs. 18.8 months; P=0.0002). A trend between albumin and lymphopenia was also detected (P=0.17). The addition of 1.0 g/dL albumin increased the ex vivo proteolytic rate of carfilzomib in apheresis plasma from healthy individuals (74% vs 57.2% unreacted carfilzomib after 24 hours at 37°C), and the addition of a non-specific protease inhibitor reduced carfilzomib metabolism in plasmaConclusions: Although they have lower grade myeloma, patients with albumin >4.0 g/dL, surprisingly, are at greater risk at having worse clinical outcomes to carfilzomib therapy; this finding appears to depend upon the ability of albumin to directly potentiate plasma protease-mediated metabolism of carfilzomib. Therefore, dose adjustments or concomitant therapy with protease inhibitors may be warranted in such patients. Efforts are underway to identify specific proteases that metabolize carfilzomib and determine potential inhibitors that may be clinically useful. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of offgel electrophoresis, electrothermal atomic absorption spectroscopy and inductively coupled plasma optical emission spectroscopy for trace metal analysis in marine plankton protein

Two-dimensional offgel and lab-on-a-chip (LOC) electrophoresis was performed for assessing proteins from marine plankton. Proteins were isolated from plankton samples by using a phenol/sodium dodecylsulfate (SDS) buffer (2:1) pH8.0 mixture as an extracting solution. The SDS buffer consisted of 30% (m/v) sucrose, 2% (m/v) SDS, 0.1M of Tris–HCl, pH8.0, 5% (v/v) β-mercaptoethanol and protease inhibitor cocktail. Before protein extraction, the plankton were subjected to different washing steps involving 10% trichloroacetic acid (TCA)/acetone and methanol. Proteins exhibiting the highest molecular weights (MWs), within the 52.6–57.3kDa range, were found in offgel fractions of isoelectric points (pIs) ranging from 4.40 to 8.60; whereas, the highest protein concentrations were found in fraction 5 (100μgg−1 for a protein of pI of 4.5 and MW of 22.0kDa), fraction 16 (55μgg−1, protein of pI/MW of 7.4/19.1kDa), and fraction 8 (51μgg−1, pI/MW of 5.3/22.2kDa). Further studies were also developed for determining the amount of trace metals associated with the isolated proteins of similar pIs by electrothermal atomic absorption spectroscopy (ETAAS) and inductively coupled plasma optical emission spectroscopy (ICP-OES). Preliminary results indicate that Fe, Ni, Mn and Zn were found to be associated with proteins of variable MW (from 14 to 54kDa) and pIs (from 3.4 to 8.2). Otherwise, Cd was found to be associated with proteins of certain pIs (6.0–6.5) and with MWs ranging from 14.1 to 53.1kDa.

Lisdexamfetamine prodrug activation by peptidase-mediated hydrolysis in the cytosol of red blood cells.

Lisdexamfetamine dimesylate (LDX) is approved as a once-daily treatment for attention-deficit/hyperactivity disorder in children, adolescents, and adults in some countries. LDX is a prodrug comprising d-amphetamine covalently linked to l-lysine via a peptide bond. Following oral administration, LDX is rapidly taken up from the small intestine by active carrier-mediated transport, probably via peptide transporter 1. Enzymatic hydrolysis of the peptide bond to release d-amphetamine has previously been shown to occur in human red blood cells but not in several other tissues. Here, we report that LDX hydrolytic activity resides in human red blood cell lysate and cytosolic extract but not in the membrane fraction. Among several inhibitors tested, a protease inhibitor cocktail, bestatin, and ethylenediaminetetra-acetic acid each potently inhibited d-amphetamine production from LDX in cytosolic extract. These results suggest that an aminopeptidase is responsible for hydrolytic cleavage of the LDX peptide bond, although purified recombinant aminopeptidase B was not able to release d-amphetamine from LDX in vitro. The demonstration that aminopeptidase-like activity in red blood cell cytosol is responsible for the hydrolysis of LDX extends our understanding of the smooth and consistent systemic delivery of d-amphetamine by LDX and the long daily duration of efficacy of the drug in relieving the symptoms of attention-deficit/hyperactivity disorder.

A nonradioactive approach to investigate the metabolism of therapeutic peptides by tagging with 127i and using inductively-coupled plasma mass spectrometry analysis.

The metabolic fate of adrenocorticotropic hormone (ACTH) fragment 4-10 (4-10) was evaluated following incorporation of a nonradioactive (127)I-tag and with selective detection of I(+) at m/z 127 by inductively coupled plasma mass spectrometry (ICP-MS). (127)I has all the advantages of radioactive (125)I as a metabolite tracer and, together with its detection in the femtogram range, has led to a successful metabolite profiling of (127)I-ACTH (4-10) in vitro. The observed metabolic stability of this peptide in tissue preparations from human was plasma > kidney S9 > liver microsomes > liver cytosol, liver S9. Metabolic turnover of (127)I-ACTH (4-10) was not NADPH-dependent and, together with inhibition by protease inhibitor cocktail and EDTA, is consistent with metabolism exclusively by proteases. Our preliminary studies using chemical inhibitors suggested the involvement of metalloprotease, serine peptidase, and aminopeptidase in (127)I-ACTH (4-10) metabolism. The liver is the primary site of metabolic clearance of (127)I-ACTH (4-10), with kidney S9 taking four times longer to produce a metabolite profile comparable to that produced by liver S9. A total of six metabolites retaining the (127)I-tag was detected by ICP-MS, and their structures were elucidated using a LTQ/Orbitrap. (127)I-ACTH (4-10) underwent both N- and C-terminal proteolysis to produce (127)I-Phe as the major metabolite. The (127)I-tag had minimal effect on the metabolic turnover and site of proteolysis of ACTH (4-10), which, together with ICP-MS providing essentially equimolar responses, suggests that the use of a (127)I-tag may have general utility as an alternative to radioiodination to investigate the metabolism of peptide therapeutics.

The Chlamydia protease CPAF: caution, precautions and function.

Recent biochemical and genetic studies have called into question many of the functions proposed for the chlamydial protease CPAF (Zhong et al ., 2001). In 2012, we demonstrated that the proteolysis of multiple reported substrates was occurring during the preparation of lysates from Chlamydia infected cells rather than in intact cells (Chen et al ., 2012). The recent report by Snavely et al . of a CPAF null mutant now provides genetic evidence that CPAF is not necessary for many Chlamydia induced phenotypes nor for a successful intracellular infection (Bavoil & Byrne, 2014; Snavely et al ., 2014). The proteolysis observed when analyzing Chlamydia infected cells must be carefully interpreted because lysates prepared under standard conditions contain CPAF proteolytic activity (Chen et al ., 2012). In general, proteolysis during lysate preparation is not a problem for protein analysis because proteases in cell lysates are typically inactive on ice and are readily inhibited with a protease inhibitor cocktail. However, CPAF is unusual because it remains active in cell lysates at 4 °C and is resistant to most protease inhibitors (Zhong et al ., 2000; Jorgensen et al ., 2011; Chen et al ., 2012). As an unfortunate consequence of this ongoing enzymatic activity, CPAF cleaves or degrades numerous host proteins during lysate preparation from infected cells. Multiple studies have reported that CPAF mediated proteolysis of specific host proteins begins at about 10–20 h postinfection (hpi) (Dong et al ., 2004; Kumar & Valdivia, 2008; Christian et al ., 2011). However, we did not detect the published proteolysis of 11 host proteins, even as late as 48 hpi, if we inhibited CPAF activity at the time of lysate preparation (Chen et al ., 2012). These doubts about the proteolysis of proposed CPAF targets challenge …

Assessment of pre-analytical and analytical variables affecting peptidome profiling of gingival crevicular fluid by MALDI-TOF mass spectrometry

BackgroundGingival crevicular fluid (GCF) may be a source of new biomarkers of periodontitis/gingivitis. However, the minute volumes of GCF harvested in healthy sites are a serious drawback. We evaluated how pre-analytical and analytical variables concerning GCF collection and processing, could significantly influence quality and reproducibility of MALDI-TOF profiles. MethodsGCF was collected from healthy sites. The use of paper strips vs paper points was compared. Peptides and proteins were extracted by centrifugal elution in different solutions, at different accelerations, with and without protease inhibitor cocktail (PIC). Finally, we evaluated how matrix composition and matrix/sample volume ratio affect the reproducibility of MALDI-TOF profiles. ResultsTrifluoroacetic acid elution generated richer gingival fingerprints compared to acetic acid, independently of the collection device. Centrifugation speed and PIC supplementation did not change GCF profiles. A fine modulation of matrix composition and matrix/sample volume ratio resulted in a satisfactory reproducibility (CV less than 10% for peak area and signal-to-noise ratio). ConclusionAn optimized procedure, enabling generation of reproducible MALDI-TOF profiles from limited volume of GCF, is proposed. These fingerprints may serve as reference for future studies oriented to the maintenance and preservation of good gingival status and to discovery biomarkers of periodontitis/gingivitis.

Quantitative detection of epidermal growth factor and interleukin-8 in whole saliva of healthy individuals

ObjectivesThis study aims to create consensus concerning the use of a methodology by which the handling of saliva is standardized and quantitative detection of IL-8 and EGF in whole saliva is achieved. Our study involves evaluating the extent to which the pre-treatment of saliva samples with an anionic detergent – sodium dodecyl sulphate (SDS) – improved detection levels for IL-8 and EGF. MethodsWhole saliva samples (n=28) were collected from healthy individuals and a protease inhibitor cocktail was added immediately. They were treated with either SDS or PBS for 20min and were then applied to a sandwich ELISA. Results and conclusionsSaliva is a complex viscous fluid that requires degrading before the analysis of salivary biomarkers. We found that pre-treatment of samples with SDS significantly increased the detection levels for both EGF (293%) and IL-8 (346%) when compared with PBS-treated pairs (⁎⁎⁎P<0.001). According to the results we recommend: (i) pre-treatment of whole saliva samples with SDS for quantitative analysis (ii) using secretory output instead of concentration in the presentation of results to avoid individual variations and (iii) taking into consideration gender, age and meal intake since these have an impact on the secretory output of salivary proteins.

Galectin-3 Regulates Desmoglein-2 and Intestinal Epithelial Intercellular Adhesion

The desmosomal cadherins, desmogleins, and desmocollins mediate strong intercellular adhesion. Human intestinal epithelial cells express the desmoglein-2 isoform. A proteomic screen for Dsg2-associated proteins in intestinal epithelial cells identified a lectin referred to as galectin-3 (Gal3). Gal3 bound to N-linked β-galactosides in Dsg2 extracellular domain and co-sedimented with caveolin-1 in lipid rafts. Down-regulation of Gal3 protein or incubation with lactose, a galactose-containing disaccharide that competitively inhibits galectin binding to Dsg2, decreased intercellular adhesion in intestinal epithelial cells. In the absence of functional Gal3, Dsg2 protein was internalized from the plasma membrane and degraded in the proteasome. These results report a novel role of Gal3 in stabilizing a desmosomal cadherin and intercellular adhesion in intestinal epithelial cells.

Dopamine D2 Receptor Relies upon PPM/PP2C Protein Phosphatases to Dephosphorylate Huntingtin Protein

Striatal dopamine D2 receptor (D2R) relies upon G protein- and β-arrestin-dependent signaling pathways to convey its action on motor control and behavior. Considering that D2R activation inhibits Akt in the striatum and that huntingtin physiological functions are affected by Akt phosphorylation, we sought to investigate whether D2R-mediated signaling could regulate huntingtin phosphorylation. We demonstrate that D2R activation decreases huntingtin phosphorylation on its Akt site. This dephosphorylation event depends upon the Gαi-dependent engagement of specific members of the protein phosphatase metallo-dependent (PPM/PP2C) family and is independent of β-arrestin 2. These observations identify the PPM/PP2C family as a mediator of G protein-coupled receptor signaling and thereby suggest a novel mechanism of dopaminergic signaling.