Amino-terminal Fragment Research Articles

NLRC3 Localizes to the Endoplasmic Reticulum via Interactions with a Novel ER-Resident Protein

Abstract Nucleotide binding domain, leucine rich repeat CARD containing protein 3 (NLRC3) is a member of the NLR gene family. Members of this family have been associated with human inflammatory diseases such as Crohn’s disease and cryopyrin-associated periodic syndrome. Although NLRC3 (and others) are not associated with human diseases, it is expressed preferentially in the immune system and functions in pathogen recognition. NLRC3 is an intracellular protein involved in the sensing of lipopolysaccharide and cytosolic nucleic acids. NLRC3 is hypothesized to act as a negative regulator in response to bacterial and viral infection, suggesting that the vertebrate immune system has evolved specific inhibitors to limit the inflammatory response. We performed an unbiased yeast two-hybrid screen using an amino terminal fragment of NLRC3 to identify putative interacting proteins that might help elucidate the mechanism by which NLRC3 might inhibit inflammatory responses. To this end, we identified several interacting proteins. One protein in particular localizes to the endoplasmic reticulum (ER) and might serve as a localization-dependent anchor for NLRC3, thus facilitating its interaction with STING and TBK1. Here we show NLRC3 is distributed to the ER where it can interact with several important signaling molecules involved in type I interferon production. Supported by a grant from the NIH (R15 GM134430)

Macrophages in epicardial adipose tissue and serum NT-proBNP in patients with stable coronary artery disease

Coronary artery disease (CAD) is widely considered a chronic inflammatory disorder, and dysfunction of epicardial adipose tissue could be an important source of the inflammation. Amino-terminal fragment of pro-B-type natriuretic peptide (NT-proBNP) is a known marker of cardiovascular disorders of cardiac origin. Recent studies show that inflammatory stimuli may influence its secretion. Our purpose was to evaluate NT-proBNP serum concentration in relation to immune cell ratios in epicardial adipose tissue (EAT), and cytokine levels in the patients with stable CAD. Patients with stable CAD and heart failure classified into classes II–III, according to the New York Heart Association (NYHA) scale, scheduled for the coronary artery bypass graft (CABG) surgery, were recruited into the study (n = 10; 59.5 (53.0-65.0) y. o.; 50% males). The EAT and subcutaneous adipose tissue (SAT) specimens were harvested in the course of CABG surgery. Immunostaining with anti-CD68, anti-CD45, antiIL-1β and anti-TNFα monoclonal antibodies was performed to evaluate cell composition by differential counts per ten fields (400 magnification). Fasting venous blood was obtained from patients before CABG. Blood was centrifuged at 1500g, aliquots were collected and stored frozen at -40 °С until final analysis. Concentrations of NT-proBNP, IL-1β, IL-6, IL-10, TNFα were determined in serum samples by enzyme-linked immunosorbent assay (ELISA). We have found increased production of IL-1β and TNFα cytokines in EAT compared to SAT. Concentrations of NT-proBNP exceeded 125 pg/ml in 4 patients, and correlations between the CD68+macrophage counts in both EAT and SAT samples (rs= 0.762; p = 0.010 and rs= 0.835; p = 0.003, respectively). NT-proBNP levels showed positive relations with CD45+leukocyte counts (rs= 0.799; p = 0.006), and with IL-1β+cell numbers (rs= 0.705; p = 0.023) in EAT samples only. As for the serum biomarkers, NT-proBNP levels showed negative correlation with fasting glucose levels (rs= -0.684; p = 0.029), and positive correlation with serum IL-6 concentrations (rs= 0.891; p = 0.001). Increased serum concentrations of NT-proBNP in CAD patients correlate with accumulation of macrophages in EAT, which is associated with increased production of IL-1β in EAT and correlates with some metabolic parameters.

Crystal structure and cellular functions of uPAR dimer

Receptor dimerization of urokinase-type plasminogen activator receptor (uPAR) was previously identified at protein level and on cell surface. Recently, a dimeric form of mouse uPAR isoform 2 was proposed to induce kidney disease. Here, we report the crystal structure of human uPAR dimer at 2.96 Å. The structure reveals enormous conformational changes of the dimer compared to the monomeric structure: D1 of uPAR opens up into a large expanded ring that captures a β-hairpin loop of a neighboring uPAR to form an expanded β-sheet, leading to an elongated, highly intertwined dimeric uPAR. Based on the structure, we identify E49P as a mutation promoting dimer formation. The mutation increases receptor binding to the amino terminal fragment of its primary ligand uPA, induces the receptor to distribute to the basal membrane, promotes cell proliferation, and alters cell morphology via β1 integrin signaling. These results reveal the structural basis for uPAR dimerization, its effect on cellular functions, and provide a basis to further study this multifunctional receptor.

Fracture healing is delayed in the absence of gasdermin-interleukin-1 signaling.

Amino-terminal fragments from proteolytically cleaved gasdermins (GSDMs) form plasma membrane pores that enable the secretion of interleukin-1β (IL-1β) and IL-18. Excessive GSDM-mediated pore formation can compromise the integrity of the plasma membrane thereby causing the lytic inflammatory cell death, pyroptosis. We found that GSDMD and GSDME were the only GSDMs that were readily expressed in bone microenvironment. Therefore, we tested the hypothesis that GSDMD and GSDME are implicated in fracture healing owing to their role in the obligatory inflammatory response following injury. We found that bone callus volume and biomechanical properties of injured bones were significantly reduced in mice lacking either GSDM compared with wild-type (WT) mice, indicating that fracture healing was compromised in mutant mice. However, compound loss of GSDMD and GSDME did not exacerbate the outcomes, suggesting shared actions of both GSDMs in fracture healing. Mechanistically, bone injury induced IL-1β and IL-18 secretion in vivo, a response that was mimicked in vitro by bone debris and ATP, which function as inflammatory danger signals. Importantly, the secretion of these cytokines was attenuated in conditions of GSDMD deficiency. Finally, deletion of IL-1 receptor reproduced the phenotype of Gsdmd or Gsdme deficient mice, implying that inflammatory responses induced by the GSDM-IL-1 axis promote bone healing after fracture.

Exploring the activity and inhibition of urokinase-type plasminogen activator (UPA)

Urokinase-type plasminogen activator (uPA) is a trypsin-like serine protease that activates plasminogen into the active serine protease, plasmin. Studying the catalytic activity of uPA gives insight to plasmin’s involvement in anticoagulation, cell adhesion, and degradation of the extracellular matrix, among others. The uPA’s catalytic activity is influenced by the orientation of the catalytic triad and the substrate binding affinity at the specificity pocket. This enzyme consists of an Amino-Terminal Fragment (ATF), a protease domain, and a disordered linker which connects the ATF and the protease domain.

Regulation of microglial transcription factor MEF2C by Alzheimer’s disease‐relevant stimuli

The transcription factor, myocyte enhancer factor-2C (MEF2C), is a master regulator of gene expression in microglia, the brain-resident immune cells. In recent years, microglia have become increasingly recognized as key players in Alzheimer's disease (AD)- they are thought to be protective in early AD and detrimental in late AD by producing pro-inflammatory cytokines that contribute to brain inflammation. Both AD and aging have been associated with increased microglial inflammatory responses and chronic inflammation. MEF2C was recently identified as an 'off-factor' helping to restrain the microglial inflammatory response and may therefore be a potential target for AD treatment. This research project aimed to understand the regulation of microglial MEF2C by AD-relevant inflammatory stimuli lipopolysaccharide (LPS) and polyinosinic:polycytidylic (polyI:C).Mouse microglial cell line, BV2, was exposed to LPS or PolyI:C for 3 h and 24 h. Levels of MEF2C protein and RNA were measured by western blot and qPCR, respectively. Furthermore, MEF2C activity in response to the inflammatory stimuli was investigated by measuring the cytoplasmic and nuclear localisation of MEF2C inhibitor histone deacetylase 4 (HDAC4) and by analysing the expression of two predicted target genes of MEF2C, Ccl3 and Mndal, by qPCR.Expression of whole-cell microglial MEF2C was reduced in BV2 cells exposed to LPS and PolyI:C for 3hrs but was increased at 24hrs LPS exposure relative to the control. Cytoplasmic HDAC4 expression was reduced with LPS exposure, which was more pronounced at the 24-hour BV2 cells exposure. In addition, the inflammatory stimuli increased the amount of a potential amino-terminal HDAC4 fragment in the nucleus. Furthermore, Ccl3 and Mndal expression in response to LPS and PolyI:C at 3 hours was reduced.This research project provided evidence that AD-relevant inflammatory stimuli downregulate microglial MEF2C expression. In addition, MEF2C transcriptional activity was reduced by these inflammatory stimuli, possibly via induction of caspase-3, leading to fragmentation of HDAC4 and subsequent translocation of HDAC4 amino-terminal fragment into the nucleus at MEF2C-targeted promoters. Overall, this provided a mechanistic insight on how the crucial function of MEF2C is lost during inflammatory conditions.

Gasdermin D deficiency attenuates arthritis induced by traumatic injury but not autoantibody-assembled immune complexes

BackgroundGasdermin D (GSDMD) is cleaved by several proteases including by caspase-1, a component of intracellular protein complexes called inflammasomes. Caspase-1 also converts pro-interleukin-1β (pro-IL-1β) and pro-IL-18 into bioactive IL-1β and IL-18, respectively. GSDMD amino-terminal fragments form plasma membrane pores, which mediate the secretion of IL-1β and IL-18 and cause the inflammatory form of cell death pyroptosis. Here, we tested the hypothesis that GSDMD contributes to joint degeneration in the K/BxN serum transfer-induced arthritis (STIA) model in which autoantibodies against glucose-6-phosphate isomerase promote the formation of pathogenic immune complexes on the surface of myeloid cells, which highly express the inflammasomes. The unexpected outcomes with the STIA model prompted us to determine the role of GSDMD in the post-traumatic osteoarthritis (PTOA) model caused by meniscus ligamentous injury (MLI) based on the hypothesis that this pore-forming protein is activated by signals released from damaged joint tissues.MethodsGsdmd+/+ and Gsdmd−/− mice were injected with K/BxN mouse serum or subjected to MLI to cause STIA or PTOA, respectively. Paw and ankle swelling and DXA scanning were used to assess the outcomes in the STIA model whereas histopathology and micro-computed tomography (μCT) were utilized to monitor joints in the PTOA model. Murine and human joint tissues were also examined for GSDMD, IL-1β, and IL-18 expression by qPCR, immunohistochemistry, or immunoblotting.ResultsGSDMD levels were higher in serum-inoculated paws compared to PBS-injected paws. Unexpectedly, ablation of GSDMD failed to reduce joint swelling and osteolysis, suggesting that GSDMD was dispensable for the pathogenesis of STIA. GSDMD levels were also higher in MLI compared to sham-operated joints. Importantly, ablation of GSDMD attenuated MLI-associated cartilage degradation (p = 0.0097), synovitis (p = 0.014), subchondral bone sclerosis (p = 0.0006), and subchondral bone plate thickness (p = 0.0174) based on histopathological and μCT analyses.ConclusionGSDMD plays a key role in the pathogenesis of PTOA, but not STIA, suggesting that its actions in experimental arthropathy are tissue context-specific.

NT-proBNP and Major Adverse Cardiovascular Events in Patients with ST-Segment Elevation Myocardial Infarction Who Received Primary Percutaneous Coronary Intervention: A Prospective Cohort Study.

Background The prognostic significance of the amino-terminal fragment of the prohormone brain-type natriuretic peptide (NT-proBNP) in patients with ST-segment elevation myocardial infarction (STEMI) after percutaneous coronary intervention (PCI) has not been fully elucidated. Major adverse cardiovascular events (MACEs) are clinically viable indicators for the accurate, rapid, and safe evaluation of patients with STEMI. This study was designed to investigate the relationship between NT-proBNP levels and the occurrence of short-term MACEs in patients with STEMI who underwent emergency PCI. Methods This prospective cohort study included 405 patients with STEMI aged 20–90 years who underwent emergency PCI at the First People's Hospital of Changde City from April 6, 2017, to May 31, 2019. Stent thrombosis, reinfarction, congestive heart failure, unstable angina, and cardiac death were considered as MACEs in this study. The target-independent and -dependent variables were NT-proBNP at baseline and MACE, respectively. Results There were 28.25% of MACEs. Age, number of implanted stents, Killip class, infarction-related artery, applied intra-aortic balloon pump (IABP), creatine kinase (CK) peak value, CK-MB peak value, TnI peak value, and ST-segment resolution were independently associated with MACE (P < 0.05). In a multivariate model, after adjusting all potential covariates, Log2 NT-proBNP levels remained significantly associated with MACE, with an inflection point of 11.66. The effect sizes and confidence intervals of the left and right sides of the inflection point were 1.07 and 0.84–1.36 (P=0.5730) and 3.47 and 2.06–5.85 (P < 0.0001), respectively. Conclusions In patients with STEMI who underwent PCI, Log2 NT-proBNP was positively correlated with MACE within 1 month when the Log2 NT-proBNP was >11.66 (NT-proBNP >3.236 pg/mL).

APC-Targeted DNA Vaccination Against Reticulocyte-Binding Protein Homolog 5 Induces Plasmodium falciparum-Specific Neutralizing Antibodies and T Cell Responses.

Targeted delivery of antigen to antigen presenting cells (APCs) is an efficient way to induce robust antigen-specific immune responses. Here, we present a novel DNA vaccine that targets the Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5), a leading blood-stage antigen of the human malaria pathogen, to APCs. The vaccine is designed as bivalent homodimers where each chain is composed of an amino-terminal single chain fragment variable (scFv) targeting unit specific for major histocompatibility complex class II (MHCII) expressed on APCs, and a carboxyl-terminal antigenic unit genetically linked by the dimerization unit. This vaccine format, named “Vaccibody”, has previously been successfully applied for antigens from other infectious diseases including influenza and HIV, as well as for tumor antigens. Recently, the crystal structure and key functional antibody epitopes for the truncated version of PfRH5 (PfRH5ΔNL) were characterized, suggesting PfRH5ΔNL to be a promising candidate for next-generation PfRH5 vaccine design. In this study, we explored the APC-targeting strategy for a PfRH5ΔNL-containing DNA vaccine. BALB/c mice immunized with the targeted vaccine induced higher PfRH5-specific IgG1 antibody responses than those vaccinated with a non-targeted vaccine or antigen alone. The APC-targeted vaccine also efficiently induced rapid IFN-γ and IL-4 T cell responses. Furthermore, the vaccine-induced PfRH5-specific IgG showed inhibition of growth of the P. falciparum 3D7 clone parasite in vitro. Finally, sera obtained after vaccination with this targeted vaccine competed for the same epitopes as PfRH5-specific mAbs from vaccinated humans. Robust humoral responses were also induced by a similar P. vivax Duffy-binding protein (PvDBP)-containing targeted DNA vaccine. Our data highlight a novel targeted vaccine platform for the development of vaccines against blood-stage malaria.

Novel antisense therapy targeting microRNA-132 in patients with heart failure

Abstract Background Cardiac microRNA-132-3p (miR-132) levels are increased in patients with heart failure (HF) and mechanistically drive cardiac remodelling processes. CDR132L, a specific antisense oligonucleotide, is a first-in-class miR-132 inhibitor that attenuates and even reverses HF in preclinical models. Purpose The aim of the current clinical Phase 1b study was to assess safety, pharmacokinetics, target engagement, and exploratory pharmacodynamic effects of CDR132L in patients on standard-of-care therapy for chronic ischaemic HF in a randomized, placebo-controlled, double-blind, dose-escalation study. Methods Patients had left ventricular ejection fraction between ≥30% and &amp;lt;50% or amino terminal fragment of pro-brain natriuretic peptide (NT-proBNP) &amp;gt;125 ng/L at screening. Twenty-eight patients were randomized to receive CDR132L (0.32, 1, 3, and 10 mg/kg body weight) or placebo (0.9% saline) in two intravenous infusions, 4 weeks apart in four cohorts of seven (five verum and two placebo) patients each. Results CDR132L was safe and well tolerated, without apparent dose-limiting toxicity. A pharmacokinetic/pharmacodynamic dose modelling approach suggested an effective dose level at ≥1 mg/kg CDR132L. CDR132L treatment resulted in a dose-dependent, sustained miR-132 reduction in plasma. Patients given CDR132L ≥1 mg/kg displayed median 23.3% NT-proBNP reduction, vs. 0.9% median increase in the control group. CDR132L treatment induced significant QRS narrowing and positive trends for cardiac fibrosis biomarkers. Conclusions This study is the first clinical trial of an antisense drug in HF patients. CDR132L was safe and well tolerated, confirmed linear plasma pharmacokinetics with no signs of accumulation, and suggests cardiac functional improvements. The indicative efficacy of this drug is very encouraging justifying additional clinical studies to confirm the beneficial CDR132L pharmacodynamic effects for the treatment of HF. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Cardior Pharmaceuticals GmbH

Fragmentation of Apolipoprotein E4 is Required for Differential Expression of Inflammation and Activation Related Genes in Microglia Cells.

The apolipoprotein E4 (APOE4) allele represents the single greatest risk factor for late-onset Alzheimer's disease (AD) and accumulating evidence suggests that fragmentation with a toxic-gain of function may be a key molecular step associated with this risk. Recently, we demonstrated strong immunoreactivity of a 151 amino-terminal fragment of apoE4 (E4-fragment) within the nucleus of microglia in the human AD brain. In vitro, this fragment led to toxicity and activation of inflammatory processes in BV2 microglia cells. Additionally, a transcriptome analysis following exogenous treatment of BV2 microglia cells with this E4 fragment led to a > 2-fold up regulation of 1,608 genes, with many genes playing a role in inflammation and microglia activation. To extend these findings, we here report a similar transcriptome analysis in BV2 microglia cells following treatment with full-length ApoE4 (FL-ApoE4). The results indicated that full-length ApoE4 had a very small effect on gene expression compared to the fragment. Only 48 differentially expressed genes (DEGs) were identified (p < 0.05, and greater than 2-fold change). A gene ontology analysis of these DEGs indicated that they are not involved in inflammatory and activation processes, in contrast to the genes up regulated by the E4-fragment. In addition, genes that showed a negative fold-change upon FL-E4 treatment typically showed a strong positive fold-change upon treatment with the fragment (Pearson's r = -0.7). Taken together, these results support the hypothesis that a key step in the conversion of microglia to an activated phenotype is proteolytic cleavage of FL-ApoE4. Therefore, the neutralization of this amino-terminal fragment of ApoE4, specifically, may serve as an important therapeutic strategy in the treatment of AD.

Abstract 114: Safety And Efficacy Of CDR132L, A Novel Antisense Therapeutic Which Targets MicroRNA-132 In Heart Failure Patients

Cardiac microRNA-132-3p (miR-132) levels are elevated in people suffering with heart failure (HF) and mechanistically drive remodelling processes in the heart. Here we present CDR132L, a specific antisense oligonucleotide. It is a first-in-class miR-132 inhibitor that attenuates and even reverses HF in preclinical models. The aim of this clinical Phase 1b study was to assess safety, pharmacokinetics, target engagement, and pharmacodynamics of CDR132L in patients on therapy for chronic ischaemic HF in a randomized, placebo-controlled, double-blind, dose-escalation study. Inclusion criteria for participant patients was left ventricular ejection fraction between 30 and 50% or levels of amino terminal fragment of pro-brain natriuretic peptide (NT-proBNP) higher than 125 ng/L at screening. Twenty-eight patients were randomized to receive CDR132L (0.32, 1, 3, and 10 mg/kg body weight) or placebo (0.9% saline) in two intravenous infusions, 4 weeks apart. Randomization separated participants into four cohorts of seven (five verum and two placebo). Results revealed that CDR132L was safe and well tolerated, and there was no detectable dose-limiting toxicity. A pharmacokinetic/pharmacodynamic dose modelling approach suggested an effective dose level of ≥1 mg/kg CDR132L. CDR132L treatment resulted in a dose-dependent, sustained miR-132 reduction in plasma. Patients who were administered ≥1 mg/kg of CDR132L displayed 23.3% NT-proBNP reduction, whereas patients receiving placebo experienced a 0.9% increase. In addition, CDR132L treatment was observed to significantly reduce duration of the QRS interval. This is the first clinical trial in which an antisense oligonucleotide was administered as a therapeutic agent to HF patients. Linear plasma pharmacokinetics were confirmed, and there were no signs of accumulation. The application of this drug suggests cardiac functional improvements and cardiac remodelling. The efficacy of this drug is encouraging, and further clinical work with larger patient populations should follow. Although the study used small patient numbers, results suggest some clinical benefit in chronic HF patients on the top of standard of care. This included an improvement in HF severity and narrowing of the QRS complex. The pharmacodynamic findings presented here are encouraging as they confirm efficacy results seen in animal studies.

Suppression of amyloid-β secretion from neurons by cis-9, trans-11-octadecadienoic acid, an isomer of conjugated linoleic acid.

Two common conjugated linoleic acids (LAs), cis-9, trans-11 CLA (c9,t11 CLA) and trans-10, cis-12 CLA (t10,c12 CLA), exert various biological activities. However, the effect of CLA on the generation of neurotoxic amyloid-β (Aβ) protein remains unclear. We found that c9,t11 CLA significantly suppressed the generation of Aβ in mouse neurons. CLA treatment did not affect the level of β-site APP-cleaving enzyme 1 (BACE1), a component of active γ-secretase complex presenilin 1 amino-terminal fragment, or Aβ protein precursor (APP) in cultured neurons. BACE1 and γ-secretase activities were not directly affected by c9,t11 CLA. Localization of BACE1 and APP in early endosomes increased in neurons treated with c9,t11 CLA; concomitantly, the localization of both proteins was reduced in late endosomes, the predominant site of APP cleavage by BACE1. The level of CLA-containing phosphatidylcholine (CLA-PC) increased dramatically in neurons incubated with CLA. Incorporation of phospholipids containing c9,t11 CLA, but not t10,c12 CLA, into the membrane may affect the localization of some membrane-associated proteins in intracellular membrane compartments. Thus, in neurons treated with c9,t11 CLA, reduced colocalization of APP with BACE1 in late endosomes may decrease APP cleavage by BACE1 and subsequent Aβ generation. Our findings suggest that the accumulation of c9,t11 CLA-PC/LPC in neuronal membranes suppresses the production of neurotoxic Aβ in neurons.

Highly Conserved C-Terminal Region of Indian Hedgehog N-Fragment Contributes to Its Auto-Processing and Multimer Formation.

Hedgehog (HH) is a highly conserved secretory signalling protein family mainly involved in embryonic development, homeostasis, and tumorigenesis. HH is generally synthesised as a precursor, which subsequently undergoes autoproteolytic cleavage to generate an amino-terminal fragment (HH-N), mediating signalling, and a carboxyl-terminal fragment (HH-C), catalysing the auto-processing reaction. The N-terminal region of HH-N is required for HH multimer formation to promote signal transduction, whilst the functions of the C-terminal region of HH-N remain ambiguous. This study focused on Indian Hedgehog (IHH), a member of the HH family, to explore the functions of the C-terminal region of the amino-terminal fragment of IHH (IHH-N) via protein truncation, cell-based assays, and 3D structure prediction. The results revealed that three amino acids, including S195, A196, and A197, were crucial for the multimer formation by inserting the mutual binding of IHH-N proteins. K191, S192, E193, and H194 had an extremely remarkable effect on IHH self-cleavage. In addition, A198, K199, and T200 evidently affected the stability of IHH-N. This work suggested that the C-terminus of IHH-N played an important role in the physiological function of IHH at multiple levels, thus deepening the understanding of HH biochemical properties.

Protective role of PhtD and its amino and carboxyl fragments against pneumococcal sepsis

The implementation of polysaccharide-based vaccines has massively reduced the incidence of invasive pneumococcal diseases. However, there is great concern regarding serotype replacement and the increase in antibiotic resistant strains expressing non-vaccine capsular types. In addition, conjugate vaccines have high production costs, a limiting factor for their implementation in mass immunization programs in developing countries. These limitations have prompted the development of novel vaccine strategies for prevention of Streptococcus pneumoniae infections. The use of conserved pneumococcal antigens such as recombinant proteins or protein fragments presents an interesting serotype-independent alternative. Pht is a family of surface-exposed proteins which have been evaluated as potential vaccine candidates with encouraging results. The present work investigated the immune responses elicited by subcutaneous immunization of mice with the polyhistidine triad protein D (PhtD) and its amino and carboxyl terminal fragments. The proteins were immunogenic and protective against pneumococcal sepsis in mice. Antibodies raised against PhtD increased complement C3b deposition on the pneumococcal surface, mainly mediated by the alternative pathway. Sera from mice immunized with PhtD and PhtD_Cter promoted an increase in bacterial uptake by mouse phagocytes. The interaction of PhtD with the complement system regulator factor H was investigated in silico and in vitro by ELISA and western blot, confirming PhtD as a factor-H binding protein. Our results support the inclusion of PhtD and more specifically, its C-terminal fragment in a multicomponent serotype independent vaccine and suggests a role for the complement system in PhtD-mediated protection.

Enhanced insulin‐responsive AS160 elicits cardioprotection during metabolic stress

Heart disease is the leading cause of death in the United States, and cardiovascular mortality rates positively correlate with the presence of diabetes and metabolic syndrome. There is a consensus that abdominal obesity is a major driver in the pathogenesis of metabolic syndrome, with severe adverse effects on cardiovascular risk factors. In the midst of a growing recognition of the effect of adipose tissue dysfunction and adipokine imbalance on cardiovascular disease, a less-well understood mechanism is the effect of cardiac metabolism and function on systemic metabolic homeostasis. There is increasing recognition that signals produced by the heart can affect distant organ function; however, little is known regarding mechanistic links. While elevated GRK2 activity and expression occur early in cardiovascular disease and contribute to progression, ongoing studies show cardiac restricted expression of a short, amino terminal fragment of GRK2 (βARKnt) preserves cardiac structure and function during chronic pressure overload. Surprisingly, these studies revealed a significant decrease in gonadal fat weight, equivalent to human abdominal fat, in male βARKnt mice at baseline and following cardiac stress. In contrast, cardiac overexpression of GRK2 produces an overall lean phenotype, while the peptide inhibitor of GRK2 (βARKct) enhances the obesogenic phenotype. Proteomic analysis to identify βARKnt binding partners that may underlie the improved cardiovascular and metabolic phenotype uncovered a selective functional interaction of both endogenous GRK2 and βARKnt with AKT substrate of 160kDa (AS160). AS160 has emerged as a key downstream regulator of insulin signaling, integrating physiological and metabolic cues to couple energy demand to membrane recruitment of Glut4. Our preliminary data indicate that in βARKnt mice, cardiomyocyte insulin sensitivity is improved during stress, without metabolic switching. These mice demonstrate an ~23% increase in phosphorylated Akt, ~75% increase in phosphorylated and total AS160, and upregulation of the mTORC1 pathway. Seahorse of dissociated adult cardiomyocytes and Oroboros of fresh, permeabilized cardiac tissue exhibited enhanced spare respiratory activity and ATP production in the presence of glucose and palmitate. Unlike control mice that develop insulin resistance in response to high fat diet stress, the βARKnt mice demonstrate enhanced glucose tolerance and improved insulin sensitivity (glucose tolerance test area under curve (AUC) is 2958 vs 2354; insulin tolerance AUC is 1245 vs 991.7 at 14 weeks), increased energy expenditure, and enhanced cardiac function at 4 and 14 weeks. These data suggest that the enhanced AS160-mediated signaling in the βARKnt mice may ameliorate pathological cardiac remodeling through direct modulation of insulin signaling within cardiomyocytes, and translate these to beneficial effects on systemic metabolism. Ongoing studies seek to elucidate the underlying mechanisms of these beneficial effects, to gain insights into development of new therapeutic strategies for metabolic syndrome and obesity-related cardiovascular disease.

A peptide of the amino-terminus of GRK2 induces hypertrophy and yet elicits cardioprotection after pressure overload

G protein-coupled receptor (GPCR) kinase 2 (GRK2) expression and activity are elevated early on in response to several forms of cardiovascular stress and are a hallmark of heart failure. Interestingly, though, in addition to its well-characterized role in regulating GPCRs, mounting evidence suggests a GRK2 “interactome” that underlies a great diversity in its functional roles. Several such GRK2 interacting partners are important for adaptive and maladaptive myocyte growth; therefore, an understanding of domain-specific interactions with signaling and regulatory molecules could lead to novel targets for heart failure therapy. Herein, we subjected transgenic mice with cardiac restricted expression of a short, amino terminal fragment of GRK2 (βARKnt) to pressure overload and found that unlike their littermate controls or previous GRK2 fragments, they exhibited an increased left ventricular wall thickness and mass prior to cardiac stress that underwent proportional hypertrophic growth to controls after acute pressure overload. Importantly, despite this enlarged heart, βARKnt mice did not undergo the expected transition to heart failure observed in controls. Further, βARKnt expression limited adverse left ventricular remodeling and increased cell survival signaling. Proteomic analysis to identify βARKnt binding partners that may underlie the improved cardiovascular phenotype uncovered a selective functional interaction of both endogenous GRK2 and βARKnt with AKT substrate of 160 kDa (AS160). AS160 has emerged as a key downstream regulator of insulin signaling, integrating physiological and metabolic cues to couple energy demand to membrane recruitment of Glut4. Our preliminary data indicate that in βARKnt mice, cardiomyocyte insulin signaling is improved during stress, with a coordinate increase in spare respiratory activity and ATP production without metabolite switching. Surprisingly, these studies also revealed a significant decrease in gonadal fat weight, equivalent to human abdominal fat, in male βARKnt mice at baseline and following cardiac stress. These data suggest that the enhanced AS160-mediated signaling in the βARKnt mice may ameliorate pathological cardiac remodeling through direct modulation of insulin signaling within cardiomyocytes, and translate these to beneficial effects on systemic metabolism.

Hdxms Reveals the Role of Dynamics on the Serine Protease Activity of the Urokinase-Type Plasminogen Activator (UPA)

The urokinase-type plasminogen activator (uPA) is a key regulator of cellular processes such wound healing, angiogenesis and anticoagulation, among others. This protein is a serine protease synthesized as inactive single-chain zymogen that becomes active after a very specific cleavage between K158 and I159. The uPA catalyzes the rate limiting step of anticoagulation: the activation of peri-cellular plasminogen into the active serine protease plasmin. Even though the chemistry behind this reaction is well understood with a Ser/Asp/His catalytic triad, the dynamics of the protein and how dynamics control the activity remain unknown. The uPA is formed by an EGF domain, a Kringle domain, a 27-residue linker region and a serine protease domain. The structure of Full-length uPA has not been solved, however, there are structures available for the EGF and Kringle domains (the Amino terminal fragment, or ATF), and the serine protease domain separately. Whether the ATF interacts through the linker with the protease domain remains unknown, but the full-length protein is 6-fold more active (kcat) towards plasminogen when the ATF is present. We hypothesized that there is through-linker dynamic allostery within uPA. To test our hypothesis, we evaluated the changes in dynamics of uPA by Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS). We compared the protease domain alone to full-length uPA, the single chain to the two chain, and receptor (uPAR)-bound to free. The HDX-MS results help to resolve which regions of the protease domain are allosterically regulated.

Development and validation of a novel LC-MS/MS assay for C-peptide in human serum

IntroductionC-peptide is used as a marker of endogenous insulin secretion in the assessment of residual β-cell function in diabetes and in the diagnostic workup of hypoglycemia. Previously developed LC-MS/MS methods to quantify serum concentrations of C-peptide have monitored intact peptide, which ionizes poorly. As a result, methods have leveraged immunoaffinity enrichment or two-dimensional chromatography. In this study, we aimed to use proteolysis during sample preparation to enhance the sensitivity of traditional LC-MS/MS. MethodsDue to the absence of arginine and lysine residues in C-peptide, we utilized Glu-C as the proteolytic enzyme in the method. After protein precipitation using acetonitrile and solid phase extraction with mixed anion exchange, lower molecular weight polypeptides were reduced, alkylated, and proteolyzed. The two amino-terminal peptide fragments, EAEDLQVGQVEand LGGGPGAGSLQPLALE, were monitored using multiple reaction monitoring in positive ion mode (Acquity ULPC-Xevo TQ-S, Waters). The former peptide was used for quantification and the latter for quality assurance. ResultsGlu-C was determined to be a reliable proteolytic enzyme with monotonic digestion kinetics.The assay was linear between 0.1 and 15 ng/mL and had a lower limit of quantification of 0.06 ng/mL. Total imprecision was 7.7 %CV and long-term imprecision at 0.16 ng/mL was 10.0%. Spike-recovery experiments demonstrated a mean recovery of 98.2 % (± 9.1 %) and the method compared favorably with a commercially available immunoassay and a reference measurement procedure. ConclusionProtein precipitation with solid phase extraction and proteolysis with Glu-C is a robust sample preparation method for quantification of C-peptide in human serum by LC-MS/MS.

The amino-terminal heptapeptide of the algesic substance P provides analgesic effect in relieving chronic neuropathic pain

Of painful conditions, somatic pain of acute nociceptive origin can be effectively managed clinically, while neuropathic pain of chronic neuropathy origin is difficult to control. For molecules involved in pain sensation, substance P (SP) is algesic, exacerbating painful sensation, while its amino-terminal fragment, heptapeptide SP(1-7), confers biological activities different from its full-length parent neuropeptide precursor. We previously demonstrated SP(1-7) interaction with pain processing to alleviate chronic pain. Here we evaluated SP(1-7) and its C-terminal amidated analogue SP(1-7)amide, together with SP and opioid agonist DAMGO. We tested mouse behaviors of both acute somatic pain in tail-flick latency assay, and neuropathic pain in sciatic nerve injury model of chronic constriction injury (CCI). DAMGO produced dose-dependent analgesia for somatic pain as expected, so did both SP(1-7) and its analogue SP(1-7)amide, while SP yielded the opposite effect of algesia, in a phenomenon we termed ‘contrintus’, meaning ‘opposite from within’ to denote that two peptides of the same origin (SP and its metabolic fragment SP(1-7)) produced opposite effects. In CCI model, DAMGO showed a general reduction in allodynia sensitivity for both nerve-injured and normal paws, without selective effect for neuropathic pain, consistent with clinical observation that opioids are less effective for chronic neuropathic pain. On the other hand, both SP(1-7) and SP(1-7)amide displayed dose-dependent anti-allodynia effect that is selective for neuropathic pain. These findings suggest that SP(1-7) and its analogue may be useful for developing pharmaceuticals to treat neuropathic pain.