Penultimate Amino Acid Research Articles

The extracellular tail of IFITM3 promotes SARS-CoV-2 entry into cells and is diversifying in primates

Abstract The interferon-induced transmembrane (IFITM) proteins confer cells with resistance to diverse virus infections by altering membrane biomechanics and disfavoring virus-cell membrane fusion. Human IFITM3 strongly restricts SARS-CoV and MERS Spike-mediated infections in endosomes. However, work from our labs demonstrated that IFITM3 has opposing effects on SARS-CoV-2 (SCV2) infection: IFITM3 in endosomes restricts SCV2 infection, but IFITM3 at the cell surface enhances SCV2 infection. In the present study, we investigated the determinants governing the enhancement of SCV2 infection by human IFITM3. Using clinical isolates of SCV2 and pseudoviruses bearing SCV2 Spike, we found that the penultimate and ultimate amino acid residues of IFITM3, which are predicted to extend into the extracellular space, are essential for enhancement of SCV2 Spike-mediated infection. Deletion of these residues in human IFITM3 resulted in loss of SCV2 infection enhancement. On the other hand, we found that certain non-human IFITM3 proteins enhanced SCV2 infection to a greater extent than human IFITM3, and this mapped to sequence divergence in the penultimate and ultimate residues of the IFITM3 extracellular tail. Transferring the last two amino acids of macaque IFITM3 to human IFITM3 resulted in further enhancement of SCV2 infection. These results indicate that certain non-human primate IFITM3 proteins may be poised to act as potent enhancers of SCV2 infection in those species. Genomic analyses revealed that the IFITM3 extracellular tail is diversifying rapidly and recurrently in primates over evolutionary time, raising the possibility that variation at these sites is adaptive in nature and allows for escape from co-option by coronaviruses. NIH Intramural Research Program NIAID Intramural Targeted Anti-COVID19 Program Award

Prolylcarboxypeptidase promotes IGF1R/HER3 signaling and is a potential target to improve endocrine therapy response in estrogen receptor positive breast cancer

ABSTRACT Prolylcarboxypeptidase (PRCP) is a lysosomal serine protease that cleaves peptide substrates when the penultimate amino acid is proline. Previous studies have linked PRCP to blood-pressure and appetite control through its ability to cleave peptide substrates such as angiotensin II and α-MSH. A potential role for PRCP in cancer has to date not been widely appreciated. Endocrine therapy resistance in breast cancer is an enduring clinical problem mediated in part by aberrant receptor tyrosine kinase (RTK) signaling. We previously found PRCP overexpression promoted 4-hydroxytamoxifen (4-OHT) resistance in estrogen receptor-positive (ER+) breast cancer cells. Currently, we tested the potential association between PRCP with breast cancer patient outcome and RTK signaling, and tumor responsiveness to endocrine therapy. We found high PRCP protein levels in ER+ breast tumors associates with worse outcome and earlier recurrence in breast cancer patients, including patients treated with TAM. We found a PRCP specific inhibitor (PRCPi) enhanced the response of ER+ PDX tumors and MCF7 tumors to endoxifen, an active metabolite of TAM in mice. We found PRCP increased IGF1R/HER3 signaling and AKT activation in ER+ breast cancer cells that was blocked by PRCPi. Thus, PRCP is an adverse prognostic marker in breast cancer and a potential target to improve endocrine therapy in ER+ breast cancers.

Prolyl Carboxypeptidase Maintains Receptor Tyrosine Kinase Signaling and Is a Potential Therapeutic Target in Triple Negative Breast Cancer.

Simple SummaryTriple negative breast cancer (TNBC) is an aggressive cancer type with limited treatment options and poor prognosis. Our research has revealed that a protein called prolylcarboxypeptidase (PRCP) is a potential therapy target for TNBC. We found that high levels of PRCP in tumors coincides with worse prognosis in TNBC patients. Inhibition of PRCP with a small molecule inhibitor blocked TNBC cell and tumor growth and inhibited the activity of several receptor tyrosine kinases (RTKs), proteins that are located on the surface of cells and that are important for cancer development and progression. Our findings suggest that PRCP is a novel prognostic factor for TNBC and that specific inhibitors of PRCP could be developed for TNBC treatment.TNBC is an aggressive cancer sub-type with limited treatment options and poor prognosis. New therapeutic targets are needed to improve outcomes in TNBC patients. PRCP is a lysosomal serine protease that cleaves peptide substrates when the penultimate amino acid is proline. A role for PRCP in TNBC or other cancers, and its potential as a therapy target has not yet been tested. In the current study, we found high tumor expression of PRCP associates with worse outcome and earlier recurrence in TNBC patients. Knockdown of PRCP or treatment with a small molecule PRCP inhibitor blocked proliferation and survival in TNBC cell lines and inhibited growth of TNBC tumors in mice. Mechanistically, we found PRCP maintains signaling from multiple receptor tyrosine kinases (RTKs), potentially by promoting crosstalk between RTKs and G-protein coupled receptors (GPCRs). Lastly, we found that the PRCP inhibitor caused synergistic killing of TNBC cells when combined with the EGFR and ErbB2 inhibitor lapatinib. Our results suggest that PRCP is potential prognostic marker for TNBC patient outcome and a novel therapeutic target for TNBC treatment.

DPP4+ exosomes in AML patients' plasma suppress proliferation of hematopoietic progenitor cells.

Mechanisms by which acute myeloid leukemia (AML) interferes with normal hematopoiesis are under intense investigation. Emerging evidence suggests that exosomes produced by leukemia blasts suppress hematopoiesis. Exosomes isolated from AML patients' plasma at diagnosis significantly and dose-dependently suppressed colony formation of normal hematopoietic progenitor cells (HPC). Levels of HPC suppression mediated by exosomes of AML patients who achieved complete remission (CR) were significantly decreased compared to those observed at AML diagnosis. Exosomes from plasma of patients who had achieved CR but with incomplete cell count recovery (CRi) after chemotherapy suppressed in vitro colony formation as effectively as did exosomes obtained at AML diagnosis. Dipeptidylpeptidase4 (DPP4/CD26), a serine protease that cleaves select penultimate amino acids of various proteins, has been previously implicated in the regulation of hematopoiesis. DPP4 was carried by exosomes from AML plasma or leukemia cell lines. Leukemia exosomes which suppressed HSC colony formation had markedly higher DPP4 functional activity than that detected in the exosomes of normal donors. Pharmacological inhibition of DPP4 activity in AML exosomes reversed the effects of exosome-mediated myelosuppression. Reversing the negative effects of exosomes on AML hematopoiesis, and thus improving cell count recovery, might emerge as a new therapeutic approach to AML.

Combined Ligand-Based and Structure-Based Virtual Screening Approach for Identification of New Dipeptidyl Peptidase 4 Inhibitors.

Dipeptidyl Peptidase 4 (DPP 4) enzyme cleaves an incretin-based glucoregulatory hormone Glucagon Like Peptide -1 from N-terminal where penultimate amino acid is either alanine or proline. Several DPP 4 inhibitors, "gliptins", are approved for the management of Type 2 Diabetes or are under clinical trial. In the present study, combined pharmacophore and docking-based virtual screening protocol were used for the identification of new hits from the Specs Database, which would inhibit DPP 4. The entire computational studies were performed using the Discovery Studio v. 4.1 software package, Pipeline Pilot v. 9.2 (Accelrys Inc.) and FRED v. 2.2.5 (OpenEye Scientific Software). Common feature pharmacophore model was generated from known DPP 4 inhibitors and validated by Receiver Operating curve analysis and GH-scoring method. Database search of Specs commercial database was performed using validated pharmacophore. Hits obtained from pharmacophore search were further docked into the binding site of DPP 4. Based on the analysis of docked poses of hits, 10 compounds were selected for in- vitro DPP 4 enzyme inhibition assay. Based on docking studies, virtual hits were predicted to form interaction with essential amino acid residues of DPP 4 and have an almost similar binding orientation as that of the reference molecule. Three compounds having Specs database ID- AN-465/42837213, AP-064/42049348 and AN- 465/43369427 were found to inhibit DPP 4 enzyme moderately. The present study demonstrates a successful utilization of in-silico tools in the identification of new DPP 4 inhibitor, which can serve as a starting point for the development of novel DPP 4 inhibitors.

Active-site directed peptide l-Phe-d-His-l-Leu inhibits angiotensin converting enzyme activity and dexamethasone-induced hypertension in rats

Hypertension is the fundamental cause of cardiovascular and cerebrovascular disorders. Several natural and synthetic peptides are being used as antihypertensive agents, which target angiotensin converting enzyme (ACE), the master regulator of angiotensin (Ang) II production. In this study, we have evaluated ACE-inhibitory potential of the tripeptide l-Phenylalanyl-d-Histidyl-l-Leucine (l-Phe-d-His-l-Leu) in vitro and its antihypertensive effect in rat model of dexamethasone-induced hypertension. l-Phe-d-His-l-Leu was custom-designed by changing the configuration of penultimate amino acid residue (histidine) from C-terminal of Ang I, the site at which ACE acts upon and generates Ang II. l-Phe-d-His-l-Leu effectively inhibited ACE activity in a dose-dependent and competitive manner with an IC50 of 53.32 ± 0.13 nmol/L. Both fluorescence spectra and circular dichroism data revealed the direct interaction between l-Phe-d-His-l-Leu and ACE. In addition, molecular docking studies revealed the strong interaction of l-Phe-d-His-l-Leu with the critical active site amino acid residues of ACE. Further, the administration of l-Phe-d-His-l-Leu resulted in decrease in blood pressure (142 ± 3 mmHg) compared to dexamethasone alone group (167 ± 2 mmHg). Besides, l-Phe-d-His-l-Leu decreased the levels of circulating Ang II, and reduced fibrosis in heart and kidney, as evidenced by decreases in collagen deposition. Thus, the strategy of incorporation of d-amino acids in ACE-inhibitory peptides could be valuable in the development of antihypertensive drugs.

Charge and Polarity Preferences for N-Glycosylation: A Genome-Wide In Silico Study and Its Implications Regarding Constitutive Proliferation and Adhesion of Carcinoma Cells.

The structural and functional diversity of the human proteome is mediated by N- and O-linked glycosylations that define the individual properties of extracellular and membrane-associated proteins. In this study, we utilized different computational tools to perform in silico based genome-wide mapping of 1,117 human proteins and unravel the contribution of both penultimate and vicinal amino acids for the asparagine-based, site-specific N-glycosylation. Our results correlate the non-canonical involvement of charge and polarity environment of classified amino acids (designated as L, O, A, P, and N groups) in the N-glycosylation process, as validated by NetNGlyc predictions, and 130 literature-reported human proteins. From our results, particular charge and polarity combinations of non-polar aliphatic, acidic, basic, and aromatic polar side chain environment of both penultimate and vicinal amino acids were found to promote the N-glycosylation process. However, the alteration in side-chain charge and polarity environment of genetic variants, particularly in the vicinity of Asn-containing epitope, may induce constitutive glycosylation (e.g., aberrant glycosylation at preferred and non-preferred sites) of membrane proteins causing constitutive proliferation and triggering epithelial-to-mesenchymal transition. The current genome-wide mapping of 1,117 proteins (2,909 asparagine residues) was used to explore charge- and polarity-based mechanistic constraints in N-glycosylation, and discuss alterations of the neoplastic phenotype that can be ascribed to N-glycosylation at preferred and non-preferred sites.

DPP4 truncated GM-CSF and IL-3 manifest distinct receptor-binding and regulatory functions compared with their full-length forms.

Dipeptidylpeptidase 4 (DPP4/CD26) enzymatically cleaves select penultimate amino acids of proteins, including colony stimulating factors (CSFs), and has been implicated in cellular regulation. To better understand the role of DPP4 regulation of hematopoiesis, we analyzed the activity of DPP4 on the surface of immature blood cells and then comparatively assessed the interactions and functional effects of full-length (FL) and DPP4 truncated factors [(T)-GM-CSF and- IL-3] on both in vitro and in vivo models of normal and leukemic cells. T-GM-CSF and T-IL-3 had enhanced receptor binding, but decreased CSF activity, compared to their FL forms. Importantly, T-GM-CSF and T-IL-3 significantly, and reciprocally, blunted receptor binding and myeloid progenitor cell proliferation activity of both FL-GM-CSF and FL-IL-3 in vitro and in vivo. Similar effects were apparent in vitro using cluster forming cells from patients with Acute Myeloid Leukemia (AML) regardless of cytogenetic or molecular alterations and in vivo utilizing animal models of leukemia. This suggests that DPP4 T-molecules have modified binding and functions compared to their FL counterparts and may serve regulatory roles in normal and malignant hematopoiesis.

Antimicrobials: Putting antibiotic action into context.

This study reveals that ribosomal antibiotics function in a context-specific manner, dependent on the penultimate amino acid in the nascent chain and the amino acid in the P-site of the peptidyl transferase center.

Phosphorylation of GENOMES UNCOUPLED 4 Alters Stimulation of Mg Chelatase Activity in Angiosperms.

GENOMES UNCOUPLED 4 (GUN4) is a positive regulator of light-dependent chlorophyll biosynthesis. GUN4 activates Mg chelatase (MgCh) that catalyzes the insertion of an Mg2+ ion into protoporphyrin IX. We show that Arabidopsis (Arabidopsis thaliana) GUN4 is phosphorylated at Ser 264 (S264), the penultimate amino acid residue at the C terminus. While GUN4 is preferentially phosphorylated in darkness, phosphorylation is reduced upon accumulation of Mg porphyrins. Expression of a phosphomimicking GUN4(S264D) results in an incomplete complementation of the white gun4-2 null mutant and a chlorotic phenotype comparable to gun4 knockdown mutants. Phosphorylated GUN4 has a reduced stimulatory effect on MgCh in vitro and in vivo but retains its protein stability and tetrapyrrole binding capacity. Analysis of GUN4 found in oxygenic photosynthetic organisms reveals the evolution of a C-terminal extension, which harbors the phosphorylation site of GUN4 expressed in angiosperms. Homologs of GUN4 from Synechocystis and Chlamydomonas lack the conserved phosphorylation site found in a C-terminal extension of angiosperm GUN4. Biochemical studies proved the importance of the C-terminal extension for MgCh stimulation and inactivation of GUN4 by phosphorylation in angiosperms. An additional mechanism regulating MgCh activity is proposed. In conjunction with the dark repression of 5-aminolevulinic acid synthesis, GUN4 phosphorylation minimizes the flow of intermediates into the Mg branch of the tetrapyrrole metabolic pathway for chlorophyll biosynthesis.

Dipeptidyl Peptidase 4 (DPP4) Is Altered By, and Modulates Sensitivity to, Extra-Physiologic Oxygen Shock Stress (EPHOSS): Implications for Hematopoietic Stem Cell Transplantation

Hematopoietic stem cells (HSC) reside in hypoxic niches (~1-4% O2), however, HSC studies are consistently performed using cells isolated in ambient air (~20% O2), regardless of subsequent processing in low oxygen tension. We recently published that by collecting/processing stem cells in physiologically native conditions of hypoxia, with all procedures performed inside a hypoxic chamber (3% O2), we enhance the recovery of phenotypic, and functional, self-renewing long-term repopulating HSC (LT-HSC) with concomitantly decreasing numbers of progenitor cells. This occurs by inhibiting damage due to brief exposure of mouse bone marrow (BM) or human cord blood (CB) cells to ambient oxygen (a phenomenon we term Extra Physiologic Oxygen Shock/Stress (EPHOSS)) which we, in part, mechanistically linked to mitochondrial permeability transition pore (MPTP), Reactive Oxygen Species (ROS) and cyclophilin D. This data suggests that true numbers of HSCs, and the transplantation potency of BM and CB, have been consistently underestimated due to rapid differentiation of LT-HSCs in ambient air (EPHOSS), but the broad effects of EPHOSS on stem cell phenotype are unknown. We hypothesized that Dipeptidyl Peptidase 4 (DPP4) may be altered by EPHOSS and involved in the effects of EPHOSS on HSC. We showed that DPP4, a serine peptidase whose enzymatic activity leads to the N terminal cleavage of select penultimate amino acids of proteins, alters homing and engraftment of HSC and the number of cytokines, chemokines and growth factors that have putative DPP4 truncation sites have been dramatically underestimated. Functional and mechanistic roles of full length (FL) versus DPP4 truncated (T) factors, the ability of DPP4 T proteins to induce signaling that FL factors cannot, and the effects of EPHOSS on DPP4 expression/activity, and vice versa, have not been investigated and may have yet unappreciated clinical application. Here we present novel data demonstrating that mouse bone marrow harvested in air in the presence of Diprotin A, a DPP4 inhibitor, or from DPP4 K/O mice, results in a significant increase in the number of phenotypic LT-HSC (p=.017), suggesting that inhibition of DPP4 can diminish the loss of phenotypic LT-HSC due to EPHOSS. Further, the percentage of DPP4+ cells is significantly increased in primitive fractions of mouse bone marrow and human cord blood (LSK ~15% DPP4+, LSKCD150+ ~40%DPP4+, CD34+CD38- of CB ~10% DPP4+, CD34+CD38-CD45RA-CD90+CD49F+ ~40% DPP4+, p=.007), the numbers of DPP4+ cells are additionally enhanced 15- 20% when cells (BM and hCB) are isolated in hypoxia, especially in the LT-HSC fraction (Air 40% DPP4+ Hypoxia 60% DPP4+, p=.005). However, DPP4 activity on lineage- bone marrow harvested in hypoxia showed a 2 fold decrease (p=.005) compared to lineage- cells harvested in air. Interestingly, this increase in the number of DPP4+ cells in hypoxia is not recapitulated when mouse BM is harvested in the presence a Cyclosporin A, a cylophilin D inhibitor, (even though the increase in numbers of LT-HSC is preserved similarly to that in hypoxia) suggesting an alternative mechanism for modulation of DPP4 other than inhibition of mitochondrial ROS/MPTP. Unexpectedly, LT-HSC ROS levels (both mitochondrial and total) were not diminished in groups with decreased DPP4 activity (DPA or DPP4 K/O) harvested in air despite the blunting of EPHOSS leading to maintenance of the phenotypic LT-HSC increase over air harvest alone. These data suggest that pathways in addition to ROS, such as DPP4 expression/activity, may be influencing LT-HSC function after, and sensitivity to, EPHOSS as well as being modulated by EPHOSS. Further investigation of these collaborative pathways may facilitate increased HSC collections to enhance HSC transplantation. DisclosuresNo relevant conflicts of interest to declare.

Study of Common Genetic Variant S447X in Lipoprotein Lipase and Its Association with Lipids and Lipoproteins in Type 2 Diabetic Patients.

Elevated plasma triglyceride and non-esterified fatty acid concentrations may cause insulin resistance and type 2 diabetes mellitus. Lipoprotein lipase (LPL) is a rate-determining enzyme in lipid metabolism. A variant in the LPL gene has been identified which alters the penultimate amino acid Serine at 447 to a stop codon (S447X), and results in a truncated LPL molecule lacking the C-terminal dipeptide Ser-Gly. The present study was designed to evaluate the frequency of S447X variant in the LPL gene and its effect on the lipid and lipoprotein levels in type 2 diabetic subjects. The genotype frequency distributions of type 2 diabetes patients and controls were in Hardy-Weinberg equilibrium. Comparison of the genotype and allelic frequencies of S447X in subjects with type 2 diabetics compared to controls demonstrated no significant difference. In subjects with type 2 diabetics having hypertriglyceridemia (TG≥150mg/dl) compared to diabetics with TG level <150mg/dl, significant difference in genotype frequency was found among these groups, while allelic frequency of X was significantly differed. Logistic regression analysis showed the negative association of LPL S447X variant with TG and VLDL cholesterol, while no association with total cholesterol, HDL cholesterol and LDL cholesterol was found. The lipid levels except for HDL cholesterol were found to be significantly lower in carriers for S447X than wild type in diabetes group. The decreased level of TG and TG rich lipoprotein in subjects with SNP S447X in LPL, predicts anti-atherogenic activity of carriers for S447X variant in general population as well as type 2 diabetic patients.

Dipeptidyl Peptidase-4 Regulation of SDF-1/CXCR4 Axis: Implications for Cardiovascular Disease.

Dipeptidyl peptidase-4 (DPP4) is a ubiquitously expressed protease that regulates diverse number of physiological functions. As a dipeptidase, it exerts its catalytic effects on proteins/peptides with proline, alanine, or serine in the penultimate (P1) amino acid residue from the amino terminus. The evidence to date supports an important effect of DPP4 in catalytic cleavage of incretin peptides and this perhaps represents the main mechanism by which DPP4 inhibition improves glycemic control. DPP4 also plays an important role in the degradation of multiple chemokines of which stromal cell-derived factor-1 (SDF-1, also known as CXCL12) is perhaps an increasingly recognized target, given its importance in processes, such as hematopoiesis, angiogenesis, and stem cell homing. In the current review, we will summarize the importance of DPP4-mediated enzymatic processing of cytokines/chemokines with an emphasis on SDF-1 and resultant implications for cardiovascular physiology and disease.

Glycemia Lowering and Risk for Heart Failure: Recent Evidence from Studies of Dipeptidyl Peptidase Inhibition.

The global epidemic of type 2 diabetes mellitus (T2DM) has substantial implications for cardiovascular disease–related morbidity and mortality.1 The prevalence of T2DM in patients with heart failure (HF) is high, with strong and independent association between T2DM and incident HF observed in multiple prospective studies and in randomized-controlled clinical trials. In the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT), which enrolled subject’s ≥55 years of age with hypertension and ≥1 risk factor, patients with T2DM had a 2-fold risk for HF hospitalization or death after adjustment for other risk factors (RR, 1.95). The association with T2DM was independent of coronary artery disease and at least equivalent in magnitude and greater than that for electrocardiographic left ventricular (LV) hypertrophy.2 All measures of glycemia including fasting, postprandial, measures of insulin resistance, and hemoglobin A1c (HbA1c) have been associated with risk of developing HF, with the association extending to both HF with preserved ejection fraction and to HF with reduced ejection fraction.3,4 A substantial body of evidence from preclinical studies, endomyocardial biopsies in humans and more recently with cardiac MRI, support increased myocardial stiffness in T2DM related to alteration in extracellular matrix. There are multiple proximate mediators that have been hypothesized to play a role including advanced glycation end product deposition and reactive oxygen species that may increase myocardial stiffness during diastole, by cross-linking collagen or by enhancing collagen formation.5,6 Another pernicious proximal mediator is the elevation in postprandial lipids, such as remnant lipoproteins, characteristic of atherogenic dyslipidemia, a highly prevalent abnormality in T2DM, that may result in direct myocellular deposition of lipid, leading to microcirculatory dysfunction, alteration in substrate use and mitochondrial dysfunction.7,8 Indeed, positron emission tomography studies show reduced myocardial glucose uptake in favor of fatty acid …

Potential role of dipeptidyl peptidase IV in the pathophysiology of heart failure.

Dipeptidyl peptidase IV (DPPIV) is a widely expressed multifunctional serine peptidase that exists as a membrane-anchored cell surface protein or in a soluble form in the plasma and other body fluids. Numerous substrates are cleaved at the penultimate amino acid by DPPIV, including glucagon-like peptide-1 (GLP-1), brain natriuretic peptide (BNP) and stromal cell-derived factor-1 (SDF-α), all of which play important roles in the cardiovascular system. In this regard, recent reports have documented that circulating DPPIV activity correlates with poorer cardiovascular outcomes in human and experimental heart failure (HF). Moreover, emerging evidence indicates that DPPIV inhibitors exert cardioprotective and renoprotective actions in a variety of experimental models of cardiac dysfunction. On the other hand, conflicting results have been found when translating these promising findings from preclinical animal models to clinical therapy. In this review, we discuss how DPPIV might be involved in the cardio-renal axis in HF. In addition, the potential role for DPPIV inhibitors in ameliorating heart disease is revised, focusing on the effects of the main DPPIV substrates on cardiac remodeling and renal handling of salt and water.

Unique and Unexpected Functional Activities of Hematopoietically Active Cytokines Truncated By Dipeptidylpeptidase-4 (DPP4)

Dipeptidylpeptidase 4 (DPP4) is a serine peptidase with enzymatic activity leading to the N terminal cleavage of select penultimate amino acids of proteins. We previously published that the number of cytokines, chemokines and growth factors that have putative DPP4 truncation sites have been dramatically underestimated. Functional and mechanistic roles of full length (FL) versus DPP4 truncated (T) factors, as well as the ability of DPP4 T proteins to induce signaling that FL factors can not, have not been previously investigated and may have yet unappreciated clinical application. Here we present novel data demonstrating the here to fore unknown ability of DPP4 cleavage of proteins to alter not only cellular function, but intracellular signaling of growth factors leading to miRNA expression, phosphorylation, and global induction of proteins that the FL form of the protein does not induce. Additionally, and unexpectedly, a 1:1 mixture of the DPP4 T protein and their FL counterpart leads to both overlapping signaling between the FL and T, as well as unique signaling, that is not induced by the FL or T protein alone. This suggests multifaceted, important, and currently unappreciated roles that the DPP4 truncation of proteins may play in the regulation of normal and malignant hematopoiesis as well as other physiologic and pathophysiologic states.From functional data, utilizing the human factor-dependent TF-1 cell line, as well as primary samples from human cord blood (CB) and patients with Acute Myeloid Leukemia (AML), we observed that DPP4 truncation of GM-CSF (T-GM-CSF) and IL-3 (T-IL-3) results in decreased colony stimulating factor (CSF) activity in normal and malignant hematopoietic progenitor cells (HPC). Receptor binding studies confirmed that both T-GM-CSF and T-IL-3 have enhanced receptor affinity compared to their FL form and each can compete to blunt the receptor binding of either FL-GM-CSF or FL-IL-3. In vivo studies demonstrated that either exogenously added T-GM-CSF or T-IL-3 suppressed the effects of exogenously added FL-GM-CSF or FL-IL-3 on progenitor cell numbers per femur and diminished HPC cycling. Investigation of FL vs T mediated signaling alterations was done with TF-1 cells using proteomic and bioinformatic analysis of miRNA expression, and phosphorylation/global protein induction. Both T-GM-CSF and T-IL-3 induced unique signaling that FL-GM-CSF and FL-IL-3 did not, as well as signaling that overlapped with their FL counterparts (Figure 1, Venn Diagram example of global induction of proteins). Additionally, treatment with a 1:1 ratio of the FL/T proteins resulted in both distinctive, and common, signaling compared to that detected with treatment of only FL or T molecules, thus revealing the complexity of the signaling interactions and heretofore unknown activities of DPP4 truncated proteins (Figure 1). Fisher’s exact test or B-H multiple testing correction in IPA software were used to statistically generate comparisons of molecules associated with specific signaling induction, and differential as well as overlapping signaling for all GM-CSF vs IL-3 groups. As examples, for both the GM-CSF and IL-3 groups, molecules associated with PI3K/Akt signaling were increased in T and 1:1 groups vs treatment with FL molecules. In contrast, molecules affiliated with CDC42 signaling were induced by treatment with a 1:1 ratio of FL:T molecules but not in the FL or T groups alone for both GM-CSF and IL-3. Interestingly, phosphorylated proteins associated with Flt3 and IL-9 signaling in hematopoietic progenitors was increased in the T and 1:1 groups for GM-CSF and IL-3 compared to the FL groups. Conversely, phosphorylated molecules involved in nucleic acid metabolism were induced by the FL, T and 1:1 of the GM-CSF groups but only by the FL version of IL-3. Further, phosphorylated molecules associated with antigen presentation were only detected in the FL IL-3 and 1:1 IL-3 groups, and molecules functionally associated with carbohydrate metabolism and cellular therapeutics were only detected in the T and 1:1 groups for both GM-CSF and IL-3, and not in the FL stimulated groups. Collectively, these data substantiate that further investigation into the roles and regulation of DPP4, in normal and malignant hematopoiesis, will allow for a better understanding of the significance, and potential clinical utility, of DPP4 activity altering compounds as well as DPP4 truncated molecules. [Display omitted] DisclosuresBroxmeyer:CordUse: Membership on an entity’s Board of Directors or advisory committees.

Study of effect of substitution of the penultimate amino acid residue on expression, structure, and functional properties of Yersinia pseudotuberculosis OmpY porin.

The purpose of the study was to compare the expression of two Yersinia pseudotuberculosis proteins, wild-type porin OmpY and the mutant porin OmpY designated as OmpY-Q having the uncharged amino acid residue Gln instead of positively charged Arg at the penultimate position in the same heterologous host. According to the literature, a similar substitution (Lys to Gln) of the penultimate amino acid residue in Neisseria meningitidis porin PorA drastically improved the assembly of the protein in the E. coli outer membrane in vivo. Site-directed mutagenesis was used to replace Arg by Gln (R338Q) in OmpY, and the conditions for optimal expression and maturation of OmpY-Q were selected. It was found that the growth rates of E. coli strains producing OmpY and OmpY-Q and the expression levels of the porins were approximately equal. Comparative analysis of recombinant OmpY and OmpY-Q did not show significant differences in structure, antigenic, and functional properties of the porins, or any noticeable effect of the R338Q substitution in OmpY on its assembly in the E. coli outer membrane in vivo. The probable causes of discrepancies between our results and the previous data on porin PorA are discussed considering the known mechanisms of biogenesis of porins at the periplasmic stage.

Kinetic analysis of the leucyl/phenylalanyl-tRNA-protein transferase with acceptor peptides possessing different N-terminal penultimate residues

The introduction of non-natural amino acids at the N-terminus of peptides/proteins using leucyl/phenylalanyl-tRNA-protein transferase (L/F-transferase) is a useful technique for protein engineering. To accelerate the chemoenzymatic reaction, here we systematically optimized the N-terminal penultimate residue of the acceptor peptide. Positively charged, small, or hydrophilic amino acids at this position show positive effects for the reaction. Kinetic analysis of peptides possessing different penultimate residues suggests that the side chain of the residue affects peptide-binding affinity towards the L/F-transferase. These findings also provide biological insight into the effect of the penultimate amino acid on substrate specificity of natural proteins to be degraded via the N-end rule pathway.

Developmental retardation, microcephaly, and peptiduria in mice without aminopeptidase P1

Cytosolic aminopeptidase P1 (APP1) is one of the three known mammalian aminopeptidase Ps (APPs) that cleave the N-terminal amino acid residue of peptides in which the penultimate amino acid is proline. In mammals, many biologically active peptides have a highly conserved N-terminal penultimate proline. However, little is known about the physiological role of APP1. In addition, there is no direct evidence to associate a deficiency in APP1 with metabolic diseases. Although two human subjects with reduced APP activity exhibited peptiduria, it is unclear which of the three APP isoforms is responsible for this disorder. In this study, we generated APP1-deficient mice by knocking out Xpnpep1. Mouse APP1 deficiency causes severe growth retardation, microcephaly, and modest lethality. In addition, imino-oligopeptide excretion was observed in urine samples from APP1-deficient mice. These results suggest an essential role for APP1-mediated peptide metabolism in body and brain development, and indicate a strong causal link between APP1 deficiency and peptiduria.

Dipeptidyl peptidase-4 expression is reduced in Crohn's disease

BackgroundDipeptidyl peptidase 4 (DP4) is a serine protease that preferentially cleaves N‐terminal dipeptides from polypeptides containing proline or alanine as the penultimate amino acid. DP4 inactivates glucagon like peptide-2 (GLP-2), a trophic peptide with cytoprotective and reparative properties in the injured gut; therefore DP4 potentially inhibits repair processes. DP4 also modulates the activity of GLP-1 and polypeptide YY (PYY) which regulate appetite and motility. No data are yet available on the tissue and plasma expression of DP4 in inflammatory bowel disease (IBD). MethodsTissue and plasma were studied from active CD and healthy controls for DP4 quantification. Experiments were also carried out in a reductionist Caco-2 cell line model of intestinal inflammation with TNFα incubation. DP4 expression was studied by tissue Western blotting and plasma enzymelinked immunosorbent assay (ELISA), in addition to quantitative polymerase chain reaction (qPCR). ResultsThere was a ~2.7-fold decrease in DP4 protein in CD tissue (p=0.05). Plasma DP4 in CD was also significantly lower than the control group. A negative correlation between plasma DP4 levels and inflammatory activity as measured by C-reactive protein was observed. In Caco-2 cells an ~18-fold increase (p<0.0001) in DP4 protein expression was seen after incubation with TNFα at a concentration of 25ng/μl for 48hours paralleled by a 2-fold increase in DP4 mRNA. DiscussionDP4 is reduced in tissue and plasma in active Crohn's disease. This is unlikely to represent simple downregulation induced by inflammation since the key proinflammatory cytokine strongly upregulated DP4 expression in Caco-2 cells. Clearly a more complex situation exists in vivo. We propose that reduced DP4 activity limits the cleavage of regulatory peptides, for example potentiating the trophic signal from GLP-2. Pharmacological DP4 inhibition may present an additional therapeutic target in IBD.