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Sample records for protease inhibitor-containing haart

  1. Discovery of MK-8718, an HIV Protease Inhibitor Containing a Novel Morpholine Aspartate Binding Group.

    PubMed

    Bungard, Christopher J; Williams, Peter D; Ballard, Jeanine E; Bennett, David J; Beaulieu, Christian; Bahnck-Teets, Carolyn; Carroll, Steve S; Chang, Ronald K; Dubost, David C; Fay, John F; Diamond, Tracy L; Greshock, Thomas J; Hao, Li; Holloway, M Katharine; Felock, Peter J; Gesell, Jennifer J; Su, Hua-Poo; Manikowski, Jesse J; McKay, Daniel J; Miller, Mike; Min, Xu; Molinaro, Carmela; Moradei, Oscar M; Nantermet, Philippe G; Nadeau, Christian; Sanchez, Rosa I; Satyanarayana, Tummanapalli; Shipe, William D; Singh, Sanjay K; Truong, Vouy Linh; Vijayasaradhi, Sivalenka; Wiscount, Catherine M; Vacca, Joseph P; Crane, Sheldon N; McCauley, John A

    2016-07-14

    A novel HIV protease inhibitor was designed using a morpholine core as the aspartate binding group. Analysis of the crystal structure of the initial lead bound to HIV protease enabled optimization of enzyme potency and antiviral activity. This afforded a series of potent orally bioavailable inhibitors of which MK-8718 was identified as a compound with a favorable overall profile. PMID:27437081

  2. Discovery of MK-8718, an HIV Protease Inhibitor Containing a Novel Morpholine Aspartate Binding Group.

    PubMed

    Bungard, Christopher J; Williams, Peter D; Ballard, Jeanine E; Bennett, David J; Beaulieu, Christian; Bahnck-Teets, Carolyn; Carroll, Steve S; Chang, Ronald K; Dubost, David C; Fay, John F; Diamond, Tracy L; Greshock, Thomas J; Hao, Li; Holloway, M Katharine; Felock, Peter J; Gesell, Jennifer J; Su, Hua-Poo; Manikowski, Jesse J; McKay, Daniel J; Miller, Mike; Min, Xu; Molinaro, Carmela; Moradei, Oscar M; Nantermet, Philippe G; Nadeau, Christian; Sanchez, Rosa I; Satyanarayana, Tummanapalli; Shipe, William D; Singh, Sanjay K; Truong, Vouy Linh; Vijayasaradhi, Sivalenka; Wiscount, Catherine M; Vacca, Joseph P; Crane, Sheldon N; McCauley, John A

    2016-07-14

    A novel HIV protease inhibitor was designed using a morpholine core as the aspartate binding group. Analysis of the crystal structure of the initial lead bound to HIV protease enabled optimization of enzyme potency and antiviral activity. This afforded a series of potent orally bioavailable inhibitors of which MK-8718 was identified as a compound with a favorable overall profile.

  3. Low level HIV viremia is more frequent under protease-inhibitor containing firstline therapy than under NNRTI-regimens

    PubMed Central

    Wiesmann, Frank; Braun, Patrick; Knickmann, Mechthild; Knechten, Heribert

    2014-01-01

    Introduction An association of persistent low level viremia (LLV) below 500 copies/mL and a higher risk of therapy failure is still point of controversial discussion. Furthermore, it seems that LLV occurs more frequently in patients with protease-inhibitor regimens than in NNRTI- / or integrase-inhibitor containing therapies. The focus of this work was to assess the prevalence of LLV (50–200 copies/mL) and weak viremia (201–500 copies/mL) in firstline-treated patients according to their therapy regimen. Methods A total of 832 and 944 patients from 23 German centres were under firstline therapy in 2012 and 2013, respectively. All patients received their therapy for more than 24 weeks. VL data was related to clinical data retrospectively including ART-composition, subdivided into NNRTIs (Efavirenz, Nevirapine), PIs (Atazanavir, Darunavir, Lopinavir) and INIs (Raltegravir). Low viremic patients were classified into two arms of 50–200 copies/mL (group A) and 201–500 copies/mL (group B). Results Success of therapy was defined as <50 copies/mL and was observed in 90.0% and 91.1% (2012/2013), respectively. An additional 2.0% and 2.3% had LLV. The amount of viremic patients with VLs <500 copies/mL differed significantly between NNRTI-based firstline regimens 1.7% and 2.5% and PI-based regimens 4.8% and 5.7% (2012/2013), respectively. LLV was clearly less often observed in EFV-based- (1.6% and 1.1% [group A] / 0.4% and 0.4% [group B]) or NVP-based firstline therapies (1.0% and 3.6% [group A] + 0% and 0% [group B]) than in ATV-based- (7.5% and 3.8% [group A] + 1.5% and 2.5% [group B]), DRV-based- (2.9% and 3.0% [group A] + 2.2% and 0% [group B]) or LPV-based firstline therapies (1.6% and 3.3% [group A] + 0.8% and 2.5% [group B]) and also in parts for RAL-based regimens (0% and 3.7% [group A] + 0% and 1.9% [group B]). Conclusions LLV is more often observed under PI-based firstline than under NNRTI-regimens. Only one NNRTI-patient of group B remained on therapy. A

  4. Increased Risk of Preterm Delivery Among HIV-Infected Women Randomized to Protease Versus Nucleoside Reverse Transcriptase Inhibitor-Based HAART During Pregnancy

    PubMed Central

    Kitch, Douglas; Ogwu, Anthony; Hughes, Michael D.; Lockman, Shahin; Leidner, Jean; van Widenfelt, Erik; Moffat, Claire; Moyo, Sikhulile; Makhema, Joseph; Essex, Max; Shapiro, Roger L.

    2011-01-01

    (See the editorial commentary by Kourtis, on pages 493–4.) Background. Protease inhibitor (PI)-based highly active antiretroviral therapy (HAART) use in pregnancy has been associated with preterm deliveries in some observational studies. Methods. HIV-infected, HAART-naive pregnant women with CD4+ counts ≥200 cells/mm3 were randomized between 26 and 34 weeks gestation to lopinavir/ritonavir/zidovudine/lamivudine (PI group) or abacavir/zidovudine/lamivudine (NRTI group) in a clinical trial to prevent mother-to-child HIV transmission. Risk factors for preterm delivery (<37 weeks) and differences by randomization arm were evaluated for live infants by logistic regression. Results. Preterm delivery rates were higher among 267 women in the PI group than 263 women in the NRTI group (21.4% vs 11.8%, P = .003). PI-based HAART was the most significant risk factor for preterm delivery [odds ratio = 2.03, 95% confidence interval 1.26–3.27, P = .004]. Mean change in maternal body mass index (BMI) 1 month after HAART initiation was lower in the PI group (P < .001); however, this was not significantly associated with preterm delivery. Neither infant hospitalizations nor mortality through 6 months of life differed by maternal regimen. Conclusions. PI-based HAART was associated with increased preterm delivery but not increased infant hospitalizations or mortality in a clinical trial setting. The association between PI use and lower increase in BMI in late pregnancy warrants further study. PMID:21791651

  5. Synthesis and biological evaluation of papain-family cathepsin L-like cysteine protease inhibitors containing a 1,4-benzodiazepine scaffold as antiprotozoal agents.

    PubMed

    Ettari, Roberta; Pinto, Andrea; Tamborini, Lucia; Angelo, Ilenia C; Grasso, Silvana; Zappalà, Maria; Capodicasa, Natale; Yzeiraj, Laura; Gruber, Esther; Aminake, Makoah N; Pradel, Gabriele; Schirmeister, Tanja; De Micheli, Carlo; Conti, Paola

    2014-08-01

    Novel papain-family cathepsin L-like cysteine protease inhibitors endowed with antitrypanosomal and antimalarial activity were developed, through an optimization study of previously developed inhibitors. In the present work, we studied the structure-activity relationships of these derivatives, with the aim to develop new analogues with a simplified and more synthetically accessible structure and with improved antiparasitic activity. The structure of the model compounds was significantly simplified by modifying or even eliminating the side chain appended at the C3 atom of the benzodiazepine scaffold. In addition, a simple methylene spacer of appropriate length was inserted between the benzodiazepine ring and the 3-bromoisoxazoline moiety. Several rhodesain and falcipain-2 inhibitors displaying single-digit micromolar or sub-micromolar antiparasitic activity against one or both parasites were identified, with activities that were one order of magnitude more potent than the model compounds. PMID:24919925

  6. Early loss of bone mineral density is correlated with a gain of fat mass in patients starting a protease inhibitor containing regimen: the prospective Lipotrip study

    PubMed Central

    2013-01-01

    Background HIV-infected patients starting antiretroviral treatment (ART) experience deep and early disorders in fat and bone metabolism, leading to concomitant changes in fat mass and bone mineral density. Methods We conducted a prospective study in treatment-naive HIV-infected patients randomized to receive two nucleoside reverse transcriptase inhibitors in combination with either a protease inhibitor (PI) or a non-nucleosidic reverse transcriptase inhibitor (NNRTI), to evaluate early changes in body composition, bone mineral density and metabolic markers as differentially induced by antiretroviral therapies. We measured changes in markers of carbohydrate, of fat and bone metabolism, and, using dual-emission X-ray absorptiometry (DXA), body composition and bone mineral density (BMD). Complete data on changes between baseline and after 21 months treatment were available for 35 patients (16 in the PI group and 19 in the NNRTI group). Results A significant gain in BMI and in total and lower limb fat mass was recorded only in patients receiving PI. A loss of lumbar BMD was observed in both groups, being higher with PI. Plasma markers of bone metabolism (alkaline phosphatase, osteocalcin, collagen crosslaps) and levels of parathormone and of 1,25diOH-vitamin D3 significantly increased in both groups, concomitant with a decline in 25OH-vitamin D3. Lipids and glucose levels increased in both groups but rise in triglyceride was more pronounced with PI. A correlation between loss of BMD and gain of fat mass is observed in patients starting PI. Conclusions We evidenced an early effect of ART on lipid and bone metabolisms. PI lead to a significant gain in fat mass correlated with a sharp drop in BMD but active bone remodelling is evident with all antiretroviral treatments, associated with low vitamin D levels and hyperparathyroidism. In parallel, signs of metabolic restoration are evident. However, early increases in lean and fat mass, triglycerides, waist circumference and

  7. Abacavir, efavirenz, didanosine, with or without hydroxyurea, in HIV-infected adults failing initial nucleoside/protease inhibitor-containing regimens

    PubMed Central

    Swindells, Susan; Cohen, Calvin J; Berger, Daniel S; Tashima, Karen T; Liao, Qiming; Pobiner, Bonnie F; Snidow, Jerry W; Pakes, Gary E; Hernandez, Jaime E

    2005-01-01

    Background Hydroxyurea (HU) is an immunomodulatory agent that has been documented to enhance the antiretroviral activity of nucleoside reverse transcriptase inhibitors, such as abacavir (ABC) and didanosine (ddI), and would be expected to improve virologic efficacy. Methods A 48-week, phase IV, multicenter, open-label, proof-of-concept clinical trial was conducted to evaluate second-line, protease inhibitor (PI)-sparing therapy with ABC/efavirenz (EFV)/ddI plus HU or without HU in HIV-infected subjects failing to achieve HIV-1 RNA ≤ 400 copies/mL after ≥ 16 weeks of treatment with lamivudine/zidovudine or lamivudine/stavudine, plus 1 or 2 PIs. Subjects were assigned to ABC (300 mg twice daily)/ EFV (600 mg once daily)/ ddI (400 mg once daily) plus HU (500 mg twice daily) (n = 30) or this regimen without HU (n = 24). Results Baseline mean HIV-1 RNA was 3.86 log10 copies/mL and CD4+ cell count was 345 cells/mm3. A similar percentage of subjects in the non-HU arm (58%) and HU arm (53%) completed the study. Intent-to-treat: missing = failure analysis showed no differences in proportions of subjects in the non-HU and HU arms achieving undetectable plasma HIV-1 RNA levels at week 24 (<400 copies/mL: 58% [14/24] vs 57% [17/30], P = 0.899; <50 copies/mL (50% [12/24] vs 47% [14/30], P = 0.780). Median change from baseline in CD4+ cell count in the non-HU and HU arms at week 48 was +114 cells/mm3 and -63 cells/mm3 (P = 0.007), respectively. Both regimens were generally well tolerated, although more subjects in the HU arm withdrew prematurely from the study due to adverse events (23% vs 4%). Four cases of possible ABC-related hypersensitivity were observed. Conclusion ABC/EFV/ddI was an effective and well-tolerated second-line regimen for nucleoside/PI-experienced HIV-infected subjects. The addition of HU blunted the CD4+ cell response, did not appear to enhance antiviral activity, and resulted in more treatment-limiting adverse events. PMID:15819974

  8. Increased VLDL-apoB and IDL-apoB production rates in nonlipodystrophic HIV-infected patients on a protease inhibitor-containing regimen: a stable isotope kinetic study.

    PubMed

    Petit, Jean Michel; Duong, Michel; Florentin, Emmanuel; Duvillard, Laurence; Chavanet, Pascal; Brun, Jean Marcel; Portier, Henri; Gambert, Philippe; Verges, Bruno

    2003-09-01

    The aim of this study was to identify the first abnormalities of apolipoprotein B (apoB) metabolism in HIV-infected patients treated by antiretroviral therapy (ART) with protease inhibitors (PIs). The influence of ART on the metabolism of apoB in VLDL, IDL, and LDL was investigated in six patients receiving dual nucleoside reverse transcriptase inhibitors (NRTIs) and PI, and in five patients receiving NRTI and nevirapine. None of the patients had lipodystrophy. The study was performed in the fed state. Each subject received an intravenous injection of a 0.7 mg.kg-1 bolus of l-[1-13C]leucine, immediately followed by a 16 h constant infusion at 0.7 mg.kg-1.h-1. The VLDL- and IDL-apoB concentrations were significantly higher in PI-treated patients compared to non-PI-treated patients. The VLDL-apoB and IDL-apoB production rates were markedly higher in PI-treated patients compared to non-PI-treated patients (54.5 +/- 30.1 vs. 30.9 +/- 8.4 mg.kg-1.d-1, P = 0.04; and 43.5 +/- 20.0 vs. 18.7 +/- 7.8 mg.kg-1.d-1, P = 0.04, respectively). In conclusion, our study shows that patients receiving ART with PI present altered metabolism of the VLDL-IDL-LDL chain compared with patients treated without PI. These data confirm that PI therapy is associated with a physiopathological mechanism for dyslipidemia in addition to the effect of lipodystrophy on lipid metabolism.

  9. Premature and accelerated aging: HIV or HAART?

    PubMed

    Smith, Reuben L; de Boer, Richard; Brul, Stanley; Budovskaya, Yelena; van Spek, Hans

    2012-01-01

    Highly active antiretroviral therapy (HAART) has significantly increased life expectancy of the human immunodeficiency virus (HIV)-positive population. Nevertheless, the average lifespan of HIV-patients remains shorter compared to uninfected individuals. Immunosenescence, a current explanation for this difference invokes heavily on viral stimulus despite HAART efficiency in viral suppression. We propose here that the premature and accelerated aging of HIV-patients can also be caused by adverse effects of antiretroviral drugs, specifically those that affect the mitochondria. The nucleoside reverse transcriptase inhibitor (NRTI) antiretroviral drug class for instance, is known to cause depletion of mitochondrial DNA via inhibition of the mitochondrial specific DNA polymerase-γ. Besides NRTIs, other antiretroviral drug classes such as protease inhibitors also cause severe mitochondrial damage by increasing oxidative stress and diminishing mitochondrial function. We also discuss important areas for future research and argue in favor of the use of Caenorhabditis elegans as a novel model system for studying these effects.

  10. Viral BLIP dynamics during HAART.

    SciTech Connect

    Markowitz, M.; Louie, M.; Hurley, A.; Ho, David D.; Perelson, Alan S.,; Di Mascio, M.

    2001-01-01

    Intermittent episodes of low-level viremia (blips) are often observed in well-suppressed, HAART-treated patients. It has been reported that viral blips do not correlate with the emergence of new HAART-related mutations; however, increased frequency of blips correlates with slower decay of latently infected cells. Since blips are transient and unpredictable, detailed knowledge about them is difficult to obtain. We present an analysis of the dynamics of viral blips from viral load (VL) measurements on 123 patients for a period of 809k480d (21-1817d) and sampled every 31{+-}12d for a total of 26{+-}15 samples per patient.

  11. Crossing the waters to the post-HAART era (or Jeanette meets Amalia).

    PubMed

    Mascolini, M

    1999-12-01

    The first time this reporter heard the term HAART was at the 1996 Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC). Now, highly active antiretroviral therapy is so common that some refer to the current state of treatment as "the post-HAART era." HAART initially meant a treatment that included one or two protease inhibitors (PIs) combined with two nucleoside reverse transcriptase inhibitors (NRTIs). Initial reports on the effectiveness of that treatment strategy led some researchers to believe HIV could be eradicated completely; but that has not happened. The history of early HAART treatment and its evolution to its current use is described in detail. Tables show prescribing patterns with PIs and non-nucleoside reverse transcriptase inhibitors (NNRTIs); retrospective drug comparisons; and results of clinical trials. An extensive table shows the clinical implications of trading a PI for a reverse transcriptase (RT) inhibitor in treatment. The role of amprenavir after failure of a first PI is discussed. The concept of drug holidays is explored, including cases of patients in Belgium. Several definitions of post-HAART are suggested. Extensive references and notes are provided.

  12. Effect of HAART on Salivary Gland Function in the Women's Interagency HIV Study (WIHS)

    PubMed Central

    Navazesh, M; Mulligan, R; Karim, R; Mack, WJ; Ram, S; Seirawan, H; Greenspan, J; Greenspan, D; Phelan, J; Alves, M

    2009-01-01

    Objective To determine the impact of highly active antiretroviral therapy (HAART) on salivary gland function in HIV positive women from the Women's Interagency HIV Study (WIHS). Design Longitudinal cohort study. Subjects and Methods A total of 668 HIV positive women from the WIHS cohort with an initial and at least 1 follow-up oral sub-study visit contributed 5358 visits. Salivary gland function was assessed based on a dry mouth questionnaire, whole unstimulated and stimulated salivary flow rates, salivary gland enlargement or tenderness and lack of saliva on palpation of the major salivary glands. Main Outcome Measures Changes in unstimulated and stimulated flow rates at any given visit from that of the immediate prior visit (continuous variables). The development of self-reported dry mouth (present/absent), enlargement or tenderness of salivary glands (present/absent), and absence of secretion on palpation of the salivary glands were binary outcomes (yes/no). Results Protease Inhibitor (PI) based HAART was a significant risk factor for developing decreased unstimulated (p=0.01) and stimulated (p=0.0004) salivary flow rates as well as salivary gland enlargement (p=0.006) as compared with non-PI based HAART. Conclusions PI-based HAART therapy is a significant risk factor for developing reduced salivary flow rates and salivary gland enlargement in HIV positive patients. PMID:19017280

  13. Nutrition and disease progression pre-highly active antiretroviral therapy (HAART) and post-HAART: can good nutrition delay time to HAART and affect response to HAART?

    PubMed

    Chandrasekhar, Aditya; Gupta, Amita

    2011-12-01

    Several studies have investigated a variety of nutritional supplementation interventions in adults with HIV. In this narrative review, we summarize the evidence from 31 clinical trials that explore clinical benefits of macronutrient and micronutrient supplementation in this population while attempting to answer the question of whether good nutrition can delay the time to highly active antiretroviral therapy (HAART) initiation and response. We focused on trials published in English between 1990 and 2010 that reported on CD4 count, viral load, and disease progression or survival. Among 9 macronutrient and 22 micronutrient trials, we found that evidence for improved CD4 count and HIV viral load with nutritional supplementation was limited; only 11.1% and 36.8% of macronutrient and micronutrient supplementation trials, respectively, reported improved CD4 count; and 33.3% and 12.5% of macronutrient and micronutrient trials, respectively, reported decreased viral load. Given their utility as surrogate markers of HIV disease progression, this suggests limited evidence for nutritional interventions having an impact on delaying HAART initiation or on improving HAART response. However, there are challenges in evaluating the effects of nutritional supplementation on clinical disease in that comparisons are difficult due to heterogeneity in study design, patient population, nutrient doses and combinations, baseline levels of deficiency, and study endpoints, including lack of clarity in defining and reporting HAART status. Future studies need to adopt a more rigorous standard design with adequate power and follow-up and require a consensus on composition and dose of nutrient interventions to be tested to more specifically answer the question on the impact of nutritional interventions on HIV disease progression and HAART response.

  14. Nutrition and disease progression pre–highly active antiretroviral therapy (HAART) and post-HAART: can good nutrition delay time to HAART and affect response to HAART?1234

    PubMed Central

    Chandrasekhar, Aditya; Gupta, Amita

    2011-01-01

    Several studies have investigated a variety of nutritional supplementation interventions in adults with HIV. In this narrative review, we summarize the evidence from 31 clinical trials that explore clinical benefits of macronutrient and micronutrient supplementation in this population while attempting to answer the question of whether good nutrition can delay the time to highly active antiretroviral therapy (HAART) initiation and response. We focused on trials published in English between 1990 and 2010 that reported on CD4 count, viral load, and disease progression or survival. Among 9 macronutrient and 22 micronutrient trials, we found that evidence for improved CD4 count and HIV viral load with nutritional supplementation was limited; only 11.1% and 36.8% of macronutrient and micronutrient supplementation trials, respectively, reported improved CD4 count; and 33.3% and 12.5% of macronutrient and micronutrient trials, respectively, reported decreased viral load. Given their utility as surrogate markers of HIV disease progression, this suggests limited evidence for nutritional interventions having an impact on delaying HAART initiation or on improving HAART response. However, there are challenges in evaluating the effects of nutritional supplementation on clinical disease in that comparisons are difficult due to heterogeneity in study design, patient population, nutrient doses and combinations, baseline levels of deficiency, and study endpoints, including lack of clarity in defining and reporting HAART status. Future studies need to adopt a more rigorous standard design with adequate power and follow-up and require a consensus on composition and dose of nutrient interventions to be tested to more specifically answer the question on the impact of nutritional interventions on HIV disease progression and HAART response. PMID:22089439

  15. Mitochondrial toxicity in HAART: an overview of in vitro evidence.

    PubMed

    Apostolova, Nadezda; Blas-García, Ana; Esplugues, Juan V

    2011-01-01

    The combined antiretroviral therapeutic approach currently employed for the treatment of HIV infection, known as Higly Active Antiretroviral Therapy (HAART), has dramatically reduced AIDS-related morbidity and mortality. However, the adverse reactions associated with the long term use of this therapy have now become a major issue and researchers have focused on understanding the cellular mechanisms underlying these drug-induced detrimental effects which englobe a large list of different events including rash and hypersensibility reactions, hepatotoxicity, metabolic disturbances including lipodystrophy, and other metabolic syndrome-like disturbances such as hyperlactatemia, hyperlipedimia, insulin resistance and pancreatitis. Other events include CNS toxic effects, peripheral neuropathies as well as nephrotoxicity and increased risk of cardiovascular diseases. Many of these reactions have been shown to develop as e result of mitochondrial dysfunction. The mitochondrial effect of N(t)RTI (Nucleos(t)ide Reverse Transcriptase Inhibitors) class of drugs, which has been widely studied, is believed to originate from the inhibitory action of these drugs on DNA polymerase gamma, the enzyme responsible for replication of mitochondrial DNA. However, additional mitochondrial targets have also been described and need to be considered. As to NNRTI (Non-Nucleoside-Transcriptase Inhibitor) or PI (Protease Inhibitors), evidence of the implication of mitochondria has also been reported, however the details of the mechanisms underlying these actions are still not fully known. This review covers the current knowledge of mitochondrial toxicities, particularly the available in vitro evidence, regarding the most commonly used groups of HIV drugs. Novel findings of mtDNA-independent mitochondrial dysfunction have received special attention. PMID:21718249

  16. Oral manifestations in the era of HAART.

    PubMed Central

    Cherry-Peppers, Gail; Daniels, Christine O.; Meeks, Valli; Sanders, Charles F.; Reznik, David

    2003-01-01

    AIDS has reached epidemic proportions in the United States, disproportionately affecting African-Americans and other minorities. As highly active antiretroviral therapy (HAART) have improved the length and quality of life for HIV-Infected people, oral health care has made similar strides. It is important that physicians and dentists recognize the earliest signs and symptoms of HIV infection in order that a timely diagnosis and patient referral can be made for early counseling testing, and treatment. At the same time, dentists have seen themselves at considerable risk from HIV Infection. Some dentists believe that they may also be more at risk from stigma then other providers if they treat HIV patients. Images p22S-a p22S-b p24S-a p25S-a p28S-a PMID:12656429

  17. Supermarket Proteases.

    ERIC Educational Resources Information Center

    Hagar, William G.; Bullerwell, Lornie D.

    2003-01-01

    Presents a laboratory activity on enzymes. Uses common items found in the supermarket that contain protease enzymes, such as contact lens cleaner and meat tenderizer. Demonstrates the digestion of gelatin proteins as part of enzymatic reactions. (Author/SOE)

  18. Conserved hydrogen bonds and water molecules in MDR HIV-1 protease substrate complexes

    SciTech Connect

    Liu, Zhigang; Wang, Yong; Yedidi, Ravikiran S.; Dewdney, Tamaria G.; Reiter, Samuel J.; Brunzelle, Joseph S.; Kovari, Iulia A.; Kovari, Ladislau C.

    2012-12-19

    Success of highly active antiretroviral therapy (HAART) in anti-HIV therapy is severely compromised by the rapidly developing drug resistance. HIV-1 protease inhibitors, part of HAART, are losing their potency and efficacy in inhibiting the target. Multi-drug resistant (MDR) 769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, 90) was selected for the present study to understand the binding to its natural substrates. The nine crystal structures of MDR769 HIV-1 protease substrate hepta-peptide complexes were analyzed in order to reveal the conserved structural elements for the purpose of drug design against MDR HIV-1 protease. Our structural studies demonstrated that highly conserved hydrogen bonds between the protease and substrate peptides, together with the conserved crystallographic water molecules, played a crucial role in the substrate recognition, substrate stabilization and protease stabilization. Additionally, the absence of the key flap-ligand bridging water molecule might imply a different catalytic mechanism of MDR769 HIV-1 protease compared to that of wild type (WT) HIV-1 protease.

  19. Characterizing retention in HAART as a recurrent event process: insights into ‘cascade churn’

    PubMed Central

    Nosyk, Bohdan; Lourenço, Lillian; Min, Jeong Eun; Shopin, Dimitry; Lima, Viviane D.; Montaner, Julio S.G.

    2015-01-01

    Objective The benefits of HAART rely on continuous lifelong treatment retention. We used linked population-level health administrative data to characterize durations of HAART retention and nonretention. Design This is a retrospective cohort study. Methods We considered individuals initiating HAART in British Columbia (1996–2012). An HAART episode was considered discontinued if individuals had a gap of at least 30 days between days in which medication was prescribed. We considered durations of HAART retention and nonretention separately, and used Cox proportional hazards frailty models to identify demographic and treatment-related factors associated with durations of HAART retention and nonretention. Results Six thousand one hundred fifty-two individuals were included in the analysis; 81.2% were male, 40.6% were people who inject drugs, and 42.8% initiated treatment with CD4 cell count less than 200 cells/μl. Overall, 29% were continuously retained on HAART through the end of follow-up. HAART episodes were a median 6.8 months (25th, 75th percentile: 2.3, 19.5), whereas off-HAART episodes lasted a median 1.9 months (1.2, 4.5). In Cox proportional hazards frailty models, durations of HAART retention improved over time. Successive treatment episodes tended to decrease in duration among those with multiple attempts, whereas off-HAART episodes remained relatively stable. Younger age, earlier stages of disease progression, and injection drug use were all associated with shorter durations of HAART retention and longer off-HAART durations. Conclusion Metrics to monitor HAART retention, dropout, and reentry should be prioritized for HIV surveillance. Clinical strategies and public health policies are urgently needed to improve HAART retention, particularly among those at earlier stages of disease progression, the young, and people who inject drugs. PMID:26372279

  20. Adipokines in the HIV/HAART-associated lipodystrophy syndrome.

    PubMed

    Paruthi, Jason; Gill, Natasha; Mantzoros, Christos S

    2013-09-01

    The use of highly active antiretroviral therapy (HAART) in the treatment of human immunodeficiency virus has dramatically altered both the landscape of this disease and the prognosis for those affected. With more patients now receiving HAART, adverse effects such as lipodystrophy and metabolic syndrome have emerged. In HIV/HAART-associated lipodystrophy syndrome (HALS), patients demonstrate fat maldistribution with dyslipidemia, insulin resistance, and other metabolic complications. Recent studies have contributed to the elucidation of the pathophysiological abnormalities seen in this syndrome and have provided guidance for the study and use of potential treatments for these patients, but widely accepted guidelines have not yet been established. Two adipokines, leptin and adiponectin, are decreased in patients with HALS and lipoatrophy or lipodystrophy. Further, recent proof-of-concept clinical trials have proven the efficacy of leptin replacement and medications that increase circulating adiponectin levels in improving the metabolic profile of HALS patients. This review article highlights recent evidence on leptin replacement and compares leptin's efficacy to that of other treatments, including metformin and thiazolidinediones, on metabolic abnormalities such as impaired insulin-glucose homeostasis associated with lipodystrophy in patients receiving HAART. It is hoped that forthcoming large phase III clinical trials will allow the addition of leptin to our therapeutic armamentarium for use in patients suffering from this disease state.

  1. Salvage solitaire (or, HAART takes a holiday).

    PubMed

    Mascolini, M

    1999-07-01

    Attendees at the Second International Workshop on Salvage Therapy heard little to make them optimistic about the immediate future of salvage therapy. Treatment veterans with only a few virologic failures stand a slim chance of ongoing viral control. Current salvage treatment, even combination therapy, only pushes viral loads to below 500 copies in 20 to 40 percent of patients, and those patients who have had viral loads suppressed to below 20 copies/mL stand the best chance of having a sustained response to the treatment. Results of several studies are reviewed. Adefovir, which had been considered an option, has performed poorly in trials. One table shows the effects of nonnucleoside reverse transcriptase inhibitor/protease inhibitor (NNRTI/PI) salvage therapy in the NNRTI-naive patient. Another table presents figures to show that salvage success correlates significantly with lower viral loads prior to treatment. A third table shows the "megasalvage" track record, while a fourth presents a proposal for a rapid-assessment salvage study. The concept of using drug holidays as a means to force the immune system to begin fighting HIV on its own remains controversial.

  2. PDT in periodontal disease of HAART resistance patients

    NASA Astrophysics Data System (ADS)

    Giovani, Elcio M.; Noro-Filho, Gilberto A.; Caputo, Bruno V.; Casarin, Renato; Costa, Claudio; Salgado, Daniela; Santos, Camila C.

    2016-03-01

    HIV/Aids patients present a change of microbiota associated with host immunodeficiency. Photodynamic therapy (PDT) showed as a promising and viable alternative in reducing microbiota. Present study evaluate effectiveness of photodynamic therapy in periodontal disease of AIDS patients with highly activity antiretroviral therapy (HAART) failure, measuring the clinical periodontal parameters and periodontal microbiota. Twelve patients with HARRT resistance (R group) divided into two groups (control and PDT) and 12 patients with no HAART resistance (NR group) divided into two groups (control and PDT). The results show the difference in baseline of CD4 cells count, NR group 640.0 +/- 176.2 cells/mm3 R group and 333.3 +/- 205.8 cells / mm3 (p<0.05), and in 8.3% detectable viral load in NR group and 75% detectable (p <0.001) in R group. As clinical periodontal parameters (PD and CAL), PDT was more effective than the control group only in the NR group (p <0.05%), moreover, there was no difference in the evaluation of clinical periodontal parameters between the both R groups (p>0.05%). Microbiological evaluation in R group presents a general reduction in the Aa at 3 and 6 months. Furthermore, demonstrated a reduction of Pg in all groups at 6 months and in R group at 3 months. The impact assessment of photodynamic therapy in patients with different levels of immunosuppression determined that the combination of mechanical periodontal treatment with photodynamic therapy in patients with HAART failure did not cause additional benefits. Therefore, PDT in this study could not been indicated in HAART resistance patients.

  3. Control of viral replication after cessation of HAART

    PubMed Central

    2011-01-01

    We describe two patients who did not experience a viral rebound after cessation of HAART which was initiated for progressive disease. CD4 T-cell count remained stable in one patient and progressively declined in the other, despite apparent viral control. We failed to identify any immune activation or genetic markers that could offer an explanation for this unusual "secondary controller" status. But their viruses are clearly less fit compared to viruses from rebounders. PMID:21314914

  4. Relationship between oral Kaposi 's sarcoma and HAART: contribution of two case reports.

    PubMed

    Campo-Trapero, Julián; Del Romero-Guerrero, Jorge; Cano-Sánchez, Jorge; Rodríguez-Martín, Carmen; Martínez-González, José Ma; Bascones-Martínez, Antonio

    2008-11-01

    Two HIV infected patients not receiving Highly Active Antiretroviral Treatment (HAART) presented with epidemic Kaposi's sarcoma of the oral cavity. One patient initially refused HAART, but when the lesion became large enough to be noticeable he agreed to HAART associated with excision of the intraoral lesion by CO2 laser. The other patient developed KS and progressed to AIDS at two years after ceasing HAART due to adverse effects; he was referred to hospital for renewed administration of HAART. In both cases, the lesions observed in the oral cavity were the first clinical manifestation of AIDS. These reports underline the close relationship between the use of HAART and the control of KS lesions, highlighting the important role of the dentist in the identification and early diagnosis of these oral lesions.

  5. The Impact of HAART on Cardiomyopathy among Children and Adolescents Perinatally Infected with HIV-1

    PubMed Central

    Patel, Kunjal; van Dyke, Russell B.; Mittleman, Murray A.; Colan, Steven D.; Oleske, James M.; Seage, George R.

    2012-01-01

    Objective Previous studies of cardiomyopathy among children perinatally infected with HIV were conducted before the routine use of highly active antiretroviral therapy (HAART). Nucleoside analogues (NRTIs), the backbone of HAART, have been associated with mitochondrial toxicity, which can lead to cardiomyopathy. We evaluated the association of HAART and specific NRTIs associated with mitochondrial toxicity, on development of cardiomyopathy among perinatally HIV-infected children. Design 3,035 perinatally HIV-infected children enrolled in a US-based multicenter prospective cohort study, were followed for cardiomyopathy, defined as a clinical diagnosis or initiation of digoxin, from 1993–2007. Methods Cox models were used to estimate the effects of HAART and NRTIs on cardiomyopathy, identify predictors of cardiomyopathy among HAART users, and estimate the association between development of cardiomyopathy and mortality. Results 99 cases of cardiomyopathy were identified over follow-up (incidence rate: 5.6 cases per 1,000 person-years) at a median age of 9.4 years. HAART was associated with a 50% lower incidence of cardiomyopathy compared to no HAART use (95% confidence interval: 20%, 70%). Zalcitabine (ddC) use, however, was associated with an 80% higher incidence of cardiomyopathy. Among HAART users, older age at HAART initiation, ddC use before HAART initiation, initiating a HAART regimen containing zidovudine (ZDV), and a nadir CD4<15% were independently associated with a higher rate of cardiomyopathy. Cardiomyopathy was associated with a 6-fold higher mortality rate. Conclusions HAART has dramatically decreased the incidence of cardiomyopathy among perinatally HIV-infected children. However, they remain at increased risk for cardiomyopathy and ongoing ZDV exposure may increase this risk. PMID:22781228

  6. Proteases as Insecticidal Agents

    PubMed Central

    Harrison, Robert L.; Bonning, Bryony C.

    2010-01-01

    Proteases from a variety of sources (viruses, bacteria, fungi, plants, and insects) have toxicity towards insects. Some of these insecticidal proteases evolved as venom components, herbivore resistance factors, or microbial pathogenicity factors, while other proteases play roles in insect development or digestion, but exert an insecticidal effect when over-expressed from genetically engineered plants or microbial pathogens. Many of these proteases are cysteine proteases, although insect-toxic metalloproteases and serine proteases have also been examined. The sites of protease toxic activity range from the insect midgut to the hemocoel (body cavity) to the cuticle. This review discusses these insecticidal proteases along with their evaluation and use as potential pesticides. PMID:22069618

  7. Design of HIV Protease Inhibitors Targeting Protein Backbone: An Effective Strategy for Combating Drug Resistance

    SciTech Connect

    Ghosh, Arun K.; Chapsal, Bruno D.; Weber, Irene T.; Mitsuya, Hiroaki

    2008-06-03

    The discovery of human immunodeficiency virus (HIV) protease inhibitors (PIs) and their utilization in highly active antiretroviral therapy (HAART) have been a major turning point in the management of HIV/acquired immune-deficiency syndrome (AIDS). However, despite the successes in disease management and the decrease of HIV/AIDS-related mortality, several drawbacks continue to hamper first-generation protease inhibitor therapies. The rapid emergence of drug resistance has become the most urgent concern because it renders current treatments ineffective and therefore compels the scientific community to continue efforts in the design of inhibitors that can efficiently combat drug resistance.

  8. Risk Behaviors of Youth Living With HIV: Pre- and Post-HAART

    ERIC Educational Resources Information Center

    Lightfoot, Marguerita; Swendeman, Dallas; Rotheram-Borus, Mary Jane; Comulada, W. Scott; Weiss, Robert

    2005-01-01

    Objective: To examine the transmission behavior among youth living with HIV (YLH), pre- and post-HAART. Methods: Two cohorts were recruited: (1) 349 YLH during 1994 to 1996 and (2) 175 YLH during 1999 to 2000, after the wide availability of HAART. Differences in sexual and substance-use risk acts and quality of life were examined. Results:…

  9. Incident Hepatitis B Virus Infection in HIV-Infected and HIV-Uninfected Men Who Have Sex With Men From Pre-HAART to HAART Periods

    PubMed Central

    Falade-Nwulia, Oluwaseun; Seaberg, Eric C.; Snider, Anna E.; Rinaldo, Charles R.; Phair, John; Witt, Mallory D.; Thio, Chloe L.

    2015-01-01

    Background Men who have sex with men (MSM) are at high risk for hepatitis B virus (HBV) infection. Data on the effect of highly active antiretroviral therapy (HAART) on incident HBV infection in HIV-infected and HIV-uninfected MSM are limited. Objective To determine predictors of incident HBV infection in MSM during pre-HAART and HAART periods. Design Observational cohort study. Setting Cohort of MSM who have, or are at risk for, HIV infection. Patients 2375 HBV-uninfected MSM in the Multicenter AIDS Cohort Study. Measurements Poisson regression was used to compare incidence rates of HBV infection in the pre-HAART and HAART eras and to identify factors associated with incidence of HBV infection. Results In 25 322 person-years of follow-up, 244 incident HBV infections occurred. The unadjusted incidence rate was higher in HIV-infected MSM than in HIV-uninfected MSM (IRR, 1.9 [95% CI, 1.5 to 2.4]) and was significantly lower in the HAART era than in the pre-HAART era among HIV-infected (IRR, 0.2 [CI, 0.1 to 0.4]) and HIV-uninfected (IRR, 0.3 [CI, 0.2 to 0.4]) MSM. Age younger than 40 years (IRR, 2.3 [CI, 1.7 to 3.0]), more than 1 recent sexual partner (IRR, 3.1 [CI, 2.3 to 4.2]), and HIV infection (IRR, 2.4 [CI, 1.8 to 3.1]) were independently associated with higher incidence of HBV infection, whereas HBV vaccination was protective (IRR, 0.3 [CI, 0.2 to 0.4]). Highly active antiretroviral therapy with HIV RNA levels less than 400 copies/mL was associated with protection (IRR, 0.2 [CI, 0.1 to 0.5]), but HAART in those with HIV RNA levels of 400 copies/mL or greater was not. Limitation The observational nature limits inferences about causality. Conclusion Effective HAART is associated with lower incidence of HBV infection; however, even in the HAART era, incidence of HBV infection remains high among MSM. Primary Funding Source National Institute of Allergy and Infectious Diseases. PMID:26457744

  10. Development of novel dipeptide-like rhodesain inhibitors containing the 3-bromoisoxazoline warhead in a constrained conformation.

    PubMed

    Ettari, Roberta; Pinto, Andrea; Previti, Santo; Tamborini, Lucia; Angelo, Ilenia C; La Pietra, Valeria; Marinelli, Luciana; Novellino, Ettore; Schirmeister, Tanja; Zappalà, Maria; Grasso, Silvana; De Micheli, Carlo; Conti, Paola

    2015-11-01

    Novel dipeptide-like rhodesain inhibitors containing the 3-bromoisoxazoline warhead in a constrained conformation were developed; some of them possess K(i) values in the micromolar range. We studied the structure-activity relationship of these derivatives and we performed docking studies, which allowed us to find out the key interactions established by the inhibitors with the target enzyme. Biological results indicate that the nature of the P2 and P3 substituents and their binding to the S2/S3 pockets is strictly interdependent. PMID:26432608

  11. Development of novel dipeptide-like rhodesain inhibitors containing the 3-bromoisoxazoline warhead in a constrained conformation.

    PubMed

    Ettari, Roberta; Pinto, Andrea; Previti, Santo; Tamborini, Lucia; Angelo, Ilenia C; La Pietra, Valeria; Marinelli, Luciana; Novellino, Ettore; Schirmeister, Tanja; Zappalà, Maria; Grasso, Silvana; De Micheli, Carlo; Conti, Paola

    2015-11-01

    Novel dipeptide-like rhodesain inhibitors containing the 3-bromoisoxazoline warhead in a constrained conformation were developed; some of them possess K(i) values in the micromolar range. We studied the structure-activity relationship of these derivatives and we performed docking studies, which allowed us to find out the key interactions established by the inhibitors with the target enzyme. Biological results indicate that the nature of the P2 and P3 substituents and their binding to the S2/S3 pockets is strictly interdependent.

  12. AIDS in the HAART era: New York's heterogeneous geography.

    PubMed

    Wallace, Robert G

    2003-03-01

    During the 1990s, the number of new AIDS cases in New York City, USA, declined precipitously. The declines, beginning before highly active antiretroviral therapy (HAART) was introduced, were geographically heterogeneous across two New York City boroughs analyzed. From 1993 to 1998, zip codes in Lower Manhattan, with large white and affluent populations, had declines as much as 55% more than the rest of Manhattan. Bronx zip codes underwent still lesser declines. Declines also differed within zip codes among subpopulations. White zip code populations tended to have greater declines than Latino populations, which in turn tended to have greater declines than black populations. According to bivariate and stepwise regressions, an array of socioeconomic and community stress variables acted in combination on the decline in New York AIDS. Manhattan's declines in total AIDS incidence were primarily defined by changes in AIDS incidence for whites and for men who have sex with men, racial segregation, and the proportions of households in upper income classes and under rent stress. Bronx declines in total AIDS are principally explained by a broader range of income classes, and social instability as marked by housing overcrowding and cirrhosis and drug mortalities. Whatever the combination of proximate causes for the decline in AIDS incidence in 1990s New York (educational campaigns, HAART, demographic stochasticity), the decline was shaped by the city's socioeconomic structure and political and ecological history. That structure and history generates the geographically defined aggregates of behaviors that promote or impede AIDS decline. Such spatial heterogeneity may provide for HIV refugia, areas where the virus can weather the epidemic's contraction, a troubling possibility with the accelerating microbicidal failures of combination therapies.

  13. Investigations with Protease.

    ERIC Educational Resources Information Center

    Yip, Din Yan

    1997-01-01

    Presents two simple and reliable ways for measuring protease activity that can be used for a variety of investigations in a range of biology class levels. The investigations use protease from a variety of sources. (DDR)

  14. Effects of long-term use of HAART on oral health status of HIV-infected subjects

    PubMed Central

    Nittayananta, Wipawee; Talungchit, Sineepat; Jaruratanasirikul, Sutep; Silpapojakul, Kachornsakdi; Chayakul, Panthip; Nilmanat, Ampaipith; Pruphetkaew, Nannapat

    2011-01-01

    BACKGROUND The aim of this study was to determine the effects of long-term use of highly active antiretroviral therapy (HAART) on oral health status of HIV-infected subjects. METHODS Oral examination and measurement of saliva flow rate of both unstimulated and wax-stimulated whole saliva were performed in HIV-infected subjects with and without HAART, and in non-HIV individuals. The following data were recorded; duration and risk of HIV infection, type and duration of HAART, CD4 cell count, viral load, presence of orofacial pain, oral dryness, oral burning sensation, oral lesions, cervical caries, and periodontal pocket. Multiple logistic regression analysis was performed to determine the effects of long-term use of HAART on oral health status of HIV-infected subjects. RESULTS: One hundred and fifty-seven HIV-infected subjects – 99 on HAART (age range 23–57 years, mean 39 years) and 58 not on HAART (age range 20–59 years, mean 34 years) – and 50 non-HIV controls (age range 19–59 years, mean 36 years) were enrolled. The most common HAART regimen was 2 NRTI + 2 NNRTI. HIV-infected subjects without HAART showed greater risks of having orofacial pain, oral dryness, oral lesions, and periodontal pockets than those with short-term HAART (P < 0.01). The subjects with long-term HAART were found to have a greater risk of having oral lesions than those with short-term HAART (P < 0.05). The unstimulated and stimulated salivary flow rates of the subjects with HAART were significantly lower than in those without HAART (P < 0.05). CONCLUSION We conclude that long-term HAART has adverse effects on oral health status of HIV-infected subjects. PMID:20202089

  15. HIV/AIDS patients' medical and psychosocial needs in the era of HAART: a cross-sectional study among HIV/AIDS patients receiving HAART in Yunnan, China.

    PubMed

    Wen, Yi; Shi, Yun; Jiang, Chengqin; Detels, Roger; Wu, Di

    2013-01-01

    Since the launch of China's Free Antiretroviral Therapy (ART) Program in 2002, more than 100,000 HIV/AIDS patients have been treated with highly actively antiretroviral therapy (HAART). However, the current evaluation system for this program mainly focused on its medical outcomes. This study aims to evaluate the medical and psychosocial needs of HIV/AIDS patients after initiating HAART. A cross-sectional study was conducted among 499 HIV/AIDS patients who were currently being treated with HAART in three designated hospitals in Luxi City, Yunnan Province. A questionnaire was used to collect information about participants' demographic characteristics, perceived HIV-related stigma, physician-patient relationship, quality of life, family functioning, etc. Patients' medical records in the National HIV Information System were linked with their questionnaire by their ART identification number. Patients on HAART who were infected with HIV through injection drug use and were current smokers typically had poorer physical health than other participants on HAART. Better financial status and better physician-patient relationship were associated with both physical and psychological well-being. Family awareness of the patient's HIV status was negatively associated with the patient's psychological well-being. Higher levels of perceived HIV-related stigma were associated with poorer psychological health and poorer family functioning. This study emphasizes the importance of assuring a caring environment in China's AIDS treatment program and re-enforces the need to combat the stigma encountered with health providers and the public.

  16. A Comparison of the Diabetes Risk Score in HIV/AIDS Patients on Highly Active Antiretroviral Therapy (HAART) and HAART-Naïve Patients at the Limbe Regional Hospital, Cameroon

    PubMed Central

    Dimala, Christian Akem; Atashili, Julius; Mbuagbaw, Josephine C.; Wilfred, Akam; Monekosso, Gottlieb L.

    2016-01-01

    Background Highly active antiretroviral therapy (HAART) has been associated with dysglycaemia. However, there is scarce data on the risk of developing diabetes mellitus (DM) in HIV/AIDS patients in Africa. Objectives Primarily to quantify and compare the risk of having diabetes mellitus in HIV/AIDS patients on HAART and HAART-naïve patients in Limbe, Cameroon; and secondarily to determine if there is an association between HAART and increased DM risk. Methods A cross-sectional study was conducted at the Limbe Regional Hospital HIV treatment center between April and June 2013, involving 200 HIV/AIDS patients (100 on first-line HAART regimens for at least 12 months matched by age and gender to 100 HAART-naïve patients). The Diabetes Risk Score (DRS) was calculated using a clinically validated model based on routinely recorded primary care parameters. A DRS ≥ 7% was considered as indicative of an increased risk of developing DM. Results The median DRS was significantly higher in patients on HAART (2.30%) than in HAART-naïve patients (1.62%), p = 0.002. The prevalence of the increased DM risk (DRS ≥ 7%) was significantly higher in patients on HAART, 31% (95% CI: 22.13–41.03) than in HAART-naïve patients, 17% (95% CI: 10.23–25.82), p = 0.020. HAART was significantly associated with an increased DM risk, the odds ratio of the HAART group compared to the HAART-naïve group was 2.19 (95% CI: 1.12–4.30, p = 0.020). However, no association was found after adjusting for BMI-defined overweight, hypertension, age, sex, family history of DM and smoking (Odds ratio = 1.22, 95% CI: 0.42–3.59, p = 0.708). Higher BMI and hypertension accounted for the increased risk of DM in patients on HAART. Also, more than 82% of the participants were receiving or had ever used Zidovudine based HAART regimens. Conclusion HIV/AIDS patients on HAART could be at a greater risk of having DM than HAART-naïve patients as a result of the effect of HAART on risk factors of DM such as BMI

  17. The changing role of HIV-associated oral candidiasis in the era of HAART.

    PubMed

    Patuwo, Christopher; Young, Keane; Lin, Meng; Pardi, Vanessa; Murata, Ramiro M

    2015-02-01

    Oral candidiasis is the most common fungal opportunistic infection to affect the oral cavity among HIV patients. The advent of highly active antiretroviral therapy (HAART) has changed the epidemiology of candidiasis, with many studies reporting a decrease in prevalence. However, some studies report rare cases of increased prevalence. This systematic review clarifies the role of oral candidiasis in the HAART era as a marker of immune status and successful therapy for the HIV-infected population.

  18. Proteases as therapeutics

    PubMed Central

    Craik, Charles S.; Page, Michael J.; Madison, Edwin L.

    2015-01-01

    Proteases are an expanding class of drugs that hold great promise. The U.S. FDA (Food and Drug Administration) has approved 12 protease therapies, and a number of next generation or completely new proteases are in clinical development. Although they are a well-recognized class of targets for inhibitors, proteases themselves have not typically been considered as a drug class despite their application in the clinic over the last several decades; initially as plasma fractions and later as purified products. Although the predominant use of proteases has been in treating cardiovascular disease, they are also emerging as useful agents in the treatment of sepsis, digestive disorders, inflammation, cystic fibrosis, retinal disorders, psoriasis and other diseases. In the present review, we outline the history of proteases as therapeutics, provide an overview of their current clinical application, and describe several approaches to improve and expand their clinical application. Undoubtedly, our ability to harness proteolysis for disease treatment will increase with our understanding of protease biology and the molecular mechanisms responsible. New technologies for rationally engineering proteases, as well as improved delivery options, will expand greatly the potential applications of these enzymes. The recognition that proteases are, in fact, an established class of safe and efficacious drugs will stimulate investigation of additional therapeutic applications for these enzymes. Proteases therefore have a bright future as a distinct therapeutic class with diverse clinical applications. PMID:21406063

  19. Dysregulated Immune Activation in Second-Line HAART HIV+ Patients Is Similar to That of Untreated Patients

    PubMed Central

    Espíndola, Milena S.; Lima, Leonardo J. G.; Soares, Luana S.; Cacemiro, Maira C.; Zambuzi, Fabiana A.; de Souza Gomes, Matheus; Amaral, Laurence R.; Bollela, Valdes R.; Martins-Filho, Olindo A.; Frantz, Fabiani G.

    2015-01-01

    Background Successful highly active antiretroviral therapy (HAART) has changed the outcome of AIDS patients worldwide because the complete suppression of viremia improves health and prolongs life expectancy of HIV-1+ patients. However, little attention has been given to the immunological profile of patients under distinct HAART regimens. This work aimed to investigate the differences in the immunological pattern of HIV-1+ patients under the first- or second-line HAART in Brazil. Methods CD4+ T cell counts, Viral load, and plasma concentration of sCD14, sCD163, MCP-1, RANTES, IP-10, IL-1β, IL-6, TNF-α, IL-12, IFN-α, IFN-γ, IL-4, IL-5, and IL-10 were assessed for immunological characterization of the following clinical groups: Non-infected individuals (NI; n = 66), HIV-1+ untreated (HIV; n = 46), HIV-1+ treated with first-line HAART (HAART 1; n = 15); and HIV-1+ treated with second-line HAART (HAART 2; n = 15). Results We found that the immunological biosignature pattern of HAART 1 is similar to that of NI individuals, especially in patients presenting slow progression of the disease, while patients under HAART 2 remain in a moderate inflammatory state, which is similar to that of untreated HIV patients pattern. Network correlations revealed that differences in IP-10, TNF-α, IL-6, IFN-α, and IL-10 interactions were primordial in HIV disease and treatment. Heat map and decision tree analysis identified that IP-10>TNF-α>IFN-α were the best respective HAART segregation biomarkers. Conclusion HIV patients in different HAART regimens develop distinct immunological biosignature, introducing a novel perspective into disease outcome and potential new therapies that consider HAART patients as a heterogeneous group. PMID:26684789

  20. The causal effect of opioid substitution treatment on HAART medication refill adherence

    PubMed Central

    Nosyk, Bohdan; Min, Jeong E.; Colley, Guillaume; Lima, Viviane D.; Yip, Benita; Milloy, M.-J.S.; Wood, Evan; Montaner, Julio S.G.

    2015-01-01

    Background People who inject drugs (PWID) account for roughly 13% of the prevalent HIV/AIDS population outside of sub-Saharan Africa, and access to opioid substitution treatment (OST) is limited in many settings globally. OST likely facilitates access to HAART, yet sparse evidence is available to support this hypothesis. Our objective was to determine the causal impact of OST exposure on HAART adherence among HIV-positive PWID in a Canadian setting. Methods We executed a retrospective cohort study using linked population-level data for British Columbia, Canada (January 1996–March 2010). We considered HIV-positive PWID after meeting HAART initiation criteria. A marginal structural model was estimated on a monthly timescale using inverse probability of treatment weights. The primary outcome was 95% HAART adherence, according to pharmacy refill compliance. Exposure to OST was defined as 95% of OST receipt, and we controlled for a range of fixed and time-varying covariates. Results Our study included 1852 (63.3%) HIV-positive PWID with a median follow-up of 5.5 years; 34% were female and 39% had previously accessed OST. The baseline covariate-adjusted odds of HAART adherence following OST exposure was 1.96 (95% confidence interval: 1.72–2.24), although the adjusted odds estimated within the marginal structural model was 1.68 (1.48–1.92). Findings were robust to sensitivity analyses on model specification. Conclusion In a setting characterized by universal healthcare and widespread access to both office-based OST and HAART, OST substantially increased the odds of HAART adherence. This underlines the need to address barriers to OST globally to reduce the disease burden of both opioid dependence and HIV/AIDS. PMID:25915170

  1. Does HAART Efficacy Translate to Effectiveness? Evidence for a Trial Effect

    PubMed Central

    Menezes, Prema; Miller, William C.; Wohl, David A.; Adimora, Adaora A.; Leone, Peter A.; Miller, William C.; Eron, Joseph J.

    2011-01-01

    Background Patients who participate in clinical trials may experience better clinical outcomes than patients who initiate similar therapy within clinical care (trial effect), but no published studies have evaluated a trial effect in HIV clinical trials. Methods To examine a trial effect we compared virologic suppression (VS) among patients who initiated HAART in a clinical trial versus in routine clinical care. VS was defined as a plasma HIV RNA ≤400 copies/ml at six months after HAART initiation and was assessed within strata of early (1996–99) or current (2000–06) HAART periods. Risk ratios (RR) were estimated using binomial models. Results Of 738 persons initiating HAART, 30.6% were women, 61.7% were black, 30% initiated therapy in a clinical trial and 67% (n = 496) had an evaluable six month HIV RNA result. HAART regimens differed between the early and current periods (p<0.001); unboosted PI regimens (55.6%) were more common in the early and NNRTI regimens (46.4%) were more common in the current period. Overall, 78% (95%CI 74, 82%) of patients achieved VS and trial participants were 16% more likely to achieve VS (unadjusted RR 1.16, 95%CI 1.06, 1.27). Comparing trial to non-trial participants, VS differed by study period. In the early period, trial participants initiating HAART were significantly more likely to achieve VS than non-trial participants (adjusted RR 1.33; 95%CI 1.15, 1.54), but not in the current period (adjusted RR 0.98; 95%CI 0.87, 1.11). Conclusions A clear clinical trial effect on suppression of HIV replication was observed in the early HAART period but not in the current period. PMID:21765918

  2. Alterations in thigh subcutaneous adipose tissue gene expression in protease inhibitor-based highly active antiretroviral therapy

    PubMed Central

    Chaparro, Juan; Reeds, Dominic N.; Wen, Weidong; Xueping, E.; Klein, Samuel; Semenkovich, Clay F.; Bae, Kyongtae T.; Quirk, Erin K.; Powderly, William G.; Yarasheski, Kevin E.; Li, Ellen

    2006-01-01

    Use of protease inhibitor (PI)–based highly active antiretroviral therapy (HAART) has been associated with altered regional fat distribution, insulin resistance, and dyslipidemias. To assess how PI-based HAART affects adipocyte gene expression in male HIV-1–infected patients, reverse transcription–polymerase chain reaction was used to quantify messenger RNA expression of adipocyte transcription factors and adipocytokines in thigh and abdominal subcutaneous adipose tissue from male (1) HIV-1 seronegative subjects (control, n = 9), (2) asymptomatic treatment-naive HIV-1–infected patients (naive, n = 6), (3) HIV-1–infected patients who were receiving antiretroviral agents but never received PIs (PI naive, n = 5), (4) HIV-1–infected patients who were receiving PI-based HAART (PI, n = 7), and (5) HIV-1–infected patients who discontinued the PI component of their antiviral therapy more than 6 months before enrollment (past PI, n =7). In the PI group, the messenger RNA expression levels of the CCAAT/enhancer–binding protein α, leptin, and adiponectin (18%, P < .01; 23%, P < .05; and 13%, P < .05, respectively) were significantly lower than the levels measured in the PI-naive group. These results are consistent with previous studies on the effects of PIs on cultured adipocytes. Prospective longitudinal studies of thigh fat adipose tissue gene expression could provide further insights on the pathogenesis of metabolic complications associated with PI-based HAART. PMID:15877283

  3. Incidence of oral lesions in HIV-1-infected women: reduction with HAART.

    PubMed

    Greenspan, D; Gange, S J; Phelan, J A; Navazesh, M; Alves, M E A F; MacPhail, L A; Mulligan, R; Greenspan, J S

    2004-02-01

    Few studies assess the effectiveness of HAART on reducing the incidence and recurrence of oral lesions. We investigated such changes among 503 HIV+ women over six years in the Women's Interagency HIV Study. The incidence of erythematous candidiasis (EC), pseudomembranous candidiasis (PC), hairy leukoplakia (HL), and warts was computed over follow-up visits after HAART initiation compared with before HAART initiation. Analysis of our data demonstrates a strong decrease in candidiasis after HAART initiation. The incidence of EC fell to 2.99% from 5.48% (RR 0.545); PC fell to 2.85% from 6.70% (RR 0.425); and EC or PC fell to 3.43% from 7.35% (RR 0.466). No changes were seen in HL or warts. Higher HIV-RNA was associated with greater incidence of candidiasis and HL, but not warts. Analysis of these data indicates that recurrence and incidence of candidiasis are reduced by HAART, and that recurrence is reduced independently of CD4 and HIV-RNA.

  4. [Cerebrospinal fluid viral load in HIV-1 positive hemophilic patients treated with HAART].

    PubMed

    Corti, M E; Villafañe, M F; Baré, P; Alves Rosa, F; Cermelj, M; Candela, M; Pérez Bianco, R; Tezanos Pinto, M

    2001-01-01

    As HIV seropositive patients with undetectable CSF viral load have a lower likelihood of developing neurologic disease, the determination of CSF viral load levels may be useful to evaluate the efficacy of HAART. We compared plasma viral load levels with HIV-1 RNA CSF levels in 18 hemophilic patients without neurocognitive involvement under HAART. We detected a significant correlation between plasma viral load levels and CSF viral load levels. Fourteen patients with undetectable plasma viral load had undetectable RNA HIV-1 CSF levels as well. Four patients with detectable plasma viral load had detectable HIV-RNA in CSF, but the latter were significantly lower. Viral load is usually lower in non-blood fluids and HAART decreases the viral load in CSF as well as in blood.

  5. Mutations Related to Antiretroviral Resistance Identified by Ultra-Deep Sequencing in HIV-1 Infected Children under Structured Interruptions of HAART.

    PubMed

    Vazquez-Guillen, Jose Manuel; Palacios-Saucedo, Gerardo C; Rivera-Morales, Lydia G; Garcia-Campos, Jorge; Ortiz-Lopez, Rocio; Noguera-Julian, Marc; Paredes, Roger; Vielma-Ramirez, Herlinda J; Ramirez, Teresa J; Chavez-Garcia, Marcelino; Lopez-Guillen, Paulo; Briones-Lara, Evangelina; Sanchez-Sanchez, Luz M; Vazquez-Martinez, Carlos A; Rodriguez-Padilla, Cristina

    2016-01-01

    Although Structured Treatment Interruptions (STI) are currently not considered an alternative strategy for antiretroviral treatment, their true benefits and limitations have not been fully established. Some studies suggest the possibility of improving the quality of life of patients with this strategy; however, the information that has been obtained corresponds mostly to studies conducted in adults, with a lack of knowledge about its impact on children. Furthermore, mutations associated with antiretroviral resistance could be selected due to sub-therapeutic levels of HAART at each interruption period. Genotyping methods to determine the resistance profiles of the infecting viruses have become increasingly important for the management of patients under STI, thus low-abundance antiretroviral drug-resistant mutations (DRM's) at levels under limit of detection of conventional genotyping (<20% of quasispecies) could increase the risk of virologic failure. In this work, we analyzed the protease and reverse transcriptase regions of the pol gene by ultra-deep sequencing in pediatric patients under STI with the aim of determining the presence of high- and low-abundance DRM's in the viral rebounds generated by the STI. High-abundance mutations in protease and high- and low-abundance mutations in reverse transcriptase were detected but no one of these are directly associated with resistance to antiretroviral drugs. The results could suggest that the evaluated STI program is virologically safe, but strict and carefully planned studies, with greater numbers of patients and interruption/restart cycles, are still needed to evaluate the selection of DRM's during STI. PMID:26807922

  6. Evidence for Reduced Drug Susceptibility without Emergence of Major Protease Mutations following Protease Inhibitor Monotherapy Failure in the SARA Trial

    PubMed Central

    Sutherland, Katherine A.; Parry, Chris M.; McCormick, Adele; Kapaata, Anne; Lyagoba, Fred; Kaleebu, Pontiano; Gilks, Charles F.; Goodall, Ruth; Spyer, Moira; Kityo, Cissy; Pillay, Deenan; Gupta, Ravindra K.

    2015-01-01

    Background Major protease mutations are rarely observed following failure with protease inhibitors (PI), and other viral determinants of failure to PI are poorly understood. We therefore characterized Gag-Protease phenotypic susceptibility in subtype A and D viruses circulating in East Africa following viral rebound on PIs. Methods Samples from baseline and treatment failure in patients enrolled in the second line LPV/r trial SARA underwent phenotypic susceptibility testing. Data were expressed as fold-change in susceptibility relative to a LPV-susceptible reference strain. Results We cloned 48 Gag-Protease containing sequences from seven individuals and performed drug resistance phenotyping from pre-PI and treatment failure timepoints in seven patients. For the six patients where major protease inhibitor resistance mutations did not emerge, mean fold-change EC50 to LPV was 4.07 fold (95% CI, 2.08–6.07) at the pre-PI timepoint. Following viral failure the mean fold-change in EC50 to LPV was 4.25 fold (95% CI, 1.39–7.11, p = 0.91). All viruses remained susceptible to DRV. In our assay system, the major PI resistance mutation I84V, which emerged in one individual, conferred a 10.5-fold reduction in LPV susceptibility. One of the six patients exhibited a significant reduction in susceptibility between pre-PI and failure timepoints (from 4.7 fold to 9.6 fold) in the absence of known major mutations in protease, but associated with changes in Gag: V7I, G49D, R69Q, A120D, Q127K, N375S and I462S. Phylogenetic analysis provided evidence of the emergence of genetically distinct viruses at the time of treatment failure, indicating ongoing viral evolution in Gag-protease under PI pressure. Conclusions Here we observe in one patient the development of significantly reduced susceptibility conferred by changes in Gag which may have contributed to treatment failure on a protease inhibitor containing regimen. Further phenotype-genotype studies are required to elucidate genetic

  7. A narrative review of cost-effectiveness analysis of people living with HIV treated with HAART: from interventions to outcomes

    PubMed Central

    Tse, Wah Fung; Yang, Weimin; Huang, Wenlong

    2015-01-01

    Background Since its introduction in 1996, highly active antiretroviral therapy (HAART), which involves the combination of antiretroviral drugs, has resulted in significant improvements in the morbidity, mortality, and life expectancy of HIV-infected patients. Numerous studies of the cost-effectiveness of HAART from different perspectives in HIV have been reported. Aim To investigate the economic outcomes and relevance of HAART for people living with HIV. Materials and methods A narrative literature review was conducted on 22 peer-reviewed full economic evaluations of people living with HIV treated with different HAART regimens and published in English between January 2005 and December 2014. Information regarding study details, such as interventions, outcomes, and modeling methods, was extracted. The high heterogeneity of the included studies rendered a meta-analysis inappropriate; therefore, we conducted a comparative analysis of studies grouped according to the similarity of the different intervention types and outcomes. Results Most of the economic evaluations of HAART focused on comparisons between the specific HAART regimens and others from the following perspectives: injecting drug users versus noninjecting drug users, HIV-infected adults without AIDS versus those with AIDS, regimens based on developed world guidelines versus those based on developing world guidelines, self-administered HAART versus directly observed HAART, and “ideal” versus “typical” regimens. Conclusion In general, HAART is more cost-effective than other therapeutic regimens adopted so far. Further investigations, especially head-to-head comparisons of “ideal” and “typical” trials of different regimen combinations, are required to identify the optimal HAART regimens. PMID:26316787

  8. HIV transmission and 24-month survival in a randomized trial of HAART to prevent MTCT during pregnancy and breastfeeding in Botswana (The Mma Bana Study)

    PubMed Central

    Shapiro, Roger L.; Kitch, Douglas; Ogwu, Anthony; Hughes, Michael D.; Lockman, Shahin; Powis, Kathleen; Souda, Sajini; Moffat, Claire; Moyo, Sikhulile; McIntosh, Kenneth; van Widenfelt, Erik; Zwerski, Sheryl; Mazhani, Loeto; Makhema, Joseph; Essex, Max

    2014-01-01

    Objectives Highly active antiretroviral therapy (HAART) for prevention of mother-to-child HIV transmission (MTCT) may impact long-term survival of mothers and children. Design Randomized clinical trial. Methods HIV–infected pregnant women with CD4 ≥200 cells/mm3 were randomly assigned to abacavir, zidovudine, lamivudine (Arm A) or lopinavir–ritonavir, zidovudine–lamivudine (Arm B) from week 26–34 gestation through planned weaning by 6 months postpartum. Women with baseline CD4 <200 received nevirapine–zidovudine–lamivudine indefinitely (Obs arm), as did randomized women later qualifying for treatment. Results Among 560 randomized and 170 observational women enrolled, there were 14 deaths (1.9%); 1 antenatally (Obs), 3 from delivery though 6 months postpartum (1 Arm A, 2 Obs), and 10 from 6–24 months postpartum (5 Arm A, 3 Arm B, 2 Obs). Time to death or CD4 <200 was shorter in Arm A vs. B (p=0.03). Of 709 live-born children, 97% breastfed for median 5.8 months. Of 37 (5.2%) deaths by 24 months, 9 were before breastfeeding initiated (3 Arm A, 2 Arm B, 4 Obs); 6 while breastfeeding (1 Arm A, 2 Arm B, 3 Obs); and 22 after weaning (9 Arm A, 11 Arm B, 2 Obs). Only 8 children (1.1%) were HIV-infected at 24 months (6 Arm A, 1 Arm B, 1 Obs), all before 6 months. Conclusions Low MTCT was maintained through extended follow-up in all arms. Disease progression appeared slower after discontinuing protease inhibitor-based HAART, but a concerning number of maternal deaths occurred after stopping either regimen. Strategies to improve maternal and child survival in the post-intervention period are required. PMID:24180000

  9. Intestinal Parasitosis in Relation to CD4+T Cells Levels and Anemia among HAART Initiated and HAART Naive Pediatric HIV Patients in a Model ART Center in Addis Ababa, Ethiopia

    PubMed Central

    Mengist, Hylemariam Mihiretie; Taye, Bineyam; Tsegaye, Aster

    2015-01-01

    Background Intestinal parasites (IPs) are major concerns in most developing countries where HIV/AIDS cases are concentrated and almost 80% of AIDS patients die of AIDS-related infections. In the absence of highly active antiretroviral therapy (HAART), HIV/AIDS patients in developing countries unfortunately continue to suffer from the consequences of opportunistic and other intestinal parasites. The aim of the study was to determine the prevalence of intestinal parasites in relation to CD4+ T cells levels and anemia among HAART initiated and HAART naïve pediatric HIV patients in a Model ART center in Addis Ababa, Ethiopia. Methods A prospective comparative cross-sectional study was conducted among HAART initiated and HAART naive pediatric HIV/AIDS patients attending a model ART center at Zewditu Memorial Hospital between August 05, 2013 and November 25, 2013. A total of 180 (79 HAART initiated and 101 HAART naïve) children were included by using consecutive sampling. Stool specimen was collected and processed using direct wet mount, formol-ether concentration and modified Ziehl-Neelsen staining techniques. A structured questionnaire was used to collect data on socio-demographic and associated risk factors. CD4+ T cells and complete blood counts were performed using BD FACScalibur and Cell-Dyn 1800, respectively. The data was analyzed by SPSS version 16 software. Logistic regressions were applied to assess any association between explanatory factors and outcome variables. P values < 0.05 were taken as statistically significant. Results The overall prevalence of IPs was 37.8% where 27.8% of HAART initiated and 45.5% of HAART naive pediatric HIV/AIDS patients were infected (p < 0.05). Cryptosporidium species, E. histolytica/dispar, Hook worm and Taenia species were IPs associated with CD4+ T cell counts <350 cells/μμL in HAART naive patients. The overall prevalence of anemia was 10% in HAART and 31.7% in non-HAART groups. Hook worm, S. stercoralis and H. nana were

  10. Discovery of MK-5172, a Macrocyclic Hepatitis C Virus NS3/4a Protease Inhibitor

    PubMed Central

    2012-01-01

    A new class of HCV NS3/4a protease inhibitors containing a P2 to P4 macrocyclic constraint was designed using a molecular modeling-derived strategy. Building on the profile of previous clinical compounds and exploring the P2 and linker regions of the series allowed for optimization of broad genotype and mutant enzyme potency, cellular activity, and rat liver exposure following oral dosing. These studies led to the identification of clinical candidate 15 (MK-5172), which is active against genotype 1–3 NS3/4a and clinically relevant mutant enzymes and has good plasma exposure and excellent liver exposure in multiple species. PMID:24900473

  11. Discovery of MK-5172, a Macrocyclic Hepatitis C Virus NS3/4a Protease Inhibitor.

    PubMed

    Harper, Steven; McCauley, John A; Rudd, Michael T; Ferrara, Marco; DiFilippo, Marcello; Crescenzi, Benedetta; Koch, Uwe; Petrocchi, Alessia; Holloway, M Katharine; Butcher, John W; Romano, Joseph J; Bush, Kimberly J; Gilbert, Kevin F; McIntyre, Charles J; Nguyen, Kevin T; Nizi, Emanuela; Carroll, Steven S; Ludmerer, Steven W; Burlein, Christine; DiMuzio, Jillian M; Graham, Donald J; McHale, Carolyn M; Stahlhut, Mark W; Olsen, David B; Monteagudo, Edith; Cianetti, Simona; Giuliano, Claudio; Pucci, Vincenzo; Trainor, Nicole; Fandozzi, Christine M; Rowley, Michael; Coleman, Paul J; Vacca, Joseph P; Summa, Vincenzo; Liverton, Nigel J

    2012-04-12

    A new class of HCV NS3/4a protease inhibitors containing a P2 to P4 macrocyclic constraint was designed using a molecular modeling-derived strategy. Building on the profile of previous clinical compounds and exploring the P2 and linker regions of the series allowed for optimization of broad genotype and mutant enzyme potency, cellular activity, and rat liver exposure following oral dosing. These studies led to the identification of clinical candidate 15 (MK-5172), which is active against genotype 1-3 NS3/4a and clinically relevant mutant enzymes and has good plasma exposure and excellent liver exposure in multiple species.

  12. Increased regression and decreased incidence of human papillomavirus-related cervical lesions among HIV-infected women on HAART

    PubMed Central

    Adler, David H.; Kakinami, Lisa; Modisenyane, Tebogo; Tshabangu, Nkeko; Mohapi, Lerato; De Bruyn, Guy; Martinson, Neil A.; Omar, Tanvier

    2013-01-01

    Objective To determine the impact of HAART on incidence, regression, and progression of cytopathological abnormalities in HIV-infected women. Design Prospective cohort. Methods HIV-infected women (N=1123) from Soweto, South Africa underwent serial cervical smears that were analyzed and reported using the Bethesda System. The results of HAART and non-HAART users were compared using two statistical approaches: a survival analysis assessing risk of incident smear abnormality among women with baseline normal smear results; and analysis with marginal models assessing for an association between HAART use and likelihood of regression/progression in consecutive smears. Results After multivariate survival analysis, women using HAART with a normal baseline smear were 38% less likely to have an incident smear abnormality during follow-up than nonusers [confidence interval (CI) 0.42–0.91; P=0.01]. Multivariate marginal models analysis identified a significantly increased likelihood (odds ratio 2.61; CI 1.75–3.89; P< 0.0001) of regression of cervical lesions among women on HAART. Conclusion Our large prospective cohort study adds significant weight to the side of the balance of clinical research supporting the positive impact of HAART on the natural history of human papillomavirus-related cervical disease in HIV-infected women. PMID:22555167

  13. Time to HAART Initiation after Diagnosis and Treatment of Opportunistic Infections in Patients with AIDS in Latin America

    PubMed Central

    Crabtree-Ramírez, Brenda; Caro-Vega, Yanink; Shepherd, Bryan E.; Grinsztejn, Beatriz; Wolff, Marcelo; Cortes, Claudia P.; Padgett, Denis; Carriquiry, Gabriela; Fink, Valeria; Jayathilake, Karu; Person, Anna K.; McGowan, Catherine; Sierra-Madero, Juan

    2016-01-01

    Background Since 2009, earlier initiation of highly active antiretroviral therapy (HAART) after an opportunistic infection (OI) has been recommended based on lower risks of death and AIDS-related progression found in clinical trials. Delay in HAART initiation after OIs may be an important barrier for successful outcomes in patients with advanced disease. Timing of HAART initiation after an OI in “real life” settings in Latin America has not been evaluated. Methods Patients in the Caribbean, Central and South America network for HIV Epidemiology (CCASAnet) ≥18 years of age at enrolment, from 2001–2012 who had an OI before HAART initiation were included. Patients were divided in an early HAART (EH) group (those initiating within 4 weeks of an OI) and a delayed HAART (DH) group (those initiating more than 4 weeks after an OI). All patients with an AIDS-defining OI were included. In patients with more than one OI the first event reported was considered. Calendar trends in the proportion of patients in the EH group (before and after 2009) were estimated by site and for the whole cohort. Factors associated with EH were estimated using multivariable logistic regression models. Results A total of 1457 patients had an OI before HAART initiation and were included in the analysis: 213 from Argentina, 686 from Brazil, 283 from Chile, 119 from Honduras and 156 from Mexico. Most prevalent OI were Tuberculosis (31%), followed by Pneumocystis pneumonia (24%), Invasive Candidiasis (16%) and Toxoplasmosis (9%). Median time from OI to HAART initiation decreased significantly from 5.7 (interquartile range [IQR] 2.8–12.1) weeks before 2009 to 4.3 (IQR 2.0–7.1) after 2009 (p<0.01). Factors associated with starting HAART within 4 weeks of OI diagnosis were lower CD4 count at enrolment (p-<0.001), having a non-tuberculosis OI (p<0.001), study site (p<0.001), and more recent years of OI diagnosis (p<0.001). Discussion The time from diagnosis of an OI to HAART initiation has

  14. Timing of HAART Initiation and Clinical Outcomes among HIV-1 Seroconverters

    PubMed Central

    Funk, Michele Jonsson; Fusco, Jennifer S; Cole, Stephen R; Thomas, James C; Porter, Kholoud; Kaufman, Jay S; Davidian, Marie; White, Alice D; Hartmann, Katherine E; Eron, Joseph J

    2013-01-01

    Background To estimate the clinical benefit of HAART initiation versus deferral in a given month among patients with CD4 counts <800 cells/µL. Methods In this observational cohort study of HIV-1 seroconverters from CASCADE, we constructed monthly sequential nested subcohorts from 1/1996 to 5/2009 including all eligible HAART-naïve, AIDS-free individuals with a CD4 count <800 cells/uL. The primary outcome was time to AIDS or death among those who initiated HAART in the baseline month compared to those who did not, pooled across subcohorts and stratified by CD4. Using inverse-probability-of-treatment-weighted survival curves and Cox proportional hazards models, we estimated the absolute and relative effect of treatment with robust 95% confidence intervals (in parentheses). Results Of 9,455 patients with 52,268 person-years of follow-up, 812 (8.6%) developed AIDS and 544 (5.8%) died. Within CD4 strata of 200–349, 350–499, and 500–799 cells/µL, HAART initiation was associated with adjusted hazard ratios for AIDS/death of 0.59 (0.43,0.81), 0.75 (0.49,1.14), and 1.10 (0.67,1.79), respectively; and with adjusted 3-year cumulative risk differences of −4.8% (−7.0%,−2.6%), −2.9% (−5.0%,−0.9%), and 0.3% (−3.7%,4.2%), respectively. In the analysis of all-cause mortality, HAART initiation was associated with adjusted hazard ratios of 0.71 (0.44,1.15), 0.51 (0.33,0.80) and 1.02 (0.49,2.12), respectively. Numbers needed to treat to prevent one AIDS event or death within 3 years were 21 (14,38) and 34 (20,115) in CD4 strata of 200–349 and 350–499 cells/µL, respectively. Conclusions Compared to deferring in a given month, HAART initiation at CD4 counts <500 (but not 500–799) cells/µL was associated with slower disease progression. PMID:21949165

  15. Study of T Cell subsets and IL-7 protein expression in HIV-1-infected patients after 7 years HAART

    PubMed Central

    2011-01-01

    Objective To study the changes in T cell subsets and IL-7 in HIV-1-infected patients after seven years of highly active antiretroviral therapy (HAART). Methods Seventy-five individuals were included in this study (25 with effective HAART, 18 with ineffective HAART, 17 untreated HIV+ patients, and 15 volunteers in the HIV negative control group). The counts of CD4+, CD8+, CD8/CD38+, and CD8/HLADR+ T cells as well as the IL-7 protein expression was measured at 5 time points during a period of seven years in patients starting HAART (baseline) and in the HIV negative control group. The expression of CD127 on CD3+ T cells was measured by flow cytometry at a single time point (after 7 years) in patients with HAART and was compared with untreated HIV+ patients and the HIV negative control group. Results At baseline CD4+ T cell counts of HIV-1-infected patients were lower than that in the control group (p < 0.01), whereas the CD8+, CD8/HLADR+ and CD8/CD38+ T cell counts were higher than those in the control group (p <0.01). After seven years of effective HAART, the CD4+ T cell counts had increased and the CD8+ T cell count had decreased, although not to the normal levels (p < 0.05). Both the CD8/HLADR+ and CD8/CD38+ T cell counts had gradually approached those of the control group (p > 0.05). In the ineffective HAART group, the CD8/CD38+ T cell count had not decreased significantly, and CD8/HLADR+ T cell count gradually decreased. Before treatment, IL-7 serum levels of patients were significantly higher than that in the control group (p < 0.01). After seven years of effective HAART, IL-7 levels had gradually decreased, but were still higher than in the control group (p < 0.01). The CD127 expression on CD3+ CD8+ T cells in effective HAART patients was higher than in untreated HIV+ patients (p < 0.05), but was lower than that in the control group (p < 0.05). CD127 expression on CD3+ CD4+ T cells was not significantly different among the control group, untreated HIV+ patients

  16. Prognosis of HIV-1-infected patients up to 5 years after initiation of HAART: collaborative analysis of prospective studies

    PubMed Central

    2012-01-01

    Objective To estimate the prognosis over 5 years of HIV-1-infected, treatment-naive patients starting HAART, taking into account the immunological and virological response to therapy. Design A collaborative analysis of data from 12 cohorts in Europe and north America on 20 379 adults who started HAART between 1995 and 2003. Methods Parametric survival models were used to predict the cumulative incidence at 5 years of a new AIDS-defining event or death, and death alone, first from the start of HAART and second from 6 months after the start of HAART. Data were analysed by intention-to-continue-treatment, ignoring treatment changes and interruptions. Results During 61 798 person-years of follow-up, 1005 patients died and an additional 1303 developed AIDS. A total of 10 046 (49%) patients started HAART either with a CD4 cell count of less than 200 cells/μl or with a diagnosis of AIDS. The 5-year risk of AIDS or death (death alone) from the start of HAART ranged from 5.6 to 77% (1.8–65%), depending on age, CD4 cell count, HIV-1-RNA level, clinical stage, and history of injection drug use. From 6 months the corresponding figures were 4.1–99% for AIDS or death and 1.3–96% for death alone. Conclusion On the basis of data collected routinely in HIV care, prognostic models with high discriminatory power over 5 years were developed for patients starting HAART in industrialized countries. A risk calculator that produces estimates for progression rates at years 1 to 5 after starting HAART is available from www.art-cohort-collaboration.org. PMID:17502729

  17. The Lon AAA+ protease.

    PubMed

    Gur, Eyal

    2013-01-01

    As the first ATP-dependent protease to be identified, Lon holds a special place in the history of cellular biology. In fact, the concept of ATP-dependent protein degradation was established through the findings that led to the discovery of Lon. Therefore, this chapter begins with a historical perspective, describing the milestones that led to the discovery of Lon and ATP-dependent proteolysis, starting from the early findings in the 1960s until the demonstration of Lon's ATP-dependent proteolytic activity in vitro, in 1981. Most of our knowledge on Lon derives from studies of the Escherichia coli Lon ortholog, and, therefore, most of this chapter relates to this particular enzyme. Nonetheless, Lon is not only found in most bacterial species, it is also found in Archaea and in the mitochondrion and chloroplast of eukaryotic cells. Therefore many of the conclusions gained from studies on the E. coli enzyme are relevant to Lon proteases in other organisms. Lon, more than any other bacterial or organellar protease, is associated with the degradation of misfolded proteins and protein quality control. In addition, Lon also degrades many regulatory proteins that are natively folded, thus it also plays a prominent role in regulation of physiological processes. Throughout the years, many Lon substrates have been identified, confirming its role in the regulation of diverse cellular processes, including cell division, DNA replication, differentiation, and adaptation to stress conditions. Some examples of these functions are described and discussed here, as is the role of Lon in the degradation of misfolded proteins and in protein quality control. Finally, this chapter deals with the exquisite sensitivity of protein degradation inside a cell. How can a protease distinguish so many substrates from cellular proteins that should not be degraded? Can the specificity of a protease be regulated according to the physiological needs of a cell? This chapter thus broadly discusses the

  18. Laundry performance of subtilisin proteases.

    PubMed

    Wolff, A M; Showell, M S; Venegas, M G; Barnett, B L; Wertz, W C

    1996-01-01

    Effective laundry protease performance against susceptible stains depends upon both the enzyme itself and the environment in which it must work. In order to technically design superior laundry proteases, a model for protease's mechanism of action in detergents was developed which has been substantiated through-the-wash. While evaluation of this model and/or a given protease's effectiveness could be judged by a variety of methods, the utility of using visual wash performance comparisons, analytical, and stain characterization studies is described. Finally, data comparing the performance of wild type Subtilisin proteases with mutants designed via the projected model are given, demonstrating possible utility of the system.

  19. Mutations Related to Antiretroviral Resistance Identified by Ultra-Deep Sequencing in HIV-1 Infected Children under Structured Interruptions of HAART

    PubMed Central

    Vazquez-Guillen, Jose Manuel; Palacios-Saucedo, Gerardo C.; Rivera-Morales, Lydia G.; Garcia-Campos, Jorge; Ortiz-Lopez, Rocio; Noguera-Julian, Marc; Paredes, Roger; Vielma-Ramirez, Herlinda J.; Ramirez, Teresa J.; Chavez-Garcia, Marcelino; Lopez-Guillen, Paulo; Briones-Lara, Evangelina; Sanchez-Sanchez, Luz M.; Vazquez-Martinez, Carlos A.; Rodriguez-Padilla, Cristina

    2016-01-01

    Although Structured Treatment Interruptions (STI) are currently not considered an alternative strategy for antiretroviral treatment, their true benefits and limitations have not been fully established. Some studies suggest the possibility of improving the quality of life of patients with this strategy; however, the information that has been obtained corresponds mostly to studies conducted in adults, with a lack of knowledge about its impact on children. Furthermore, mutations associated with antiretroviral resistance could be selected due to sub-therapeutic levels of HAART at each interruption period. Genotyping methods to determine the resistance profiles of the infecting viruses have become increasingly important for the management of patients under STI, thus low-abundance antiretroviral drug-resistant mutations (DRM’s) at levels under limit of detection of conventional genotyping (<20% of quasispecies) could increase the risk of virologic failure. In this work, we analyzed the protease and reverse transcriptase regions of the pol gene by ultra-deep sequencing in pediatric patients under STI with the aim of determining the presence of high- and low-abundance DRM’s in the viral rebounds generated by the STI. High-abundance mutations in protease and high- and low-abundance mutations in reverse transcriptase were detected but no one of these are directly associated with resistance to antiretroviral drugs. The results could suggest that the evaluated STI program is virologically safe, but strict and carefully planned studies, with greater numbers of patients and interruption/restart cycles, are still needed to evaluate the selection of DRM’s during STI. PMID:26807922

  20. From proteases to proteomics.

    PubMed

    Neurath, H

    2001-04-01

    This personal and professional autobiography covers the 50-yr period of 1950-2000 and includes the following topics: History of the University of Washington School of Medicine and its Department of Biochemistry (Mount Rainier and the University of Washington, recruiting faculty, biology, research programs); scientific editing (publication, Biochemistry, Protein Science, electronic publication); Europe revisited (Heidelberg, approaching retirement, the German Research Center, reunion in Vienna); and 50 yr of research on proteolytic enzymes (trypsin, carboxypeptidases, mast cell proteases, future developments).

  1. From proteases to proteomics

    PubMed Central

    Neurath, Hans

    2001-01-01

    This personal and professional autobiography covers the 50-yr period of 1950–2000 and includes the following topics: History of the University of Washington School of Medicine and its Department of Biochemistry (Mount Rainier and the University of Washington, recruiting faculty, biology, research programs); scientific editing (publication, Biochemistry, Protein Science, electronic publication); Europe revisited (Heidelberg, approaching retirement, the German Research Center, reunion in Vienna); and 50 yr of research on proteolytic enzymes (trypsin, carboxypeptidases, mast cell proteases, future developments). PMID:11274481

  2. Protease signalling: the cutting edge

    PubMed Central

    Turk, Boris; Turk, Dus̆an; Turk, Vito

    2012-01-01

    Protease research has undergone a major expansion in the last decade, largely due to the extremely rapid development of new technologies, such as quantitative proteomics and in-vivo imaging, as well as an extensive use of in-vivo models. These have led to identification of physiological substrates and resulted in a paradigm shift from the concept of proteases as protein-degrading enzymes to proteases as key signalling molecules. However, we are still at the beginning of an understanding of protease signalling pathways. We have only identified a minor subset of true physiological substrates for a limited number of proteases, and their physiological regulation is still not well understood. Similarly, links with other signalling systems are not well established. Herein, we will highlight current challenges in protease research. PMID:22367392

  3. Community-based treatment of advanced HIV disease: introducing DOT-HAART (directly observed therapy with highly active antiretroviral therapy).

    PubMed Central

    Farmer, P.; Léandre, F.; Mukherjee, J.; Gupta, R.; Tarter, L.; Kim, J. Y.

    2001-01-01

    In 2000, acquired immunodeficiency syndrome (AIDS) overtook tuberculosis (TB) as the world's leading infectious cause of adult deaths. In affluent countries, however, AIDS mortality has dropped sharply, largely because of the use of highly active antiretroviral therapy (HAART). Antiretroviral agents are not yet considered essential medications by international public health experts and are not widely used in the poor countries where human immunodeficiency virus (HIV) takes its greatest toll. Arguments against the use of HAART have mainly been based on the high cost of medications and the lack of the infrastructure necessary for using them wisely. We re- examine these arguments in the setting of rising AIDS mortality in developing countries and falling drug prices, and describe a small community-based treatment programme based on lessons gained in TB control. With the collaboration of Haitian community health workers experienced in the delivery of home-based and directly observed treatment for TB, an AIDS-prevention project was expanded to deliver HAART to a subset of HIV patients deemed most likely to benefit. The inclusion criteria and preliminary results are presented. We conclude that directly observed therapy (DOT) with HAART, "DOT-HAART", can be delivered effectively in poor settings if there is an uninterrupted supply of high-quality drugs. PMID:11799447

  4. Protease-mediated drug delivery

    NASA Astrophysics Data System (ADS)

    Dickson, Eva F.; Goyan, Rebecca L.; Kennedy, James C.; Mackay, M.; Mendes, M. A. K.; Pottier, Roy H.

    2003-12-01

    Drugs used in disease treatment can cause damage to both malignant and normal tissue. This toxicity limits the maximum therapeutic dose. Drug targeting is of high interest to increase the therapeutic efficacy of the drug without increasing systemic toxicity. Certain tissue abnormalities, disease processes, cancers, and infections are characterized by high levels of activity of specific extracellular and/or intracellular proteases. Abnormally high activity levels of specific proteases are present at sites of physical or chemical trauma, blood clots, malignant tumors, rheumatoid arthritis, inflammatory bowel disease, gingival disease, glomerulonerphritis, and acute pancreatitis. Abnormal protease activity is suspected in development of liver thrombosis, pulmonary emphysema, atherosclerosis, and muscular dystrophy. Inactiviating disease-associated proteases by the administration of appropriate protease inhibitors has had limited success. Instead, one could use such proteases to target drugs to treat the condition. Protease mediated drug delivery offers such a possibility. Solubilizing groups are attached to insoluble drugs via a polypeptide chain which is specifically cleavable by certian proteases. When the solubilized drug enounters the protease, the solubilizing moieties are cleaved, and the drug precipitates at the disease location. Thus, a smaller systemic dosage could result in a therapeutic drug concentration at the treatment site with less systemic toxicity.

  5. Brain viral burden, neuroinflammation and neurodegeneration in HAART-treated HIV positive injecting drug users.

    PubMed

    Smith, Donald B; Simmonds, Peter; Bell, Jeanne E

    2014-02-01

    The long-term impact of chronic human immunodeficiency virus (HIV) infection on brain status in injecting drug users (IDU) treated with highly active antiretroviral therapy (HAART) is unknown. Viral persistence in the brain with ongoing neuroinflammation may predispose to Alzheimer-like neurodegeneration. In this study, we investigated the brains of ten HAART-treated individuals (six IDU and four non-DU), compared with ten HIV negative controls (six IDU and four non-DU). HIV DNA levels in brain tissue were correlated with plasma and lymphoid tissue viral loads, cognitive status, microglial activation and Tau protein and amyloid deposition. Brain HIV proviral DNA levels were low in most cases but higher in HIV encephalitis (n = 2) and correlated significantly with levels in lymphoid tissue (p = 0.0075), but not with those in plasma. HIV positive subjects expressed more Tau protein and amyloid than HIV negative controls (highest in a 58 year old), as did IDU, but brain viral loads showed no relation to Tau and amyloid. Microglial activation linked significantly to HIV positivity (p = 0.001) and opiate abuse accentuated these microglial changes (p = 0.05). This study confirms that HIV DNA persists in brains despite HAART and that opiate abuse adds to the risk of brain damage in HIV positive subjects. Novel findings in this study show that (1) plasma levels are not a good surrogate indicator of brain status, (2) viral burden in brain and lymphoid tissues is related, and (3) while Tau and amyloid deposition is increased in HIV positive IDU, this is not specifically related to increased HIV burden within the brain.

  6. HIV-Associated Lung Cancer in the Era of Highly Active Antiretroviral Therapy (HAART)

    PubMed Central

    Pakkala, Suchita; Chen, Zhengjia; Rimland, David; Owonikoko, Taofeek K.; Gunthel, Clifford; Brandes, Johann R.; Saba, Nabil R.; Shin, Dong M.; Curran, Walter J.; Khuri, Fadlo R.; Ramalingam, Suresh S.

    2011-01-01

    Background Lung cancer is the leading cause of death among non-acquired immunodeficiency syndrome (AIDS) defining malignancies. Since highly active anti-retroviral therapy (HAART) has improved survival for human immunodeficiency virus (HIV) patients, we evaluated lung cancer outcomes in the HAART era. Methods HIV-positive patients diagnosed with lung cancer in our institution during the HAART era (1995-2008) were analyzed. Patient charts were reviewed for clinical and laboratory data. CD4 count at diagnosis was treated as a continuous variable and subcategorized into distinct variables with 3 cut-off points (50, 200, & 500 μl). Pearson’s correlation coefficients were estimated for each covariate studied. Survival was determined by the Kaplan-Meier method. Results Out of 80 patients, 73 had non-small cell lung cancer. Baseline characteristics were: median age-52 yrs; male-80%; African American-84%; injection drug use-25%; smokers-100%; and prior exposure to antiretroviral agents-55%. Mean CD4 count and viral load were 304 μL and 82,420 copies/ml, respectively at cancer diagnosis. The latency between diagnosis of HIV and lung cancer was significantly shorter in women (4.1 yrs vs. 7.7 yrs, P=0.02) and 71% of the patients received anti-cancer therapy. The 1- and 3-year survival rates were 31% and 4% overall. Grade 3/4 toxicities occurred in 60% with chemo-radiation vs. 36% with chemotherapy. Cancer-related survival was better for patients with CD4 count >200 (P=0.0298) and >500 (P=0.0076). Conclusions The latency from diagnosis of HIV to lung cancer was significantly shorter for women. Although outcomes for lung cancer patients with HIV remain poor, high CD4 count is associated with an improved lung cancer-related survival. PMID:21713759

  7. Social grants, welfare, and the incentive to trade-off health for income among individuals on HAART in South Africa.

    PubMed

    Venkataramani, Atheendar S; Maughan-Brown, Brendan; Nattrass, Nicoli; Ruger, Jennifer Prah

    2010-12-01

    South Africa's government disability grants are considered important in providing income support to low-income AIDS patients. Indeed, anecdotal evidence suggests that some individuals may opt to compromise their health by foregoing Highly Active Antiretroviral Treatment (HAART) to remain eligible for the grant. In this study, we examined the disability grant's importance to individual and household welfare, and the impact of its loss using a unique longitudinal dataset of HAART patients in Khayelitsha, Cape Town. We found that grant loss was associated with sizeable declines in income and changes in household composition. However, we found no evidence of individuals choosing poor health over grant loss. Our analysis also suggested that though the grants officially target those too sick to work, some people were able to keep grants longer than expected, and others received grants while employed. This has helped cushion people on HAART, but other welfare measures need consideration.

  8. Impact of HAART and CNS-penetrating antiretroviral regimens on HIV encephalopathy among perinatally infected children and adolescents

    PubMed Central

    Patel, Kunjal; Ming, Xue; Williams, Paige L.; Robertson, Kevin R.; Oleske, James M.; Seage, George R.

    2010-01-01

    Objectives Prior to antiretroviral treatment, HIV-infected children frequently developed encephalopathy, resulting in debilitating morbidity and mortality. This is the first large study to evaluate the impact of HAART and central nervous system (CNS)-penetrating antiretroviral regimens on the incidence of HIV encephalopathy and survival after diagnosis of HIV encephalopathy among perinatally infected children. Design A total of 2398 perinatally HIV-infected children with at least one neurological examination were followed in a US-based prospective cohort study conducted from 1993 to 2007. Methods Trends in incidence rates over calendar time were described and Cox regression models were used to estimate the effects of time-varying HAART and CNS-penetrating antiretroviral regimens on HIV encephalopathy and on survival after diagnosis of HIV encephalopathy. Results During a median of 6.4 years of follow-up, 77 incident cases of HIV encephalopathy occurred [incidence rate 5.1 cases per 1000 person-years, 95% confidence interval (CI) 4.0–6.3]. A 10-fold decline in incidence was observed beginning in 1996, followed by a stable incidence rate after 2002. HAART regimens were associated with a 50% decrease (95% CI 14–71%) in the incidence of HIV encephalopathy compared with non-HAART regimens. High CNS-penetrating regimens were associated with a substantial survival benefit (74% reduction in the risk of death, 95% CI 39–89%) after HIV encephalopathy diagnosis compared with low CNS-penetrating regimens. Conclusion A dramatic decrease in the incidence of HIV encephalopathy occurred after the introduction of HAART. The use of HAART was highly effective in reducing the incidence of HIV encephalopathy among perinatally infected children and adolescents. Effective CNS-penetrating antiretroviral regimens are important in affecting survival after diagnosis of HIV encephalopathy. PMID:19644348

  9. Protease degradable electrospun fibrous hydrogels

    PubMed Central

    Wade, Ryan J.; Bassin, Ethan J.; Rodell, Christopher B.; Burdick, Jason A.

    2015-01-01

    Electrospun nanofibers are promising in biomedical applications to replicate features of the natural extracellular matrix (ECM). However, nearly all electrospun scaffolds are either non-degradable or degrade hydrolytically, whereas natural ECM degrades proteolytically, often through matrix metalloproteinases (MMPs). Here, we synthesize reactive macromers that contain protease-cleavable and fluorescent peptides and are able to form both isotropic hydrogels and electrospun fibrous hydrogels through a photoinitiated polymerization. These biomimetic scaffolds are susceptible to protease-mediated cleavage in vitro in a protease dose dependent manner and in vivo in a subcutaneous mouse model using transdermal fluorescent imaging to monitor degradation. Importantly, materials containing an alternate and non-protease-cleavable peptide sequence are stable in both in vitro and in vivo settings. To illustrate the specificity in degradation, scaffolds with mixed fiber populations support selective fiber degradation based on individual fiber degradability. Overall, this represents a novel biomimetic approach to generate protease-sensitive fibrous scaffolds for biomedical applications. PMID:25799370

  10. Viral Load in Spanish HIV patients: trends since the introduction of HAART.

    PubMed

    Eiros, J M; Sánchez-Padilla, E; Luquero, F J; Nogueira, B; Rojo, S; Atienza-Herrero, J; Ortiz de Lejarazu, R

    2009-03-01

    The aim of this study is to describe trends in the percentage of samples with undetectable HIV viral load in Spain after the implementation of HAART. A descriptive observational study of HIV-VL measurements carried out in the microbiology department of the Hospital Clínico Universitario de Valladolid (HCUV) was conducted over a 9-year period (1996-2004). Regarding the trend over the study period, the 30-39 years age group accounted for most of the samples, although the percentage decreased from 65.5% to 59.6% over the study period. In contrast, the 40-49 years group increased from 9.1% to 14.5%. The preponderance of men, with percentages above 70%, was observed during the whole period. Although the purpose of this treatment is to maintain undetectable viral loads, since 1999 more than 60% of nonfirst samples had detectable levels. Based on the results of the VL trend among HIV/AIDS patients observed in this study, a large number of patients maintain elevated detectable VL years after HAART was implemented. Although different factors may be the cause of this and should be delimited in future studies, the phenomenon observed demonstrates the usefulness of monitoring VL and analyzing its time trend to gain further knowledge about the therapeutic results and care of HIV patients as a whole, also serving as the basis for corrective measures.

  11. [The in vitro HAART pharmacodynamics study with dolutegravir as the "anchor"].

    PubMed

    Guo, Jia-mei; Guo, Ying

    2015-01-01

    This study is to evaluate the HAART pharmacodynamics with dolutegravir as the "anchor" in vitro. A nucleoside reverse transcriptase inhibitors (NRTIs) resistant recombinant virus model (VSVG/HIV-1(RT-D67N,K70R,T215F)) and an integrase inhibitors (INIs) resistant recombinant virus model (VSVG/HIV-1(IN-G140S,QI48H)) were constructed and established. The anti-viral pharmacodynamics was evaluated with drug combinations including two NRTIs along with one INI or one NNRTI. The results showed that the combination with an INI gave a stronger synergism on wild type HIV-1 replication comparing to that with an NNRTI. Comparing the two INIs as the "anchor" for HAART, DTG exhibited an equivalent CI to that of RAL on wild type HIV-1 replication; but a greater synergy than RAL on INI-resistant HIV-1 replication. Besides of the pharmacodynamics results of DTG-based drug combination, the results may contribute to clinical antiviral therapy.

  12. Crystal Structures of a Multidrug-Resistant Human Immunodeficiency Virus Type 1 Protease Reveal an Expanded Active-Site Cavity

    SciTech Connect

    Logsdon, Bradley C.; Vickrey, John F.; Martin, Philip; Proteasa, Gheorghe; Koepke, Jay I.; Terlecky, Stanley R.; Wawrzak, Zdzislaw; Winters, Mark A.; Merigan, Thomas C.; Kovari, Ladislau C.

    2010-03-08

    The goal of this study was to use X-ray crystallography to investigate the structural basis of resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors. We overexpressed, purified, and crystallized a multidrug-resistant (MDR) HIV-1 protease enzyme derived from a patient failing on several protease inhibitor-containing regimens. This HIV-1 variant contained codon mutations at positions 10, 36, 46, 54, 63, 71, 82, 84, and 90 that confer drug resistance to protease inhibitors. The 1.8-{angstrom} crystal structure of this MDR patient isolate reveals an expanded active-site cavity. The active-site expansion includes position 82 and 84 mutations due to the alterations in the amino acid side chains from longer to shorter (e.g., V82A and I84V). The MDR isolate 769 protease 'flaps' stay open wider, and the difference in the flap tip distances in the MDR 769 variant is 12 {angstrom}. The MDR 769 protease crystal complexes with lopinavir and DMP450 reveal completely different binding modes. The network of interactions between the ligands and the MDR 769 protease is completely different from that seen with the wild-type protease-ligand complexes. The water molecule-forming hydrogen bonds bridging between the two flaps and either the substrate or the peptide-based inhibitor are lacking in the MDR 769 clinical isolate. The S1, S1', S3, and S3' pockets show expansion and conformational change. Surface plasmon resonance measurements with the MDR 769 protease indicate higher k{sub off} rates, resulting in a change of binding affinity. Surface plasmon resonance measurements provide k{sub on} and k{sub off} data (K{sub d} = k{sub off}/k{sub on}) to measure binding of the multidrug-resistant protease to various ligands. This MDR 769 protease represents a new antiviral target, presenting the possibility of designing novel inhibitors with activity against the open and expanded protease forms.

  13. Substance use and HIV disease progression in the HAART era: implications for the primary prevention of HIV.

    PubMed

    Carrico, Adam W

    2011-05-23

    Prior to the era of highly active anti-retroviral therapy (HAART), cohort studies provided equivocal evidence to support the hypothesis that substance use predicts more rapid HIV disease progression. The present review examined the effects of substance use on HIV disease progression in cohort studies with follow-up that continued into the HAART era. Of the 20 studies included in this review, 16 observed that substance use predicted at least one indicator of HIV disease progression. Ten of the 11 studies that followed participants exclusively in the HAART era observed an effect of substance use on HIV disease progression. Findings across studies indicate that stimulant use promotes more rapid HIV disease progression and the effects of substance use on HIV disease progression can persist after controlling for self-reported HAART non-adherence. Future investigations that examine the bio-behavioral pathways whereby substance use promotes HIV disease progression should include: measures of HIV genotypic and phenotypic resistance, multi-method assessment of adherence, and assessment of co-morbid infections that are more prevalent among substance users. Although further mechanistic research is needed, findings from existing cohort studies have clear clinical implications. Implementing screening, brief intervention and referral to substance abuse treatment in HIV medical care could optimize health outcomes and decrease HIV transmission rates by boosting the effectiveness of "Test and Treat" approaches to HIV prevention.

  14. The neurodevelopment of HIV-infected infants on HAART compared to HIV-exposed but uninfected infants.

    PubMed

    Whitehead, Nicole; Potterton, Joanne; Coovadia, Ashraf

    2014-04-01

    The aim of this study was to compare the neurodevelopment of HIV-infected (HI) infants in combination with antiretroviral therapy also known as HAART (highly active antiretroviral therapy) to HIV-exposed uninfected (HEU) infants. Twenty-seven HIV infected and 29 HEU infants under the age of one year attending the Empilweni Clinic at Rahima Moosa Mother and Child Hospital were studied. HI infants were assessed prior to initiating HAART and then for six months whilst on HAART. Neurodevelopment was assessed using the Bayley Scales of Infant and Toddler Development, 3rd ed (Bayley III). The HI infants scored significantly lower when compared to HEU infants for motor and language development at baseline, three months and six months follow up. No significant improvement in language (p = 0.46) and motor function (p = 0.91) occurred over time; however, developmental scores did not decrease. Cognitive development in the HI group was significantly lower when compared to the HEU group at visit one (p = 0.003). By six months follow-up, there were no significant differences between the two groups for cognitive development (p = 0.18). This study suggests that HIV-positive infants are delayed when compared to HEU infants. HAART may help to prevent further delay; however, it does not reverse the neurological damage already present. There is a need for therapists to be involved in pediatric HIV clinical services in order to provide early developmental screening as well as rehabilitative services to those children in need. PMID:24125015

  15. Second-line protease inhibitor-based highly active antiretroviral therapy after failing non-nucleoside reverse transcriptase inhibitors-based regimens in Asian HIV-infected children

    PubMed Central

    Bunupuradah, Torsak; Puthanakit, Thanyawee; Fahey, Paul; Kariminia, Azar; Yusoff, Nik Khairulddin Nik; Khanh, Truong Huu; Sohn, Annette H.; Chokephaibulkit, Kulkanya; Lumbiganon, Pagakrong; Hansudewechakul, Rawiwan; Razali, Kamarul; Kurniati, Nia; Huy, Bui Vu; Sudjaritruk, Tavitiya; Kumarasamy, Nagalingeswaran; Fong, Siew Moy; Saphonn, Vonthanak; Ananworanich, Jintanat

    2013-01-01

    Background The WHO recommends boosted protease inhibitor (bPI)-based highly active antiretroviral therapy (HAART) after failing non-nucleoside reverse transcriptase inhibitor (NNRTI) treatment. We examined outcomes of this regimen in Asian HIV-infected children. Methods Children from five Asian countries in the TREAT Asia Pediatric HIV Observational Database (TApHOD) with ≥24 weeks of NNRTI-based HAART followed by ≥24 weeks of bPI-based HAART were eligible. Primary outcomes were the proportions with virologic suppression (HIV-RNA <400 copies/ml) and immune recovery (CD4% ≥25% if age <5 years and CD4 count ≥500 cells/mm3 if age ≥5 years) at 48 and 96 weeks. Results Of 3422 children, 153 were eligible; 52% were female. At switch, median age was 10 years, 26% were in WHO stage 4. Median weight-for-age z-score (WAZ) was −1.9 (n=121), CD4% was 12.5% (n=106), CD4 count was 237 (n=112) cells/mm3, and HIV-RNA was 4.6 log10copies/ml (n=61). The most common PI was lopinavir/ritonavir (83%). At 48 weeks, 61% (79/129) had immune recovery, 60% (26/43) had undetectable HIV-RNA and 73% (58/79) had fasting triglycerides ≥130mg/dl. By 96 weeks, 70% (57/82) achieved immune recovery, 65% (17/26) virologic suppression, and hypertriglyceridemia occurred in 66% (33/50). Predictors for virologic suppression at week 48 were longer duration of NNRTI-based HAART (p=0.006), younger age (p=0.007), higher WAZ (p=0.020), and HIV-RNA at switch <10,000 copies/ml (p=0.049). Conclusion In this regional cohort of Asian children on bPI-based second-line HAART, 60% of children tested had immune recovery by one year, and two-thirds had hyperlipidemia, highlighting difficulties in optimizing second-line HAART with limited drug options. PMID:23296119

  16. Design, Synthesis, Biological and Structural Evaluations of Novel HIV-1 Protease Inhibitors to Combat Drug Resistance

    PubMed Central

    Parai, Maloy Kumar; Huggins, David J.; Cao, Hong; Nalam, Madhavi N. L.; Ali, Akbar; Schiffer, Celia A.; Tidor, Bruce; Rana, Tariq M.

    2012-01-01

    A series of new HIV-1 protease inhibitors (PIs) were designed using a general strategy that combines computational structure-based design with substrate-envelope constraints. The PIs incorporate various alcohol-derived P2 carbamates with acyclic and cyclic heteroatomic functionalities into the (R)-hydroxyethylamine isostere. Most of the new PIs show potent binding affinities against wild-type HIV-1 protease and three multidrug resistant (MDR) variants, in particular inhibitors containing 2,2-dichloroacetamide, pyrrolidinone, imidazolidinone, and oxazolidinone moieties at P2 are the most potent with Ki values in the picomolar range. Several new PIs exhibit nanomolar antiviral potencies against patient-derived wild-type viruses from HIV-1 clades A, B, and C and two MDR variants. Crystal structure analyses of four potent inhibitors revealed that carbonyl groups of the new P2 moieties promote extensive hydrogen bond interactions with the invariant Asp-29 residue of the protease. These structure-activity relationship findings can be utilized to design new PIs with enhanced enzyme inhibitory and antiviral potencies. PMID:22708897

  17. Access to HAART in HIV-infected immigrants: a retrospective multicenter Italian study.

    PubMed

    Saracino, A; El-Hamad, I; Prato, R; Cibelli, D C; Tartaglia, A; Palumbo, E; Pezzoli, M C; Angarano, G; Scotto, G

    2005-09-01

    Since 1996, AIDS has declined in the Italian population, but cases in foreign patients, including both recent immigrants and long-term residents, have increased from 3.9% in 1995-1996 to 15.4% in 2001-2002. This increase can only be partly explained by a higher migratory flow and might reflect a delayed access to health facilities and to antiretroviral therapy in migrants. We performed a survey for the year 2003 of HIV-infected immigrants to Italy from countries outside the European Union to verify which factors might influence a lack of access to highly active antiretroviral therapy (HAART). Italian centers of infectious diseases were requested to send sociodemographic and clinical data of HIV-infected immigrant patients. A total of 553 HIV-infected immigrants (49.9% women) were evaluated, representing 6.5% of all HIV-infected patients from these centers. The mean duration of residency in Italy was 6.6 +/- 5.0 years. The country of origin was Africa (64.5%), North and South America (24.2%), Eastern Europe (7.0%), and Asia (3.8%). A total of 407 of 553 patients (73.6%) were taking antiretroviral drugs at the time of screening. Females presented a younger age (p = 0.001), a lower frequency of Centers for Disease Control (CDC) stage B/C (p = 0.008) and a more frequent heterosexual exposure to HIV (p < 0.001), while no differences were observed for time of first positive serology (p = 0.7). CD4 cell count (p = 0.9) and log plasma HIV-RNA (p = 0.1). Characteristics of HAART patients were compared to those of nontreated patients, despite a CD4 cell count less than 350 cells/mm(3). No significant difference was found for gender, country of origin, risk factor, and years of Italian residence, while legal immigrants (p = 0.018) and registered in the National Health Service (p = 0.014) were significantly more likely to receive HAART compared to illegal immigrants.

  18. Serine proteases of parasitic helminths.

    PubMed

    Yang, Yong; Wen, Yun jun; Cai, Ya Nan; Vallée, Isabelle; Boireau, Pascal; Liu, Ming Yuan; Cheng, Shi Peng

    2015-02-01

    Serine proteases form one of the most important families of enzymes and perform significant functions in a broad range of biological processes, such as intra- and extracellular protein metabolism, digestion, blood coagulation, regulation of development, and fertilization. A number of serine proteases have been identified in parasitic helminths that have putative roles in parasite development and nutrition, host tissues and cell invasion, anticoagulation, and immune evasion. In this review, we described the serine proteases that have been identified in parasitic helminths, including nematodes (Trichinella spiralis, T. pseudospiralis, Trichuris muris, Anisakis simplex, Ascaris suum, Onchocerca volvulus, O. lienalis, Brugia malayi, Ancylostoma caninum, and Steinernema carpocapsae), cestodes (Spirometra mansoni, Echinococcus granulosus, and Schistocephalus solidus), and trematodes (Fasciola hepatica, F. gigantica, and Schistosoma mansoni). Moreover, the possible biological functions of these serine proteases in the endogenous biological phenomena of these parasites and in the host-parasite interaction were also discussed. PMID:25748703

  19. Serine Proteases of Parasitic Helminths

    PubMed Central

    Yang, Yong; Wen, Yun jun; Cai, Ya Nan; Vallée, Isabelle; Boireau, Pascal; Liu, Ming Yuan; Cheng, Shi Peng

    2015-01-01

    Serine proteases form one of the most important families of enzymes and perform significant functions in a broad range of biological processes, such as intra- and extracellular protein metabolism, digestion, blood coagulation, regulation of development, and fertilization. A number of serine proteases have been identified in parasitic helminths that have putative roles in parasite development and nutrition, host tissues and cell invasion, anticoagulation, and immune evasion. In this review, we described the serine proteases that have been identified in parasitic helminths, including nematodes (Trichinella spiralis, T. pseudospiralis, Trichuris muris, Anisakis simplex, Ascaris suum, Onchocerca volvulus, O. lienalis, Brugia malayi, Ancylostoma caninum, and Steinernema carpocapsae), cestodes (Spirometra mansoni, Echinococcus granulosus, and Schistocephalus solidus), and trematodes (Fasciola hepatica, F. gigantica, and Schistosoma mansoni). Moreover, the possible biological functions of these serine proteases in the endogenous biological phenomena of these parasites and in the host-parasite interaction were also discussed. PMID:25748703

  20. Decreased sexual risk behavior in the era of HAART among HIV-infected urban and rural South Africans attending primary care clinics

    PubMed Central

    Venkatesh, Kartik K; de Bruyn, Guy; Lurie, Mark N; Mohapi, Lerato; Pronyk, Paul; Moshabela, Mosa; Marinda, Edmore; Gray, Glenda E; Triche, Elizabeth W; Martinson, Neil A

    2011-01-01

    Objective In light of increasing access to highly active antiretroviral treatment (HAART) in sub-Saharan Africa, we conducted a longitudinal study to assess the impact of HAART on sexual risk behaviors among HIV-infected South Africans in urban and rural primary care clinics. Design Prospective observational cohort study. Methods We conducted a cohort study at rural and urban primary care HIV clinics in South Africa consisting of 1544 men and 4719 women enrolled from 2003–2010, representing 19703 clinic visits. The primary outcomes were being sexually active, unprotected sex, and >1 sex partner and were evaluated at six monthly intervals. Generalized estimated equations assessed the impact of HAART on sexual risk behaviors. Results Among 6263 HIV-infected men and women, over a third (37.2%) initiated HAART during study follow-up. In comparison to pre-HAART follow-up, visits while receiving HAART were associated with a decrease in those reporting being sexually active (AOR: 0.86 [95% CI: 0.78–0.95]). Unprotected sex and having >1 sex partner were reduced at visits following HAART initiation compared to pre-HAART visits (AOR: 0.40 [95% CI: 0.34–0.46] and AOR: 0.20 [95% CI: 0.14–0.29], respectively). Conclusions Sexual risk behavior significantly decreased following HAART initiation among HIV-infected South African men and women in primary care programs. The further expansion of antiretroviral treatment programs could enhance HIV prevention efforts in Africa. PMID:20808202

  1. Hybrid data capture for monitoring patients on highly active antiretroviral therapy (HAART) in urban Botswana.

    PubMed Central

    Bussmann, Hermann; Wester, C. William; Ndwapi, Ndwapi; Vanderwarker, Chris; Gaolathe, Tendani; Tirelo, Geoffrey; Avalos, Ava; Moffat, Howard; Marlink, Richard G.

    2006-01-01

    Individual patient care and programme evaluation are pivotal for the success of antiretroviral treatment programmes in resource-limited countries. While computer-aided documentation and data storage are indispensable for any large programme, several important issues need to be addressed including which data are to be collected, who collects it and how it is entered into an electronic database. We describe a patient-monitoring approach, which uses patient encounter forms (in hybrid paper + electronic format) based on optical character recognition, piloted at Princess Marina Hospital in Gaborone, Botswana's first public highly active antiretroviral therapy (HAART) outpatient clinic. Our novel data capture approach collects "key" data for tracking patient and programme outcomes. It saves physician time and does not detract from clinical care. PMID:16501730

  2. PPARgamma Pro12Ala polymorphism in HIV-1-infected patients with HAART-related lipodystrophy.

    PubMed

    Saumoy, Maria; Veloso, Sergi; Alonso-Villaverde, Carlos; Domingo, Pere; Chacón, Matilde R; Miranda, Merce; Aragonès, Gerard; Gutiérrez, Maria Mar; Viladés, Consuelo; Peraire, Joaquim; Sirvent, Joan-Josep; López-Dupla, Miguel; Aguilar, Carmen; Richart, Cristóbal; Vidal, Francesc

    2009-09-01

    Peroxisome proliferator-activated receptor gamma (PPARgamma) is involved in obesity and in some components of the metabolic syndrome in unselected population. To determine whether PPARgamma genetic variants are associated with the risk of developing lipodystrophy and its associated metabolic disturbances in HIV-1-infected patients treated with HAART and to assess PPARgamma mRNA expression in subcutaneous adipose tissue (SAT). The study group comprised 278 patients infected with HIV-1 and treated with antiretroviral drugs (139 with lipodystrophy and 139 without) and 105 uninfected controls (UC). The PPARgamma Pro12Ala (C%>G) single nucleotide polymorphism (SNP) was assessed using PCR-RFLPs on white cell DNA. PPARgamma mRNA expression in SAT was assessed in 38 patients (25 with lipodystrophy and 13 without) and in 21 UC by real-time PCR. Statistical analysis was based on Student's T tests, Chi(2) tests, Spearman's correlations tests and logistic regression tests. PPARgamma Pro12Ala genotype distribution and allele frequencies were non-significantly different between both HIV-1-infected categories, lipodystrophy vs non-lipodystrophy (p=0.9 and p=0.87, respectively). Lipodystrophic patients harbouring the rare X/Ala genotype (Ala/Ala plus Pro/Ala) had significantly greater plasma total and LDL cholesterol levels compared with carriers of the common Pro/Pro genotype (p=0.029 and p=0.016, respectively) at univariate analyses. At multivariate analyses these associations were no longer significant. There was a near-significant decreased SAT PPARgamma mRNA expression in patients with lipodystrophy compared to UC (p=0.054). PPARgamma Pro12Ala SNP has no effect on the risk of developing lipodystrophy in HIV-1-infected patients treated with HAART. PPARgamma mRNA SAT expression appears decreased in lipodystrophy.

  3. Comparative genomics of mycobacterial proteases.

    PubMed

    Ribeiro-Guimarães, Michelle Lopes; Pessolani, Maria Cristina Vidal

    2007-01-01

    Although proteases are recognized as important virulent factors in pathogenic microorganisms, little information is available so far regarding the potential role of these enzymes in diseases caused by mycobacteria. Here we use bioinformatic tools to compare the protease-coding genes present in the genome of Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium avium paratuberculosis. This analysis allowed a review of the nomenclature of the protease family present in mycobacteria. A special attention was devoted to the 'decaying genome' of M. leprae where a relatively high level of conservation of protease-coding genes was observed when compared to other genes families. A total of 39 genes out of the 49 found in M. bovis were identified in M. leprae. Of relevance, a core of well-conserved 38 protease genes shared by the four species was defined. This set of proteases is probably essential for survival in the host and disease outcome and may constitute novel targets for drug development leading to a more effective control of mycobacterial diseases.

  4. Effect of GB virus C co-infection on response to generic HAART in African patients with HIV-1 clade C infection.

    PubMed

    Mosam, Anisa; Sathar, Mahomed A; Dawood, Halima; Cassol, Edana; Esterhuizen, Tonya M; Coovadia, Hoosen M

    2007-06-19

    In 38 African AIDS patients initiating generic HAART, GB virus C (GBV-C) RNA-positive patients retained GBV-C viraemia during 52 weeks of HAART, had a faster decline in HIV viral load (P = 0.03), fewer opportunistic infections (14.3 versus 50%, P = 0.18), and suffered no serious adverse events (none versus 61%, P = 0.008) compared with patients without GBV-C. GBV-C co-infection may be associated with a beneficial effect on African AIDS patients treated with generic HAART. PMID:17545721

  5. In Silico Identification and Evaluation of Leads for the Simultaneous Inhibition of Protease and Helicase Activities of HCV NS3/4A Protease Using Complex Based Pharmacophore Mapping and Virtual Screening

    PubMed Central

    Wadood, Abdul; Riaz, Muhammad; Uddin, Reaz; ul-Haq, Zaheer

    2014-01-01

    Hepatitis C virus (HCV) infection is an alarming and growing threat to public health. The present treatment gives limited efficacy and is poorly tolerated, recommending the urgent medical demand for novel therapeutics. NS3/4A protease is a significant emerging target for the treatment of HCV infection. This work reports the complex-based pharmacophore modeling to find out the important pharmacophoric features essential for the inhibition of both protease and helicase activity of NS3/4A protein of HCV. A seven featured pharmacophore model of HCV NS3/4A protease was developed from the crystal structure of NS3/4A protease in complex with a macrocyclic inhibitor interacting with both protease and helicase sites residues via MOE pharmacophore constructing tool. It consists of four hydrogen bond acceptors (Acc), one hydrophobic (Hyd), one for lone pair or active hydrogen (Atom L) and a heavy atom feature (Atom Q). The generated pharmacophore model was validated by a test database of seventy known inhibitors containing 55 active and 15 inactive/least active compounds. The validated pharmacophore model was used to virtually screen the ChemBridge database. As a result of screening 1009 hits were retrieved and were subjected to filtering by Lipinski’s rule of five on the basis of which 786 hits were selected for further assessment using molecular docking studies. Finally, 15 hits of different scaffolds having interactions with important active site residues were predicted as lead candidates. These candidates having unique scaffolds have a strong likelihood to act as further starting points in the development of novel and potent NS3/4A protease inhibitors. PMID:24551230

  6. Boosted protease inhibitor monotherapy in HIV-infected adults: outputs from a pan-European expert panel meeting

    PubMed Central

    2013-01-01

    While the introduction of combination highly active antiretroviral therapy (HAART) regimens represents an important advance in the management of human immunodeficiency virus (HIV)-infected patients, tolerability can be an issue and the use of several different agents may produce problems. The switch of combination HAART to ritonavir-boosted protease inhibitor (PI) monotherapy may offer the opportunity to maintain antiviral efficacy while reducing treatment complexity and the risks of toxicity. Current European AIDS Clinical Society (EACS) guidelines recognise ritonavir-boosted PI monotherapy with twice-daily lopinavir/ritonavir or once-daily darunavir/ritonavir as a possible option in patients who have intolerance to nucleoside reverse transcriptase inhibitors, or for treatment simplification. Clinical trials data for PI boosted monotherapy are encouraging, showing substantial efficacy in the majority of patients; however, further data are required before this approach can be recommended as a routine treatment. Available data indicate that the most suitable candidates for the use of boosted PI monotherapy are long-term virologically suppressed patients who have demonstrated good adherence to antiretroviral therapy, who do not have chronic hepatitis B, have no history of treatment failure on PIs and are able to tolerate low-dose ritonavir. PMID:23347595

  7. Peptidomimetic inhibitors of HIV protease.

    PubMed

    Randolph, John T; DeGoey, David A

    2004-01-01

    There are currently (July, 2002) six protease inhibitors approved for the treatment of HIV infection, each of which can be classified as peptidomimetic in structure. These agents, when used in combination with other antiretroviral agents, produce a sustained decrease in viral load, often to levels below the limits of quantifiable detection, and a significant reconstitution of the immune system. Therapeutic regimens containing one or more HIV protease inhibitors thus provide a highly effective method for disease management. The important role of protease inhibitors in HIV therapy, combined with numerous challenges remaining in HIV treatment, have resulted in a continued effort both to optimize regimens using the existing agents and to identify new protease inhibitors that may provide unique properties. This review will provide an overview of the discovery and clinical trials of the currently approved HIV protease inhibitors, followed by an examination of important aspects of therapy, such as pharmacokinetic enhancement, resistance and side effects. A description of new peptidomimetic compounds currently being investigated in the clinic and in preclinical discovery will follow. PMID:15193140

  8. Microbial inhibitors of cysteine proteases.

    PubMed

    Kędzior, Mateusz; Seredyński, Rafał; Gutowicz, Jan

    2016-08-01

    Cysteine proteases are one of the major classes of proteolytic enzymes involved in a number of physiological and pathological processes in plants, animals and microorganisms. When their synthesis, activity and localization in mammalian cells are altered, they may contribute to the development of many diseases, including rheumatoid arthritis, osteoporosis and cancer. Therefore, cysteine proteases have become promising drug targets for the medical treatment of these disorders. Inhibitors of cysteine proteases are also produced by almost every group of living organisms, being responsible for the control of intracellular proteolytic activity. Microorganisms synthesize cysteine protease inhibitors not only to regulate the activity of endogenous, often virulent enzymes, but also to hinder the host's proteolytic defense system and evade its immune responses against infections. Present work describes known to date microbial inhibitors of cysteine proteases in terms of their structure, enzyme binding mechanism, specificity and pathophysiological roles. The overview of both proteinaceous and small-molecule inhibitors produced by all groups of microorganisms (bacteria, archaea, fungi, protists) and viruses is provided. Subsequently, possible applications of microbial inhibitors in science, medicine and biotechnology are also highlighted. PMID:27048482

  9. Microbial inhibitors of cysteine proteases.

    PubMed

    Kędzior, Mateusz; Seredyński, Rafał; Gutowicz, Jan

    2016-08-01

    Cysteine proteases are one of the major classes of proteolytic enzymes involved in a number of physiological and pathological processes in plants, animals and microorganisms. When their synthesis, activity and localization in mammalian cells are altered, they may contribute to the development of many diseases, including rheumatoid arthritis, osteoporosis and cancer. Therefore, cysteine proteases have become promising drug targets for the medical treatment of these disorders. Inhibitors of cysteine proteases are also produced by almost every group of living organisms, being responsible for the control of intracellular proteolytic activity. Microorganisms synthesize cysteine protease inhibitors not only to regulate the activity of endogenous, often virulent enzymes, but also to hinder the host's proteolytic defense system and evade its immune responses against infections. Present work describes known to date microbial inhibitors of cysteine proteases in terms of their structure, enzyme binding mechanism, specificity and pathophysiological roles. The overview of both proteinaceous and small-molecule inhibitors produced by all groups of microorganisms (bacteria, archaea, fungi, protists) and viruses is provided. Subsequently, possible applications of microbial inhibitors in science, medicine and biotechnology are also highlighted.

  10. Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination1

    PubMed Central

    Lu, Haibin; Chandrasekar, Balakumaran; Oeljeklaus, Julian; Misas-Villamil, Johana C.; Wang, Zheming; Shindo, Takayuki; Bogyo, Matthew; Kaiser, Markus; van der Hoorn, Renier A.L.

    2015-01-01

    Cysteine proteases are an important class of enzymes implicated in both developmental and defense-related programmed cell death and other biological processes in plants. Because there are dozens of cysteine proteases that are posttranslationally regulated by processing, environmental conditions, and inhibitors, new methodologies are required to study these pivotal enzymes individually. Here, we introduce fluorescence activity-based probes that specifically target three distinct cysteine protease subfamilies: aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes. We applied protease activity profiling with these new probes on Arabidopsis (Arabidopsis thaliana) protease knockout lines and agroinfiltrated leaves to identify the probe targets and on other plant species to demonstrate their broad applicability. These probes revealed that most commercially available protease inhibitors target unexpected proteases in plants. When applied on germinating seeds, these probes reveal dynamic activities of aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes, coinciding with the remobilization of seed storage proteins. PMID:26048883

  11. Molecular mechanisms of tolerance to cyanobacterial protease inhibitors revealed by clonal differences in Daphnia magna.

    PubMed

    Schwarzenberger, Anke; Kuster, Christian J; Von Elert, Eric

    2012-10-01

    Protease inhibitors of primary producers are a major food quality constraint for herbivores. In nutrient-rich freshwater ecosystems, the interaction between primary producers and herbivores is mainly represented by Daphnia and cyanobacteria. Protease inhibitors have been found in many cyanobacterial blooms. These inhibitors have been shown (both in vitro and in situ) to inhibit the most important group of digestive proteases in the daphnid's gut, that is, trypsins and chymotrypsins. In this study, we fed four different Daphnia magna genotypes with the trypsin-inhibitor-containing cyanobacterial strain Microcystis aeruginosa PCC 7806 Mut. Upon exposure to dietary trypsin inhibitors, all D. magna genotypes showed increased gene expression of digestive trypsins and chymotrypsins. Exposure to dietary trypsin inhibitors resulted in increased activity of chymotrypsins and reduced activity of trypsin. Strong intraspecific differences in tolerance of the four D. magna genotypes to the dietary trypsin inhibitors were found. The degree of tolerance depended on the D. magna genotype. The genotypes' tolerance was positively correlated with the residual trypsin activity and the different IC(50) values of the trypsins. On the genetic level, the different trypsin loci varied between the D. magna genotypes. The two tolerant Daphnia genotypes that both originate from the same lake, which frequently produces cyanobacterial blooms, clustered in a neighbour-joining phylogenetic tree based on the three trypsin loci. This suggests that the genetic variability of trypsin loci was an important cause for the observed intraspecific variability in tolerance to cyanobacterial trypsin inhibitors. Based on these findings, it is reasonable to assume that such genetic variability can also be found in natural populations and thus constitutes the basis for local adaptation of natural populations to dietary protease inhibitors.

  12. HIV-1 Drug Resistance Mutations: an Updated Framework for the Second Decade of HAART

    PubMed Central

    Shafer, Robert W.; Schapiro, Jonathan M.

    2008-01-01

    More than 200 mutations are associated with antiretroviral resistance to drugs belonging to six licensed antiretroviral classes. More than 50 reverse transcriptase mutations are associated with nucleoside reverse transcriptase inhibitor resistance including M184V, thymidine analog mutations, mutations associated with non-thymidine analog containing regimens, multi-nucleoside resistance mutations, and several recently identified accessory mutations. More than 40 reverse transcriptase mutations are associated with nonnucleoside reverse transcriptase inhibitor resistance including major primary and secondary mutations, non-polymorphic minor mutations, and polymorphic accessory mutations. More than 60 mutations are associated with protease inhibitor resistance including major protease, accessory protease, and protease cleavage site mutations. More than 30 integrase mutations are associated with the licensed integrase inhibitor raltegravir and the investigational inhibitor elvitegravir. More than 15 gp41 mutations are associated with the fusion inhibitor enfuvirtide. CCR5 inhibitor resistance results from mutations that promote gp120 binding to an inhibitor-bound CCR5 receptor or CXCR4 tropism; however, the genotypic correlates of these processes are not yet well characterized. PMID:18615118

  13. CHAGASIC MENINGOENCEPHALITIS IN AN HIV INFECTED PATIENT WITH MODERATE IMMUNOSUPPRESSION: PROLONGED SURVIVAL AND CHALLENGES IN THE HAART ERA.

    PubMed

    Buccheri, Renata; Kassab, Maria José; Freitas, Vera Lucia Teixeira de; Silva, Sheila Cristina Vicente da; Bezerra, Rita C; Khoury, Zarifa; Shikanai-Yasuda, Maria Aparecida; Vidal, José E

    2015-12-01

    The reactivation of Chagas disease in HIV infected patients presents high mortality and morbidity. We present the case of a female patient with confirmed Chagasic meningoencephalitis as AIDS-defining illness. Interestingly, her TCD4+ lymphocyte cell count was 318 cells/mm3. After two months of induction therapy, one year of maintenance with benznidazol, and early introduction of highly active antiretroviral therapy (HAART), the patient had good clinical, parasitological and radiological evolution. We used a qualitative polymerase chain reaction for the monitoring of T. cruzi parasitemia during and after the treatment. We emphasize the potential value of molecular techniques along with clinical and radiological parameters in the follow-up of patients with Chagas disease and HIV infection. Early introduction of HAART, prolonged induction and maintenance of antiparasitic therapy, and its discontinuation are feasible, in the current management of reactivation of Chagas disease. PMID:27049711

  14. CHAGASIC MENINGOENCEPHALITIS IN AN HIV INFECTED PATIENT WITH MODERATE IMMUNOSUPPRESSION: PROLONGED SURVIVAL AND CHALLENGES IN THE HAART ERA

    PubMed Central

    BUCCHERI, Renata; KASSAB, Maria José; de FREITAS, Vera Lucia Teixeira; da SILVA, Sheila Cristina Vicente; BEZERRA, Rita C.; KHOURY, Zarifa; SHIKANAI-YASUDA, Maria Aparecida; VIDAL, José E.

    2015-01-01

    The reactivation of Chagas disease in HIV infected patients presents high mortality and morbidity. We present the case of a female patient with confirmed Chagasic meningoencephalitis as AIDS-defining illness. Interestingly, her TCD4+ lymphocyte cell count was 318 cells/mm3. After two months of induction therapy, one year of maintenance with benznidazol, and early introduction of highly active antiretroviral therapy (HAART), the patient had good clinical, parasitological and radiological evolution. We used a qualitative polymerase chain reaction for the monitoring of T. cruzi parasitemia during and after the treatment. We emphasize the potential value of molecular techniques along with clinical and radiological parameters in the follow-up of patients with Chagas disease and HIV infection. Early introduction of HAART, prolonged induction and maintenance of antiparasitic therapy, and its discontinuation are feasible, in the current management of reactivation of Chagas disease. PMID:27049711

  15. Differential survival benefit of universal HAART access in Brazil: A Nation-wide Comparison of Injecting Drug Users versus Men who Have Sex with Men

    PubMed Central

    Malta, Monica; Bastos, Francisco I.; da Silva, Cosme MFP; Pereira, Gerson Fernando Mendes; Lucena, Francisca FA; Fonseca, Maria GP; Strathdee, Steffanie A.

    2009-01-01

    Objective Brazil accounts for ∼70% of injection drug users (IDU) receiving HAART in low/middle income countries. We evaluated the impact of HAART availability/access on AIDS-related mortality among IDU versus men who have sex with men (MSM). Design Nationwide analysis on Brazilian IDU and MSM diagnosed with AIDS in 2000-2006. Methods Four national information systems were linked and Cox regression was used to assess impact of HAART availability/access on differential AIDS-related mortality. Results Among 28,426 patients, 6,777 died during 87,792 person-years of follow-up. Compared to MSM, IDU were significantly less likely to be receiving HAART, to have ever had determinations for CD4 or viral load. After controlling for confounders, IDU had a significantly higher risk of death (AHR: 1.94; 95% CI: 1.84-2.05). Among the subset that had at least one CD4 and viral load determination, higher risk of death among IDU persisted (HR: 1.82; 95% CI: 1.58-2.11). Non-white ethnicity significantly increased this risk, while prompt HAART uptake after AIDS diagnosis reduced the risk of death. After controlling for spatially-correlated survival data, AIDS-related mortality remained higher in IDU than in MSM. Conclusions Despite free/universal HAART access, differential AIDS-related mortality exists in Brazil. Efforts are needed to identify and eliminate these health disparities. PMID:19675464

  16. Targeted therapies to treat Non-AIDS Defining Cancers in patients with HIV on HAART therapy – treatment considerations and research outlook

    PubMed Central

    Deeken, John F.; Pantanowitz, Liron; Dezube, Bruce J.

    2012-01-01

    Purpose of review Highly active antiretroviral therapy (HAART) has led to a dramatic improvement in the prognosis of patients diagnosed with HIV and AIDS. This includes a significant decline in the rates of AIDS-related cancers, including Kaposi Sarcoma and Non-Hodgkin's Lymphoma. Unfortunately, rates of Non-AIDS Defining Cancers (NADCs) are on the rise, and now exceed the rates of AIDS-related cancers in patients with HIV. Treating NADCs in patients who are on HAART therapy is an open and complicated clinical question. Recent findings Newer targeted therapies are now available to treat cancers which were historically refractory to traditional cytotoxic chemotherapy. HAART agents are notorious for causing drug-drug interactions. The co-administration of targeted chemotherapies with HAART could well impede the efficacy or increase the toxicity of these targeted therapies. Unfortunately little is known about possible drug-drug interactions because HIV patients are typically excluded from clinical trials. Summary We highlight what is known about how and why HAART agents can affect drug metabolism. We then present the clinical and pharmacological data for nine recently approved targeted therapies – imatinib, dasatinib, nilotinib, erlotinib, sunitinib, lapatinib, bortezomib, sorafenib, and temsirolimus. We conclude with considerations on how to use these new agents to treat NADCs, and discuss a future research agenda to better understand and predict potential HAART-targeted therapy interactions. PMID:19606034

  17. Hypertension among HIV-Infected Adults Receiving Highly Active Antiretroviral Therapy (HAART) in Malaysia

    PubMed Central

    Hejazi, Nazisa; MSL, Huang; Lin, Khor Geok; Choong, Lee Christopher Kwok

    2014-01-01

    There are increasing researches about non-communicable disease such as elevated blood pressure among people living with HIV before and after initiation of highly active antiretroviral therapy (HAART). This cross-sectional study was designed to determine the prevalence of hypertension and associated risk factors among 340 HIV-infected patients on antiretroviral therapy at a Malaysian public hospital providing HIV-related treatment. Data on socioeconomic background, anthropometry, medical history and dietary intake of the patients were collected. Hypertension is defined as blood pressure ≥130/85 (mm Hg). Prevalence of hypertension was 45.60% (n=155) of which 86.5% of the hypertensive group were male (n=134). The results showed that increase in age (OR 1.051, 95% confidence interval (CI) 1.024-1.078), higher body mass index (OR 1.18, 95% CI 1.106-2.71), bigger waist circumference (OR 1.18, 95%CI 1.106-2.71), higher waist-hip ratio (OR 1.070, 95%CI 1.034-1.106), higher fasting plasma glucose (OR 1.332, 95% CI 0.845-2.100) and percentage energy intake from protein >15 (OR 2.519, 95%CI 1.391-4.561) were significant risk factors for hypertension (p<0.001). After adjusting for other variables, increasing age (adjusted odds ratio (aOR) 1.069 95%CI 1.016-1.124, p=0.010), being male (aOR 3.026, 95%CI 1.175-7.794, p=0.022) and higher body mass index (aOR 1.26, 95%CI 1.032-1.551, p=0.024) were independently associated with hypertension. None of the antiretroviral therapy and immunologic factors was linked to hypertension. In conclusion hypertension among PLHIV was linked to the well-known risk factors such as age, gender and body mass index. With HAART, people can live longer by making monitoring and control of some reversible factors, especially excessive weight gain for maintaining quality of life. PMID:24576366

  18. Lipodystrophy in Human Immunodeficiency Virus (HIV) Patients on Highly Active Antiretroviral Therapy (HAART)

    PubMed Central

    Kumar, N. Sunil; Shashibhushan, J.; Venugopal, K.; Vishwanatha, Huggi; Menon, Mahesh

    2015-01-01

    Background In recent years, abnormal lipid deposition (both lipoatrophy and fat redistribution) and its related complications have changed from an anecdotal issue into a major problem for HIV (Human Immunodeficiency Virus) infected patients on HAART (Highly Active Anti-Retroviral Therapy). Lipoatrophy and fat redistribution are potentially stigmatizing complications of HAART and leads to poor adherence among patients. Hence we conducted this study to determine the pattern and to assess various risk factors for maldeposition of lipids in HIV patients. Materials and Methods A cross-sectional case series study was conducted in ART PLUS centre, Bellary over a period of 8 months from January to August 2014 in HIV patients on ART to determine risk factors associated with and epidemiological pattern of fat redistribution or atrophy. Results A total of 50 patients with LD {lipodystrophy} (26 with fat redestribution and 24 with lipoatrophy {LA} were diagnosed in this period. Most of them belonged to younger age and was commonly seen in females (76%). Patients with LA had a significantly lower BMI (18.73 ± 7.4), {the p-value being 0.19} compared to LH group (21.54 ± 7.62). The duration of disease was comparable among both groups (6.96 years in LH and 5.79 years in LA group) {p-value is 0.29}. There was a relatively good immunity among these patients with mean CD4 count was 509.23 in LH and 545.91 in LA group {single CD4 count was taken and the p-value was 0.001}. Most of the patients were in TLN (Tenofovir, Lamivudine, Nevirapine) regimen (58%).The duration that patient was on ART before commencement of study varied from patient to patient, but the mean duration was approximately five years in fat redistribution group and 4.5 years in LA group. There were no derangements in lipid and sugar levels among them. Conclusion This study shows the need to identify and impact of LD with respect to treatment adherence in young patients especially female patients. Early community

  19. Excess apoptosis of mononuclear cells contributes to the depressed cytomegalovirus-specific immunity in HIV-infected patients on HAART

    SciTech Connect

    Weinberg, Adriana . E-mail: Adriana.Weinberg@uchsc.edu; Jesser, Renee D.; Edelstein, Charles L.; Bill, Jerome R.; Wohl, David A.

    2004-12-05

    HIV-infected patients on highly active antiretroviral therapy (HAART) have persistently decreased cytomegalovirus (CMV)-specific proliferative responses [lymphocyte proliferation assay (LPA)] in spite of increases in CD4+ T cell counts. Here we demonstrate an association between apoptosis of unstimulated peripheral blood mononuclear cells (uPBMC) and decreased CMV-LPA. HAART recipients had more apoptosis of uPBMC than controls when measured by caspases 3, 8, and 9 activities and by annexin V binding. Patients with undetectable HIV replication maintained significantly higher apoptosis of CD4+ and CD14+ cells compared to controls. CMV-LPA decreased with higher apoptosis of uPBMC in patients only. This association was independent of CD4+ cell counts or HIV replication. Furthermore, rescuing PBMC from apoptosis with crmA, but not with TRAIL- or Fas-pathway blocking agents or with other caspase inhibitors, increased CMV-LPA in HAART recipients. This effect was not observed in uninfected controls, further indicating that the down regulatory effect of apoptosis on cell-mediated immunity (CMI) was specifically associated with the HIV-infected status.

  20. Curcumin derivatives as HIV-1 protease inhibitors

    SciTech Connect

    Sui, Z.; Li, J.; Craik, C.S.; Ortiz de Montellano, P.R.

    1993-12-31

    Curcumin, a non-toxic natural compound from Curcuma longa, has been found to be an HIV-1 protease inhibitor. Some of its derivatives were synthesized and their inhibitory activity against the HIV-1 protease was tested. Curcumin analogues containing boron enhanced the inhibitory activity. At least of the the synthesized compounds irreversibly inhibits the HIV-1 protease.

  1. Proteases in Fas-mediated apoptosis.

    PubMed

    Zhivotovsky, B; Burgess, D H; Schlegel, J; Pörn, M I; Vanags, D; Orrenius, S

    1997-01-01

    Involvement of a unique family of cysteine proteases in the multistep apoptotic process has been documented. Cloning of several mammalian genes identifies some components of this cellular response. However, it is currently unclear which protease plays a role as a signal and/or effector of apoptosis. We summarize contributions to the data concerning proteases in Fas-mediated apoptosis.

  2. Renal Function Impairment and Associated Factors among HAART Naïve and Experienced Adult HIV Positive Individuals in Southwest Ethiopia: A Comparative Cross Sectional Study

    PubMed Central

    Mekuria, Yewulsew; Yilma, Daniel; Mekonnen, Zeleke; Kassa, Tesfaye; Gedefaw, Lealem

    2016-01-01

    Background Human immunodeficiency virus (HIV) infection and its treatment cause renal diseases. Renal disease is associated with an increasing cause of morbidity and mortality in HIV positive individuals than in the general population. It has been also associated with adverse outcomes, such as complications of decreased renal functions and progression to renal failure. Objective To determine the prevalence and factors associated with renal function impairment among highly active antiretroviral therapy (HAART) naive and HAART experienced adult HIV positive individuals. Methods A facility based comparative cross-sectional study was conducted in Jimma University Specialized Hospital (JUSH) from June to September 2014. HIV positive individuals who visited JUSH during the study period were included in the study. Sociodemographic and clinical data were collected using a structured questionnaire. Blood specimen was analyzed for renal function tests. Descriptive statistics, Mann-Whitney U test and logistic regression analysis were done using SPSS version 16 software. Results A total of 446 HIV positive individuals, 223 HAART naïve and 223 HAART experienced, were recruited. The overall prevalence of renal function impairment was 18.2% [95%CI: 14.6–21.7]. The prevalence of renal impairment in HAART naive and HAART experienced persons was 28.7% [95%CI: 23.1–34.4] and 7.6% [95%CI: 4.6–11.6], respectively. Age ≥ 50 years (AOR = 3.6; 95% CI 1.4, 9.6), advanced WHO stage (AOR = 2.3; 95% CI 1.1, 4.7), and CD4 count <200 (AOR = 6.9; 95% CI 3.3, 14.2) were independent risk factors among HAART naive participants. Female gender (AOR = 6.6; 95 CI % 1.2, 34), age ≥ 50 years (AOR = 12.1; 95% CI 1.7, 84) and CD4 count <200 (AOR = 17; 95% CI 5.2, 58) were independent risk factors among HAART experienced participants. Conclusion The prevalence of renal function impairment was higher among HAART naïve than HAART experienced HIV positive individuals. Renal function impairment was

  3. Monitoring of HAART regime antiretrovirals in serum of acquired immunodeficiency syndrome patients by micellar liquid chromatography.

    PubMed

    Casas-Breva, I; Peris-Vicente, J; Rambla-Alegre, M; Carda-Broch, S; Esteve-Romero, J

    2012-09-21

    A methodology based on micellar liquid chromatography to monitor five antiretroviral drugs (lamivudine, stavudine, tenofovir, zidovudine and efavirenz) was proposed. Antiretrovirals were studied in sets of three, corresponding to each highly active antiretroviral therapy (HAART) regime, prescribed to acquired immunodeficiency syndrome (AIDS)-infected patients. Four aqueous micellar mobile phases buffered at pH 7 were optimized to separate these compounds, using sodium dodecyl sulfate as the tensioactive, and 1-propanol or 1-pentanol as the organic modifier. The composition of each mobile phase was optimized for each antiretroviral. The common separation conditions were: C18 apolar column (125 × 4.6 mm, 5 μm particle size), UV detection set at 214 nm, and mobile phase running at 1 mL min(-1) without controlling the temperature. The finally suggested method was validated for five analysed antiretroviral drugs following the US Food and Drug Administration guidelines in terms of: linearity between 0.5 and 50 ppm (r(2) > 0.9995), sensitivity (LOD lower than 0.25 ppm), intra- and inter-day precision (<7.1 and <5.2%, respectively) and accuracy (recovery 88.5-105.3% and 93.5-101.3%, respectively), as well as robustness (<6.5%). The proposed method was used to monitor the level of antiretrovirals in the serum of AIDS patients. The suggested methodology was found to be useful in the routine analysis of antiretrovirals in serum samples.

  4. HIV Infection and Primary Prevention of Cardiovascular Disease: Lights and Shadows in the HAART Era.

    PubMed

    Ballocca, Flavia; Gili, Sebastiano; D'Ascenzo, Fabrizio; Marra, Walter Grosso; Cannillo, Margherita; Calcagno, Andrea; Bonora, Stefano; Flammer, Andreas; Coppola, John; Moretti, Claudio; Gaita, Fiorenzo

    2016-01-01

    With the progressive increase in life-expectancy of human immunodeficiency virus (HIV)-positive patients in the "highly active antiretroviral therapy" (HAART) era, co-morbidities, particularly cardiovascular (CV) diseases (CVD) are emerging as an important concern. The pathophysiology of CVD in this population is complex, due to the interaction of classical CV risk factors, viral infection and the effects of antiretroviral therapy (ARV). The role of ARV drugs in HIV is double edged. While these drugs reduce systemic inflammation, an important factor in CV development, they may at the same time be proatherogenic by inducing dyslipidemia, body fat redistribution and insulin resistance. In these patients primary prevention is challenging, considering the lower median age at which acute coronary syndromes occur. Furthermore prevention is still limited by the lack of robust evidence-based, HIV-specific recommendations. Therefore we performed a comprehensive evaluation of the literature to analyze current knowledge on CVD prevalence in HIV-infected patients, traditional and HIV-specific risk factors and risk stratification, and to summarize the recommendations for primary prevention of CVD in this HIV population. PMID:26943980

  5. Exogenous proteases for meat tenderization.

    PubMed

    Bekhit, Alaa A; Hopkins, David L; Geesink, Geert; Bekhit, Adnan A; Franks, Philip

    2014-01-01

    The use of exogenous proteases to improve meat tenderness has attracted much interest recently, with a view to consistent production of tender meat and added value to lower grade meat cuts. This review discusses the sources, characteristics, and use of exogenous proteases in meat tenderization to highlight the specificity of the proteases toward meat proteins and their impact on meat quality. Plant enzymes (such as papain, bromelain, and ficin) have been extensively investigated as meat tenderizers. New plant proteases (actinidin and zingibain) and microbial enzyme preparations have been of recent interest due to controlled meat tenderization and other advantages. Successful use of these enzymes in fresh meat requires their enzymatic kinetics and characteristics to be determined, together with an understanding of the impact of the surrounding environmental conditions of the meat (pH, temperature) on enzyme function. This enables the optimal conditions for tenderizing fresh meat to be established, and the elimination or reduction of any negative impacts on other quality attributes. PMID:24499119

  6. Network Analyses Reveal Pervasive Functional Regulation Between Proteases in the Human Protease Web

    PubMed Central

    Fortelny, Nikolaus; Cox, Jennifer H.; Kappelhoff, Reinhild; Starr, Amanda E.; Lange, Philipp F.; Pavlidis, Paul; Overall, Christopher M.

    2014-01-01

    Proteolytic processing is an irreversible posttranslational modification affecting a large portion of the proteome. Protease-cleaved mediators frequently exhibit altered activity, and biological pathways are often regulated by proteolytic processing. Many of these mechanisms have not been appreciated as being protease-dependent, and the potential in unraveling a complex new dimension of biological control is increasingly recognized. Proteases are currently believed to act individually or in isolated cascades. However, conclusive but scattered biochemical evidence indicates broader regulation of proteases by protease and inhibitor interactions. Therefore, to systematically study such interactions, we assembled curated protease cleavage and inhibition data into a global, computational representation, termed the protease web. This revealed that proteases pervasively influence the activity of other proteases directly or by cleaving intermediate proteases or protease inhibitors. The protease web spans four classes of proteases and inhibitors and so links both recently and classically described protease groups and cascades, which can no longer be viewed as operating in isolation in vivo. We demonstrated that this observation, termed reachability, is robust to alterations in the data and will only increase in the future as additional data are added. We further show how subnetworks of the web are operational in 23 different tissues reflecting different phenotypes. We applied our network to develop novel insights into biologically relevant protease interactions using cell-specific proteases of the polymorphonuclear leukocyte as a system. Predictions from the protease web on the activity of matrix metalloproteinase 8 (MMP8) and neutrophil elastase being linked by an inactivating cleavage of serpinA1 by MMP8 were validated and explain perplexing Mmp8 −/− versus wild-type polymorphonuclear chemokine cleavages in vivo. Our findings supply systematically derived and

  7. Biotechnology of Cold-Active Proteases

    PubMed Central

    Joshi, Swati; Satyanarayana, Tulasi

    2013-01-01

    The bulk of Earth’s biosphere is cold (<5 °C) and inhabited by psychrophiles. Biocatalysts from psychrophilic organisms (psychrozymes) have attracted attention because of their application in the ongoing efforts to decrease energy consumption. Proteinases as a class represent the largest category of industrial enzymes. There has been an emphasis on employing cold-active proteases in detergents because this allows laundry operations at ambient temperatures. Proteases have been used in environmental bioremediation, food industry and molecular biology. In view of the present limited understanding and availability of cold-active proteases with diverse characteristics, it is essential to explore Earth’s surface more in search of an ideal cold-active protease. The understanding of molecular and mechanistic details of these proteases will open up new avenues to tailor proteases with the desired properties. A detailed account of the developments in the production and applications of cold-active proteases is presented in this review. PMID:24832807

  8. Design, Synthesis, and Biological and Structural Evaluations of Novel HIV-1 Protease Inhibitors To Combat Drug Resistance

    SciTech Connect

    Parai, Maloy Kumar; Huggins, David J.; Cao, Hong; Nalam, Madhavi N.L.; Ali, Akbar; Schiffer, Celia A.; Tidor, Bruce; Rana, Tariq M.

    2012-09-11

    A series of new HIV-1 protease inhibitors (PIs) were designed using a general strategy that combines computational structure-based design with substrate-envelope constraints. The PIs incorporate various alcohol-derived P2 carbamates with acyclic and cyclic heteroatomic functionalities into the (R)-hydroxyethylamine isostere. Most of the new PIs show potent binding affinities against wild-type HIV-1 protease and three multidrug resistant (MDR) variants. In particular, inhibitors containing the 2,2-dichloroacetamide, pyrrolidinone, imidazolidinone, and oxazolidinone moieties at P2 are the most potent with Ki values in the picomolar range. Several new PIs exhibit nanomolar antiviral potencies against patient-derived wild-type viruses from HIV-1 clades A, B, and C and two MDR variants. Crystal structure analyses of four potent inhibitors revealed that carbonyl groups of the new P2 moieties promote extensive hydrogen bond interactions with the invariant Asp29 residue of the protease. These structure-activity relationship findings can be utilized to design new PIs with enhanced enzyme inhibitory and antiviral potencies.

  9. Malignancies in human immunodeficiency virus infected patients in India: Initial experience in the HAART era

    PubMed Central

    Sharma, Surendra K.; Soneja, Manish; Ranjan, Sanjay

    2015-01-01

    Background & objectives: Limited data are available on malignancies in human immunodeficiency virus (HIV)-infected patients from India. We undertook this study to assess the frequency and spectrum of malignancies in HIV-infected adult patients during the first eight years of highly active antiretroviral therapy (HAART) rollout under the National ART Programme at a tertiary care centre in New Delhi, India. Methods: Retrospective analysis of records of patients registered at the ART clinic between May 2005 and December 2013 was done. Results: The study included 2598 HIV-infected adult patients with 8315 person-years of follow up. Malignancies were diagnosed in 26 patients with a rate of 3.1 (IQR 2.1-4.5) cases per 1000 person-years. The median age for those diagnosed with malignancy was 45 (IQR 36-54) yr, which was significantly (P<0.01) higher compared with those not developing malignancies 35 (IQR 30-40) yr. The median baseline CD4+ T-cell count in patients with malignancy was 135 (IQR 68-269) cells/µl compared to 164 (IQR 86-243) cells/µl in those without malignancies. AIDS-defining cancers (ADCs) were seen in 19 (73%) patients, while non-AIDS-defining cancers (NADCs) were observed in seven (27%) patients. Malignancies diagnosed included non-Hodgkin's lymphoma (16), carcinoma cervix (3), Hodgkin's lymphoma (2), carcinoma lung (2), hepatocellular carcinoma (1), and urinary bladder carcinoma (1). One patient had primary central nervous system lymphoma. There was no case of Kaposi's sarcoma. Interpretation & conclusions: Malignancies in HIV-infected adult patients were infrequent in patients attending the clinic. Majority of the patients presented with advanced immunosuppression and the ADCs, NHL in particular, were the commonest malignancies. PMID:26658591

  10. Type II Transmembrane Serine Proteases*

    PubMed Central

    Bugge, Thomas H.; Antalis, Toni M.; Wu, Qingyu

    2009-01-01

    Analysis of genome and expressed sequence tag data bases at the turn of the millennium unveiled a new protease family named the type II transmembrane serine proteases (TTSPs) in a Journal of Biological Chemistry minireview (Hooper, J. D., Clements, J. A., Quigley, J. P., and Antalis, T. M. (2001) J. Biol. Chem. 276, 857–860). Since then, the number of known TTSPs has more than doubled, and more importantly, our understanding of the physiological functions of individual TTSPs and their contribution to human disease has greatly increased. Progress has also been made in identifying molecular substrates and endogenous inhibitors. This minireview summarizes the current knowledge of the rapidly advancing TTSP field. PMID:19487698

  11. Molecular Imaging of Proteases in Cancer

    PubMed Central

    Yang, Yunan; Hong, Hao; Zhang, Yin; Cai, Weibo

    2010-01-01

    Proteases play important roles during tumor angiogenesis, invasion, and metastasis. Various molecular imaging techniques have been employed for protease imaging: optical (both fluorescence and bioluminescence), magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), and positron emission tomography (PET). In this review, we will summarize the current status of imaging proteases in cancer with these techniques. Optical imaging of proteases, in particular with fluorescence, is the most intensively validated and many of the imaging probes are already commercially available. It is generally agreed that the use of activatable probes is the most accurate and appropriate means for measuring protease activity. Molecular imaging of proteases with other techniques (i.e. MRI, SPECT, and PET) has not been well-documented in the literature which certainly deserves much future effort. Optical imaging and molecular MRI of protease activity has very limited potential for clinical investigation. PET/SPECT imaging is suitable for clinical investigation; however the optimal probes for PET/SPECT imaging of proteases in cancer have yet to be developed. Successful development of protease imaging probes with optimal in vivo stability, tumor targeting efficacy, and desirable pharmacokinetics for clinical translation will eventually improve cancer patient management. Not limited to cancer, these protease-targeted imaging probes will also have broad applications in other diseases such as arthritis, atherosclerosis, and myocardial infarction. PMID:20234801

  12. Experiences of stigma and access to HAART in children and adolescents living with HIV/AIDS in Brazil.

    PubMed

    Abadía-Barrero, César Ernesto; Castro, Arachu

    2006-03-01

    This study describes and conceptualizes the experiences of stigma in a group of children living with HIV in São Paulo, Brazil, and evaluates the impact of access to highly active antiretroviral therapy (HAART) over the social course of AIDS and over the children's experiences of stigma. Through ethnographic research in São Paulo from 1999 to 2001, the life trajectories of 50 children ages 1-15 living with or affected by HIV were studied. Data were collected via participant observation and semi-structured informal interviews and analyzed using social theories on illness experience and social inequality. Our results demonstrate that AIDS-related stigma occurs within complex discrimination processes that change as children reach adolescence. We found that structural violence in the forms of poverty, racism, and inequalities in social status, gender, and age fuels children's experiences of stigma. We also describe how access to HAART changes the lived experience of children, reduces stigma, and brings new challenges in AIDS care such as adolescents' sexuality and treatment adherence. Based on these results, we propose structural violence as the framework to study stigma and argue that interventions to reduce stigma that solely target the perception and attitudes toward people living with HIV are limited. In contrast universal access to HAART in Brazil is a powerful intervention that reduces stigma, in that it transforms AIDS from a debilitating and fatal disease to a chronic and manageable one, belongs to a broader mechanism to assure citizens' rights, and reduces social inequalities in access to health care.

  13. Advances in protease engineering for laundry detergents.

    PubMed

    Vojcic, Ljubica; Pitzler, Christian; Körfer, Georgette; Jakob, Felix; Ronny Martinez; Maurer, Karl-Heinz; Schwaneberg, Ulrich

    2015-12-25

    Proteases are essential ingredients in modern laundry detergents. Over the past 30 years, subtilisin proteases employed in the laundry detergent industry have been engineered by directed evolution and rational design to tailor their properties towards industrial demands. This comprehensive review discusses recent success stories in subtilisin protease engineering. Advances in protease engineering for laundry detergents comprise simultaneous improvement of thermal resistance and activity at low temperatures, a rational strategy to modulate pH profiles, and a general hypothesis for how to increase promiscuous activity towards the production of peroxycarboxylic acids as mild bleaching agents. The three protease engineering campaigns presented provide in-depth analysis of protease properties and have identified principles that can be applied to improve or generate enzyme variants for industrial applications beyond laundry detergents.

  14. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, David B.; Lao, Guifang

    1998-01-01

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

  15. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, D.B.; Lao, G.

    1998-01-06

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium. 3 figs.

  16. Crystal structures of Bacillus subtilis Lon protease.

    PubMed

    Duman, Ramona E; Löwe, Jan

    2010-08-27

    Lon ATP-dependent proteases are key components of the protein quality control systems of bacterial cells and eukaryotic organelles. Eubacterial Lon proteases contain an N-terminal domain, an ATPase domain, and a protease domain, all in one polypeptide chain. The N-terminal domain is thought to be involved in substrate recognition, the ATPase domain in substrate unfolding and translocation into the protease chamber, and the protease domain in the hydrolysis of polypeptides into small peptide fragments. Like other AAA+ ATPases and self-compartmentalising proteases, Lon functions as an oligomeric complex, although the subunit stoichiometry is currently unclear. Here, we present crystal structures of truncated versions of Lon protease from Bacillus subtilis (BsLon), which reveal previously unknown architectural features of Lon complexes. Our analytical ultracentrifugation and electron microscopy show different oligomerisation of Lon proteases from two different bacterial species, Aquifex aeolicus and B. subtilis. The structure of BsLon-AP shows a hexameric complex consisting of a small part of the N-terminal domain, the ATPase, and protease domains. The structure shows the approximate arrangement of the three functional domains of Lon. It also reveals a resemblance between the architecture of Lon proteases and the bacterial proteasome-like protease HslUV. Our second structure, BsLon-N, represents the first 209 amino acids of the N-terminal domain of BsLon and consists of a globular domain, similar in structure to the E. coli Lon N-terminal domain, and an additional four-helix bundle, which is part of a predicted coiled-coil region. An unexpected dimeric interaction between BsLon-N monomers reveals the possibility that Lon complexes may be stabilised by coiled-coil interactions between neighbouring N-terminal domains. Together, BsLon-N and BsLon-AP are 36 amino acids short of offering a complete picture of a full-length Lon protease.

  17. Biased Signaling of Protease-Activated Receptors

    PubMed Central

    Zhao, Peishen; Metcalf, Matthew; Bunnett, Nigel W.

    2014-01-01

    In addition to their role in protein degradation and digestion, proteases can also function as hormone-like signaling molecules that regulate vital patho-physiological processes, including inflammation, hemostasis, pain, and repair mechanisms. Certain proteases can signal to cells by cleaving protease-activated receptors (PARs), a family of four G protein-coupled receptors. PARs are expressed by almost all cell types, control important physiological and disease-relevant processes, and are an emerging therapeutic target for major diseases. Most information about PAR activation and function derives from studies of a few proteases, for example thrombin in the case of PAR1, PAR3, and PAR4, and trypsin in the case of PAR2 and PAR4. These proteases cleave PARs at established sites with the extracellular N-terminal domains, and expose tethered ligands that stabilize conformations of the cleaved receptors that activate the canonical pathways of G protein- and/or β-arrestin-dependent signaling. However, a growing number of proteases have been identified that cleave PARs at divergent sites to activate distinct patterns of receptor signaling and trafficking. The capacity of these proteases to trigger distinct signaling pathways is referred to as biased signaling, and can lead to unique patho-physiological outcomes. Given that a different repertoire of proteases are activated in various patho-physiological conditions that may activate PARs by different mechanisms, signaling bias may account for the divergent actions of proteases and PARs. Moreover, therapies that target disease-relevant biased signaling pathways may be more effective and selective approaches for the treatment of protease- and PAR-driven diseases. Thus, rather than mediating the actions of a few proteases, PARs may integrate the biological actions of a wide spectrum of proteases in different patho-physiological conditions. PMID:24860547

  18. Abuse and resilience in relation to HAART medication adherence and HIV viral load among women with HIV in the United States.

    PubMed

    Dale, Sannisha; Cohen, Mardge; Weber, Kathleen; Cruise, Ruth; Kelso, Gwendolyn; Brody, Leslie

    2014-03-01

    Abuse is highly prevalent among HIV+ women, leading to behaviors, including lower adherence to highly active antiretroviral therapy (HAART) that result in poor health outcomes. Resilience (functioning competently despite adversity) may buffer the negative effects of abuse. This study investigated how resilience interacted with abuse history in relation to HAART adherence, HIV viral load (VL), and CD4+ cell count among a convenience sample of 138 HIV+ women from the Ruth M. Rothstein CORE Center/Cook County Health and Hospital Systems site of the Women's Interagency HIV Study (WIHS). Resilience was measured by the 10-item Connor-Davidson Resilience Scale (CD-RISC). HAART adherence (≥95% vs. <95% self reported usage of prescribed medication) and current or prior sexual, physical, or emotional/domestic abuse, were reported during structured interviews. HIV viral load (≥20 vs. <20 copies/mL) and CD4+ count (200 vs. <200 cells/mm) were measured with blood specimens. Multiple logistic regressions, controlling for age, race, income, enrollment wave, substance use, and depressive symptoms, indicated that each unit increase in resilience was significantly associated with an increase in the odds of having ≥95% HAART adherence and a decrease in the odds of having a detectable viral load. Resilience-Abuse interactions showed that only among HIV+ women with sexual abuse or multiple abuses did resilience significantly relate to an increase in the odds of ≥95% HAART adherence. Interventions to improve coping strategies that promote resilience among HIV+ women may be beneficial for achieving higher HAART adherence and viral suppression.

  19. HIV-1 Reverse Transcriptase Drug-Resistance Mutations in Chronically Infected Individuals Receiving or Naïve to HAART in Cameroon

    PubMed Central

    Burda, Sherri T.; Viswanath, Ragupathy; Zhao, Jiangqin; Kinge, Thompson; Anyangwe, Christopher; Tinyami, Erick T.; Haldar, Bijayesh; Powell, Rebecca L.R.; Jarido, Veronica; Hewlett, Indira K.; Nyambi, Phillipe N.

    2010-01-01

    The most common first-line, highly active anti-retroviral therapy (HAART) received by individuals infected with HIV-1 in Cameroon is the combination therapy Triomune, comprised of two nucleoside reverse transcriptase inhibitors (NRTI) and one non-NRTI (NNRTI). To examine the efficacy of these drugs in Cameroon, where diverse non-B HIV-1 subtypes and recombinant viruses predominate, the reverse transcriptase (RT) viral sequences in patient plasma were analyzed for the presence of mutations that confer drug resistance. Forty-nine HIV-1-positive individuals were randomly selected from those receiving care in HIV/AIDS outpatient clinics in the South-West and North-West Regions of Cameroon. Among the 28 patients receiving HAART, 39% (11/28) had resistance to NRTIs, and 46% (13/28) to NNRTIs after a median of 12 months from the start of therapy. Among those with drug-resistance mutations, there was a median of 14 months from the start of HAART, versus 9 months for those without; no difference was observed in the average viral load (10,997 copies/ml vs. 8,056 copies/ml). In contrast, drug-naïve individuals had a significantly higher average viral load (27,929 copies/ml) than those receiving HAART (9,527 copies/ml). Strikingly, among the 21 drug-naïve individuals, 24% harbored viruses with drug-resistance mutations, suggesting that HIV-1 drug-resistant variants are being transmitted in Cameroon. Given the high frequency of resistance mutations among those on first-line HAART, coupled with the high prevalence of HIV-1 variants with drug-resistance mutations among drug-naïve individuals, this study emphasizes the need for extensive monitoring of resistance mutations and the introduction of a second-line HAART strategy in Cameroon. PMID:20029816

  20. Proteolytic crosstalk in multi-protease networks

    NASA Astrophysics Data System (ADS)

    Ogle, Curtis T.; Mather, William H.

    2016-04-01

    Processive proteases, such as ClpXP in E. coli, are conserved enzyme assemblies that can recognize and rapidly degrade proteins. These proteases are used for a number of purposes, including degrading mistranslated proteins and controlling cellular stress response. However, proteolytic machinery within the cell is limited in capacity and can lead to a bottleneck in protein degradation, whereby many proteins compete (‘queue’) for proteolytic resources. Previous work has demonstrated that such queueing can lead to pronounced statistical relationships between different protein counts when proteins compete for a single common protease. However, real cells contain many different proteases, e.g. ClpXP, ClpAP, and Lon in E. coli, and it is not clear how competition between proteins for multiple classes of protease would influence the dynamics of cellular networks. In the present work, we theoretically demonstrate that a multi-protease proteolytic bottleneck can substantially couple the dynamics for both simple and complex (oscillatory) networks, even between substrates with substantially different affinities for protease. For these networks, queueing often leads to strong positive correlations between protein counts, and these correlations are strongest near the queueing theoretic point of balance. Furthermore, we find that the qualitative behavior of these networks depends on the relative size of the absolute affinity of substrate to protease compared to the cross affinity of substrate to protease, leading in certain regimes to priority queue statistics.

  1. [Pontine reversible leucopathy in an AIDS patient associated with highly active antiretroviral therapy (HAART): Report of one case].

    PubMed

    Cartier, Luis; Matamala, José Manuel; Yáñez, Alonso

    2016-05-01

    Posterior reversible encephalopathy (PRES) is a condition characterized by T2 and FLAIR hyperintensities in magnetic resonance imaging (MRI) studies, localized preferentially in the occipital-parietal white matter regions. Pathological MRI images located in midbrain, pons, medulla and spinal cord, that could be asymptomatic, were recently included in this entity. These images are interpreted as vasogenic edema, which is caused by arterial hypertension or eclampsia, neurotoxicity related to immunosuppressive agents or chemotherapy, among other causes. We report a 25 years old asymptomatic male with AIDS, with normal blood pressure who after initiating highly active antiretroviral therapy (HAART) reported vertigo. The MRI showed a central pontine T2 hyperintensity with diffusion restriction, which was interpreted as a central pontine myelinolysis (CPM), but the lack of motor symptoms made improbable a real demyelination of the pons. The follow-up MRI revealed complete regression of the images. To our knowledge, this case could be the second report of a reversible leucopathy of the pons in a patient with AIDS, were the MRI images also simulated a CPM. This report extends the knowledge around the variability of the pathogenic interpretation of CPM images and their association with HAART. PMID:27552021

  2. Late presentation and loss to follow-up of immigrants newly diagnosed with HIV in the HAART era.

    PubMed

    Saracino, A; Tartaglia, A; Trillo, G; Muschitiello, C; Bellacosa, C; Brindicci, G; Monno, L; Angarano, G

    2014-08-01

    To compare clinical characteristics and therapeutic management of newly HIV-diagnosed immigrants to natives. Patients with a first HIV diagnosis from 1996 to 2010 were included. Of 716 new diagnoses, 85 (12 %) were immigrants. Migrants were younger, more frequently females and sexually infected, less likely to voluntarily request testing, and less HCV-coinfected. Late presenters (CD4 <350 or AIDS) were 76 % among migrants versus 56 % in natives (p = 0.006) with an increasing trend over time. HAART was initiated in 76.5 % of natives and 72.4 % of immigrants; the number/type of adverse events and treatment discontinuation were similar. Immigrants received more NNRTIs-based regimens. A similar proportion of patients reached virological suppression at month 1-3-6 after HAART initiation, but 43 % of immigrants versus 27 % of natives resulted lost to follow-up (p < 0.001). Diagnosis of HIV was often delayed among migrants, who also presented a higher rate of lost to follow-up.

  3. Viral persistence, latent reservoir, and blips: a review on HIV-1 dynamics and modeling during HAART and related treatment implications

    SciTech Connect

    Rong, Libin; Perelson, Alan

    2008-01-01

    HIV-1 eradication from infected individuals has not been achieved with the use of highly active antiretroviral therapy (HAART) for a prolonged period of time. The cellular reservoir for HIV-1 in resting memory CD4{sup +} T cells remains a major obstacle to viral elimination. The reservoir does not decay significantly over long periods of time as is able to release replication competent HIV-1 upon cell activation. Residual ongoing viral replication may likely occur in many patients because low levels of virus can be detected in plasma by sensitive assays and transient episodes of viremia, or HIV-1 blips, are often observed in patients even with successful viral suppression for many years. Here we review our current knowledge of the factors contributing to viral persistence, the latent reservoir, and blips, and mathematical models developed to explore them and their relationships. We show how mathematical modeling can help improve our understanding of HIV-1 dynamics in patients on HAART and the quantitative events underlying HIV-1 latency, reservoir stability, low-level viremic persistence, and emergence of intermittent viral blips. We also discuss treatment implications related to these studies.

  4. Protease-degradable electrospun fibrous hydrogels

    NASA Astrophysics Data System (ADS)

    Wade, Ryan J.; Bassin, Ethan J.; Rodell, Christopher B.; Burdick, Jason A.

    2015-03-01

    Electrospun nanofibres are promising in biomedical applications to replicate features of the natural extracellular matrix (ECM). However, nearly all electrospun scaffolds are either non-degradable or degrade hydrolytically, whereas natural ECM degrades proteolytically, often through matrix metalloproteinases. Here we synthesize reactive macromers that contain protease-cleavable and fluorescent peptides and are able to form both isotropic hydrogels and electrospun fibrous hydrogels through a photoinitiated polymerization. These biomimetic scaffolds are susceptible to protease-mediated cleavage in vitro in a protease dose-dependent manner and in vivo in a subcutaneous mouse model using transdermal fluorescent imaging to monitor degradation. Importantly, materials containing an alternate and non-protease-cleavable peptide sequence are stable in both in vitro and in vivo settings. To illustrate the specificity in degradation, scaffolds with mixed fibre populations support selective fibre degradation based on individual fibre degradability. Overall, this represents a novel biomimetic approach to generate protease-sensitive fibrous scaffolds for biomedical applications.

  5. Early HAART Initiation May Not Reduce Actual Reproduction Number and Prevalence of MSM Infection: Perspectives from Coupled within- and between-Host Modelling Studies of Chinese MSM Populations.

    PubMed

    Sun, Xiaodan; Xiao, Yanni; Tang, Sanyi; Peng, Zhihang; Wu, Jianhong; Wang, Ning

    2016-01-01

    Having a thorough understanding of the infectivity of HIV, time of initiating treatment and emergence of drug resistant virus variants is crucial in mitigating HIV infection. There are many challenges to evaluating the long-term effect of the Highly Active Antiretroviral Therapy (HAART) on disease transmission at the population level. We proposed an individual based model by coupling within-host dynamics and between-host dynamics and conduct stochastic simulation in the group of men who have sex with men (MSM). The mean actual reproduction number is estimated to be 3.6320 (95% confidence interval: [3.46, 3.80]) for MSM group without treatment. Stochastic simulations show that given relatively high (low) level of drug efficacy after emergence of drug resistant variants, early initiation of treatment leads to a less (greater) actual reproduction number, lower (higher) prevalence and less (more) incidences, compared to late initiation of treatment. This implies early initiation of HAART may not always lower the actual reproduction number and prevalence of infection, depending on the level of treatment efficacy after emergence of drug resistant virus variants, frequency of high-risk behaviors and etc. This finding strongly suggests early initiation of HAART should be implemented with great care especially in the settings where the effective drugs are limited. Coupling within-host dynamics with between-host dynamics can provide critical information about impact of HAART on disease transmission and thus help to assist treatment strategy design and HIV/AIDS prevention and control.

  6. Early HAART Initiation May Not Reduce Actual Reproduction Number and Prevalence of MSM Infection: Perspectives from Coupled within- and between-Host Modelling Studies of Chinese MSM Populations

    PubMed Central

    Sun, Xiaodan; Xiao, Yanni; Tang, Sanyi; Peng, Zhihang; Wu, Jianhong; Wang, Ning

    2016-01-01

    Having a thorough understanding of the infectivity of HIV, time of initiating treatment and emergence of drug resistant virus variants is crucial in mitigating HIV infection. There are many challenges to evaluating the long-term effect of the Highly Active Antiretroviral Therapy (HAART) on disease transmission at the population level. We proposed an individual based model by coupling within-host dynamics and between-host dynamics and conduct stochastic simulation in the group of men who have sex with men (MSM). The mean actual reproduction number is estimated to be 3.6320 (95% confidence interval: [3.46, 3.80]) for MSM group without treatment. Stochastic simulations show that given relatively high (low) level of drug efficacy after emergence of drug resistant variants, early initiation of treatment leads to a less (greater) actual reproduction number, lower (higher) prevalence and less (more) incidences, compared to late initiation of treatment. This implies early initiation of HAART may not always lower the actual reproduction number and prevalence of infection, depending on the level of treatment efficacy after emergence of drug resistant virus variants, frequency of high-risk behaviors and etc. This finding strongly suggests early initiation of HAART should be implemented with great care especially in the settings where the effective drugs are limited. Coupling within-host dynamics with between-host dynamics can provide critical information about impact of HAART on disease transmission and thus help to assist treatment strategy design and HIV/AIDS prevention and control. PMID:26930406

  7. Causes of Death among People Living with AIDS in the Pre- and Post-HAART Eras in the City of São Paulo, Brazil

    PubMed Central

    Domingues, Carmen-Silvia Bruniera; Waldman, Eliseu Alves

    2014-01-01

    Objective We examine the trend in causes of death among people living with AIDS in the city of São Paulo, Brazil, in the periods before and after the introduction of highly active antiretroviral therapy (HAART), and we investigate potential disparities across districts of residence. Methods Descriptive study of three periods: pre-HAART (1991–1996); early post-HAART (1997–1999); and late post-HAART (2000–2006). The data source was the São Paulo State STD/AIDS Program and São Paulo State Data Analysis Foundation. Causes of death were classified by the ICD-9 (1991–1995) and ICD-10 (1996–2006). We estimated age-adjusted mortality rates for leading underlying causes of death and described underlying and associated causes of death according to sociodemographic characteristics and area of residence. We used Pearson's chi-square test or Fisher's exact test to compare categorical variables. Areas of residence were categorized using a socioeconomic index. To analyze trends we apply generalized linear model with Poisson regression. Results We evaluated 32,808 AIDS-related deaths. Between the pre- and late post-HAART periods, the proportion of deaths whose underlying causes were non-AIDS-related diseases increased from 0.2% to 9.6% (p<0.001): from 0.01% to 1.67% (p<0.001) for cardiovascular diseases; 0.01% to 1.62% (p<0.001) for bacterial/unspecified pneumonia; and 0.03% to 1.46% (p<0.001) for non-AIDS-defining cancers. In the late post-HAART period, the most common associated causes of death were bacterial/unspecified pneumonia (35.94%), septicemia (33.46%), cardiovascular diseases (10.11%) and liver diseases (8.0%); and common underlying causes, besides AIDS disease, included non-AIDS-defining cancers in high-income areas, cardiovascular diseases in middle-income areas and assault in low-income areas. Conclusions The introduction of HAART has shifted the mortality profile away from AIDS-related conditions, suggesting changes in the pattern of morbidity, but

  8. Mast cell proteases as pharmacological targets.

    PubMed

    Caughey, George H

    2016-05-01

    Mast cells are rich in proteases, which are the major proteins of intracellular granules and are released with histamine and heparin by activated cells. Most of these proteases are active in the granule as well as outside of the mast cell when secreted, and can cleave targets near degranulating mast cells and in adjoining tissue compartments. Some proteases released from mast cells reach the bloodstream and may have far-reaching actions. In terms of relative amounts, the major mast cell proteases include the tryptases, chymases, cathepsin G, carboxypeptidase A3, dipeptidylpeptidase I/cathepsin C, and cathepsins L and S. Some mast cells also produce granzyme B, plasminogen activators, and matrix metalloproteinases. Tryptases and chymases are almost entirely mast cell-specific, whereas other proteases, such as cathepsins G, C, and L are expressed by a variety of inflammatory cells. Carboxypeptidase A3 expression is a property shared by basophils and mast cells. Other proteases, such as mastins, are largely basophil-specific, although human basophils are protease-deficient compared with their murine counterparts. The major classes of mast cell proteases have been targeted for development of therapeutic inhibitors. Also, a human β-tryptase has been proposed as a potential drug itself, to inactivate of snake venins. Diseases linked to mast cell proteases include allergic diseases, such as asthma, eczema, and anaphylaxis, but also include non-allergic diseases such as inflammatory bowel disease, autoimmune arthritis, atherosclerosis, aortic aneurysms, hypertension, myocardial infarction, heart failure, pulmonary hypertension and scarring diseases of lungs and other organs. In some cases, studies performed in mouse models suggest protective or homeostatic roles for specific proteases (or groups of proteases) in infections by bacteria, worms and other parasites, and even in allergic inflammation. At the same time, a clearer picture has emerged of differences in the

  9. Gene expression and activity of digestive proteases in Daphnia: effects of cyanobacterial protease inhibitors

    PubMed Central

    2010-01-01

    Background The frequency of cyanobacterial blooms has increased worldwide, and these blooms have been claimed to be a major factor leading to the decline of the most important freshwater herbivores, i.e. representatives of the genus Daphnia. This suppression of Daphnia is partly attributed to the presence of biologically active secondary metabolites in cyanobacteria. Among these metabolites, protease inhibitors are found in almost every natural cyanobacterial bloom and have been shown to specifically inhibit Daphnia's digestive proteases in vitro, but to date no physiological responses of these serine proteases to cyanobacterial protease inhibitors in Daphnia have been reported in situ at the protein and genetic levels. Results Nine digestive proteases were detected in D. magna using activity-stained SDS-PAGE. Subsequent analyses by LC-MS/MS and database search led to the identification of respective protease genes. D. magna responded to dietary protease inhibitors by up-regulation of the expression of these respective proteases at the RNA-level and by the induction of new and less sensitive protease isoforms at the protein level. The up-regulation in response to dietary trypsin- and chymotrypsin-inhibitors ranged from 1.4-fold to 25.6-fold. These physiological responses of Daphnia, i.e. up-regulation of protease expression and the induction of isoforms, took place even after feeding on 20% cyanobacterial food for only 24 h. These physiological responses proved to be independent from microcystin effects. Conclusion Here for the first time it was shown in situ that a D. magna clone responds physiologically to dietary cyanobacterial protease inhibitors by phenotypic plasticity of the targets of these specific inhibitors, i.e. Daphnia gut proteases. These regulatory responses are adaptive for D. magna, as they increase the capacity for protein digestion in the presence of dietary protease inhibitors. The type and extent of these responses in protease expression might

  10. Neutralizing monoclonal antibodies to an extracellular Pseudomonas cepacia protease.

    PubMed Central

    Kooi, C; Cox, A; Darling, P; Sokol, P A

    1994-01-01

    Pseudomonas cepacia produces at least two extracellular proteases with apparent molecular masses of 36,000 and 40,000 Da. The 36-kDa protease has high proteolytic activity and the 40-kDa protease has low proteolytic activity with hide powder azure as a substrate. Monoclonal antibodies (MAbs) were raised against the purified 36- and 40-kDa proteases. Several MAbs directed against the 36-kDa protease were found to recognize the 40-kDa protease by Western immunoblot analysis. Similarly, a MAb directed against the 40-kDa protease recognized the 36-kDa protease, suggesting that these two proteases may be immunologically related. A MAb directed against the 36-kDa protease, designated 36-6-8, and a MAb directed against the 40-kDa protease (MAb G-11) cross-reacted with other extracellular proteases, such as Pseudomonas aeruginosa elastase and alkaline protease, Pseudomonas pseudomallei protease, and the Vibrio cholerae hemagglutinin/protease. MAb 36-6-8 neutralized the P. cepacia 36-kDa protease, P. aeruginosa elastase, P. pseudomallei protease, and V. cholerae hemagglutinin/protease but did not affect P. aeruginosa alkaline protease activity. In contrast, MAb G-11 to the 40-kDa protease neutralized only the P. cepacia 36-kDa protease. This evidence suggests that the neutralizing MAb, 36-6-8, recognizes an epitope conserved among some metalloproteases. This epitope may lie at or near the active site of the P. cepacia 36-kDa protease and P. aeruginosa elastase. Images PMID:7516312

  11. Epstein-Barr virus and human immunodeficiency virus serological responses and viral burdens in HIV-infected patients treated with HAART

    NASA Technical Reports Server (NTRS)

    O'Sullivan, Cathal E.; Peng, RongSheng; Cole, Kelly Stefano; Montelaro, Ronald C.; Sturgeon, Timothy; Jenson, Hal B.; Ling, Paul D.; Butel, J. S. (Principal Investigator)

    2002-01-01

    Epstein-Barr virus (EBV) associated non-Hodgkin lymphoma is recognized as a complication of human immunodeficiency virus (HIV) infection. Little is known regarding the influence of highly active antiretroviral therapy (HAART) on the biology of EBV in this population. To characterize the EBV- and HIV-specific serological responses together with EBV DNA levels in a cohort of HIV-infected adults treated with HAART, a study was conducted to compare EBV and HIV serologies and EBV DNA copy number (DNAemia) over a 12-month period after the commencement of HAART. All patients were seropositive for EBV at baseline. Approximately 50% of patients had detectable EBV DNA at baseline, and 27/30 had detectable EBV DNA at some point over the follow-up period of 1 year. Changes in EBV DNA copy number over time for any individual were unpredictable. Significant increases in the levels of Epstein-Barr nuclear antigen (EBNA) and Epstein-Barr early antigen (EA) antibodies were demonstrated in the 17 patients who had a good response to HAART. Of 29 patients with paired samples tested, four-fold or greater increases in titers were detected for EA in 12/29 (41%), for EBNA in 7/29 (24%), for VCA-IgG in 4/29 (14%); four-fold decreases in titers were detected in 2/29 (7%) for EA and 12/29 (41%) for EBNA. A significant decline in the titer of anti-HIV antibodies was also demonstrated. It was concluded that patients with advanced HIV infection who respond to HAART have an increase in their EBV specific antibodies and a decrease in their HIV-specific antibodies. For the cohort overall, there was a transient increase in EBV DNA levels that had declined by 12 months. Copyright 2002 Wiley-Liss, Inc.

  12. Regulation of protease production in Clostridium sporogenes.

    PubMed Central

    Allison, C; Macfarlane, G T

    1990-01-01

    The physiological and nutritional factors that regulate protease synthesis in Clostridium sporogenes C25 were studied in batch and continuous cultures. Formation of extracellular proteases occurred at the end of active growth and during the stationary phase in batch cultures. Protease production was inversely related to growth rate in glucose-excess and glucose-limited chemostats over the range D = 0.05 to 0.70 h-1. In pulse experiments, glucose, ammonia, phosphate, and some amino acids (tryptophan, proline, tyrosine, and isoleucine) strongly repressed protease synthesis. This repression was not relieved by addition of 4 mM cyclic AMP, cyclic GMP, or dibutyryl cyclic AMP. Protease formation was markedly inhibited by 4 mM ATP and ADP, but GTP and GDP had little effect on the process. It is concluded that protease production by C. sporogenes is strongly influenced by the amount of energy available to the cells, with the highest levels of protease synthesis occurring under energy-limiting conditions. PMID:2268158

  13. A biotechnology perspective of fungal proteases

    PubMed Central

    de Souza, Paula Monteiro; Bittencourt, Mona Lisa de Assis; Caprara, Carolina Canielles; de Freitas, Marcela; de Almeida, Renata Paula Coppini; Silveira, Dâmaris; Fonseca, Yris Maria; Ferreira, Edivaldo Ximenes; Pessoa, Adalberto; Magalhães, Pérola Oliveira

    2015-01-01

    Proteases hydrolyze the peptide bonds of proteins into peptides and amino acids, being found in all living organisms, and are essential for cell growth and differentiation. Proteolytic enzymes have potential application in a wide number of industrial processes such as food, laundry detergent and pharmaceutical. Proteases from microbial sources have dominated applications in industrial sectors. Fungal proteases are used for hydrolyzing protein and other components of soy beans and wheat in soy sauce production. Proteases can be produced in large quantities in a short time by established methods of fermentation. The parameters such as variation in C/N ratio, presence of some sugars, besides several other physical factors are important in the development of fermentation process. Proteases of fungal origin can be produced cost effectively, have an advantage faster production, the ease with which the enzymes can be modified and mycelium can be easily removed by filtration. The production of proteases has been carried out using submerged fermentation, but conditions in solid state fermentation lead to several potential advantages for the production of fungal enzymes. This review focuses on the production of fungal proteases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications. PMID:26273247

  14. Extracellular proteases of Trichoderma species. A review.

    PubMed

    Kredics, L; Antal, Zsuzsanna; Szekeres, A; Hatvani, L; Manczinger, L; Vágvölgyi, Cs; Nagy, Erzsébet

    2005-01-01

    Cellulolytic, xylanolytic, chitinolytic and beta-1,3-glucanolytic enzyme systems of species belonging to the filamentous fungal genus Trichoderma have been investigated in details and are well characterised. The ability of Trichoderma strains to produce extracellular proteases has also been known for a long time, however, the proteolytic enzyme system is relatively unknown in this genus. Fortunately, in the recent years more and more attention is focused on the research in this field. The role of Trichoderma proteases in the biological control of plant pathogenic fungi and nematodes has been demonstrated, and it is also suspected that they may be important for the competitive saprophytic ability of green mould isolates and may represent potential virulence factors of Trichoderma strains as emerging fungal pathogens of clinical importance. The aim of this review is to summarize the information available about the extracellular proteases of Trichoderma. Numerous studies are available about the extracellular proteolytic enzyme profiles of Trichoderma strains and about the effect of abiotic environmental factors on protease activities. A number of protease enzymes have been purified to homogeneity and some protease encoding genes have been cloned and characterized. These results will be reviewed and the role of Trichoderma proteases in biological control as well as their advantages and disadvantages in biotechnology will be discussed. PMID:16003937

  15. A biotechnology perspective of fungal proteases.

    PubMed

    de Souza, Paula Monteiro; Bittencourt, Mona Lisa de Assis; Caprara, Carolina Canielles; de Freitas, Marcela; de Almeida, Renata Paula Coppini; Silveira, Dâmaris; Fonseca, Yris Maria; Ferreira Filho, Edivaldo Ximenes; Pessoa Junior, Adalberto; Magalhães, Pérola Oliveira

    2015-06-01

    Proteases hydrolyze the peptide bonds of proteins into peptides and amino acids, being found in all living organisms, and are essential for cell growth and differentiation. Proteolytic enzymes have potential application in a wide number of industrial processes such as food, laundry detergent and pharmaceutical. Proteases from microbial sources have dominated applications in industrial sectors. Fungal proteases are used for hydrolyzing protein and other components of soy beans and wheat in soy sauce production. Proteases can be produced in large quantities in a short time by established methods of fermentation. The parameters such as variation in C/N ratio, presence of some sugars, besides several other physical factors are important in the development of fermentation process. Proteases of fungal origin can be produced cost effectively, have an advantage faster production, the ease with which the enzymes can be modified and mycelium can be easily removed by filtration. The production of proteases has been carried out using submerged fermentation, but conditions in solid state fermentation lead to several potential advantages for the production of fungal enzymes. This review focuses on the production of fungal proteases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications. PMID:26273247

  16. Serostatus disclosure to sexual partners by HIV-infected women before and after the advent of HAART.

    PubMed

    Siegel, Karolynn; Lekas, Helen-Marie; Schrimshaw, Eric W

    2005-01-01

    HIV-positive individuals have been encouraged by public health officials to disclose their HIV status to sexual partners. In deciding what to do, however, they must weigh what they see as the potential costs and benefits of disclosing or not disclosing. In the present report we examine the reasons women offer for disclosing or not disclosing their serostatus and the reactions to that disclosure among two matched samples of HIV-infected women. The first sample was interviewed in 1994-1996, before the widespread availability of HAART; while the second sample was interviewed from 2000-2003 after these medications were widely in use. The findings reveal striking similarities between the two time periods in women's reasons for sharing or not sharing their status with partners, and the reactions to disclosure they experienced. The reconceptualization of AIDS as a chronic illness, rather than an acute fatal one, did not appear to have diminished women's felt responsibility to share their diagnosis with potential sexual partners nor their fear that disclosure would be met with rejection. The data revealed that disclosure remains a highly stressful event for HIV-infected women and that they experience considerable emotional suffering as a result of the diminished sense of self-worth and physical attractiveness brought about by their diagnosis. These findings suggest that intervention efforts toward reducing the stigmatization of HIV/AIDS and for assisting women to manage the stress of disclosure and non-disclosure and address women's feelings of self-worth continue to be needed despite the advent of HAART. PMID:16260414

  17. Cysteine Proteases from Bloodfeeding Arthropod Ectoparasites

    PubMed Central

    Sojka, Daniel; Francischetti, Ivo M. B.; Calvo, Eric; Kotsyfakis, Michalis

    2012-01-01

    Cysteine proteases have been discovered in various bloodfeeding ectoparasites. Here, we assemble the available information about the function of these peptidases and reveal their role in hematophagy and parasite development. While most of the data shed light on key proteolytic events that play a role in arthropod physiology, we also report on the association of cysteine proteases with arthropod vectorial capacity. With emphasis on ticks, specifically Ixodes ricinus, we finally propose a model about the contribution of cysteine peptidases to blood digestion, and how their concerted action with other tick midgut proteases leads to the absorbance of nutrients by the midgut epithelial cells. PMID:21660665

  18. HIV-1 Protease: Structure, Dynamics and Inhibition

    SciTech Connect

    Louis, John M.; Ishima, R.; Torchia, D.A.; Weber, Irene T.

    2008-06-03

    The HIV-1 protease is synthesized as part of a large Gag-Pol precursor protein. It is responsible for its own release from the precursor and the processing of the Gag and Gag-Pol polyproteins into the mature structural and functional proteins required for virus maturation. Because of its indispensable role, the mature HIV-1 protease dimer has proven to be a successful target for the development of antiviral agents. In the last 5 years, a major emphasis in protease research has been to improve inhibitor design and treatment regimens.

  19. Evaluation of virulence factors of Candida albicans isolated from HIV-positive individuals using HAART.

    PubMed

    de Paula Menezes, Ralciane; de Melo Riceto, Érika Bezerra; Borges, Aércio Sebastião; de Brito Röder, Denise Von Dolingër; dos Santos Pedroso, Reginaldo

    2016-06-01

    The colonization by Candida species is one of the most important factors related to the development of oral candidiasis in HIV-infected individuals. The aim of the study was to evaluate and discuss the phospholipase, proteinase, DNAse and haemolytic activities of Candida albicans isolated from the oral cavity of HIV individuals with high efficiency antiretroviral therapy. Seventy-five isolates of C. albicans obtained from saliva samples of patients with HIV and 41 isolates from HIV-negative individuals were studied. Haemolytic activity was determined in Sabouraud dextrose agar plates containing 3% glucose and 7% sheep red cells. Culture medium containing DNA base-agar, egg yolk, and bovine albumin were used to determine DNase, phospholipase and proteinase activities, respectively. All isolates from the HIV patients group had haemolytic activity, 98% showed phospholipase activity, 92% were positive for proteinase and 32% DNAse activity. Regarding the group of indivídios HIV negative, all 41 isolates presented hemolytic activity, 90.2% showed phospholipase and proteinase activity and 12.2% were positive for DNAse. The phospholipase activity was more intense for the group of HIV positive individuals. DNase production was more frequently observed in the group of HIV-positive individuals. The percentage of isolates having DNAse activity was also significantly different between the groups of patients not using any antiretroviral therapy, those using transcriptase inhibitors and those using transcriptase inhibitor and protease inhibitor in combination. PMID:26913969

  20. Flexible Cyclic Ethers/Polyethers as Novel P2-Ligands for HIV-1 Protease Inhibitors: Design, Synthesis, Biological Evaluation, and Protein-Ligand X-Ray Studies

    SciTech Connect

    Ghosh, Arun; Gemma, Sandra; Baldridge, Abigal; Wang, Yuan-Fang; Kovalevsky, Andrey; Koh, Yashiro; Weber, Irene; Mitsuya, Hiroaki

    2008-12-05

    We report the design, synthesis, and biological evaluation of a series of novel HIV-1 protease inhibitors. The inhibitors incorporate stereochemically defined flexible cyclic ethers/polyethers as high affinity P2-ligands. Inhibitors containing small ring 1,3-dioxacycloalkanes have shown potent enzyme inhibitory and antiviral activity. Inhibitors 3d and 3h are the most active inhibitors. Inhibitor 3d maintains excellent potency against a variety of multi-PI-resistant clinical strains. Our structure-activity studies indicate that the ring size, stereochemistry, and position of oxygens are important for the observed activity. Optically active synthesis of 1,3-dioxepan-5-ol along with the syntheses of various cyclic ether and polyether ligands have been described. A protein-ligand X-ray crystal structure of 3d-bound HIV-1 protease was determined. The structure revealed that the P2-ligand makes extensive interactions including hydrogen bonding with the protease backbone in the S2-site. In addition, the P2-ligand in 3d forms a unique water-mediated interaction with the NH of Gly-48.

  1. Protease and protease inhibitory activity in pregnant and postpartum involuting uterus

    SciTech Connect

    Milwidsky, A.; Beller, U.; Palti, Z.; Mayer, M.

    1982-08-15

    The presence of two distinct proteolytic activities in the rat uterus was confirmed with /sup 14/C-labeled globin used as a sensitive protein substrate and following release of label into the trichloroacetic acid-soluble supernatant fraction. Protease I is a cytoplasmic acid protease while protease II is associated with the pellet fraction, can be extracted by 0.6 M sodium chloride, and is active at pH 7.0. Protease I activity is low during pregnancy and markedly increases at term achieving maximal activity at day 3 post partum with a subsequent decline to preterm activity values. Lactation did not affect the uterine protease I activity. Protease II activity is not significantly different during pregnancy, at term, and post partum. The presence of an inhibitor of protease I was suggested by a decrease in enzyme activity with an increased cytosolic protein concentration. The inhibitor also lessened bovine trypsin activity but had no effect on protease II. Although its inhibitory potency on trypsin fluctuated during the various uterine physiologic stages, these changes appeared to be statistically insignificant. Human uterine samples were also found to contain the two protease activities with similar changes in protease I post partum. It is suggested that, both in the rat and in man, uterine involution post partum is associated with a marked increase in activity of acid cytosolic protease, while a particulate neutral protease and a soluble inhibitor of trypsin, which are also present in uterine cells, do not appear to play a significant role in the dissolution of uterine tissues after parturition.

  2. Development of Broad-Spectrum Halomethyl Ketone Inhibitors Against Coronavirus Main Protease 3CL(pro)

    SciTech Connect

    Bacha,U.; Barilla, J.; Gabelli, S.; Kiso, Y.; Amzel, L.; Freire, E.

    2008-01-01

    Coronaviruses comprise a large group of RNA viruses with diverse host specificity. The emergence of highly pathogenic strains like the SARS coronavirus (SARS-CoV), and the discovery of two new coronaviruses, NL-63 and HKU1, corroborates the high rate of mutation and recombination that have enabled them to cross species barriers and infect novel hosts. For that reason, the development of broad-spectrum antivirals that are effective against several members of this family is highly desirable. This goal can be accomplished by designing inhibitors against a target, such as the main protease 3CLpro (Mpro), which is highly conserved among all coronaviruses. Here 3CLpro derived from the SARS-CoV was used as the primary target to identify a new class of inhibitors containing a halomethyl ketone warhead. The compounds are highly potent against SARS 3CLpro with Ki's as low as 300 nm. The crystal structure of the complex of one of the compounds with 3CLpro indicates that this inhibitor forms a thioether linkage between the halomethyl carbon of the warhead and the catalytic Cys 145. Furthermore, Structure Activity Relationship (SAR) studies of these compounds have led to the identification of a pharmacophore that accurately defines the essential molecular features required for the high affinity.

  3. Human immunodeficiency virus 1 protease expressed in Escherichia coli behaves as a dimeric aspartic protease.

    PubMed Central

    Meek, T D; Dayton, B D; Metcalf, B W; Dreyer, G B; Strickler, J E; Gorniak, J G; Rosenberg, M; Moore, M L; Magaard, V W; Debouck, C

    1989-01-01

    Recombinant human immunodeficiency virus 1 (HIV-1) protease, purified from a bacterial expression system, processed a recombinant form of its natural substrate, Pr55gag, into protein fragments that possess molecular weights commensurate with those of the virion gag proteins. Molecular weights of the protease obtained under denaturing and nondenaturing conditions (11,000 and 22,000, respectively) and chemical crosslinking studies were consistent with a dimeric structure for the active enzyme. The protease appropriately cleaved the nonapeptide Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 between the tyrosine and proline residues. HIV-1 protease was sensitive to inactivators of the aspartic proteases. The aspartic protease inactivator 1,2-epoxy-3-(4-nitrophenoxy)propane produced irreversible, time-dependent inactivation of the protease. The pH-dependent kinetics of this inactivator were consistent with the requirement of an unprotonated carboxyl group in the active site of the enzyme, suggesting that HIV-1 protease is also an aspartic protease. Images PMID:2648384

  4. Secreted fungal aspartic proteases: A review.

    PubMed

    Mandujano-González, Virginia; Villa-Tanaca, Lourdes; Anducho-Reyes, Miguel Angel; Mercado-Flores, Yuridia

    2016-01-01

    The aspartic proteases, also called aspartyl and aspartate proteases or acid proteases (E.C.3.4.23), belong to the endopeptidase family and are characterized by the conserved sequence Asp-Gly-Thr at the active site. These enzymes are found in a wide variety of microorganisms in which they perform important functions related to nutrition and pathogenesis. In addition, their high activity and stability at acid pH make them attractive for industrial application in the food industry; specifically, they are used as milk-coagulating agents in cheese production or serve to improve the taste of some foods. This review presents an analysis of the characteristics and properties of secreted microbial aspartic proteases and their potential for commercial application. PMID:27137097

  5. Secreted fungal aspartic proteases: A review.

    PubMed

    Mandujano-González, Virginia; Villa-Tanaca, Lourdes; Anducho-Reyes, Miguel Angel; Mercado-Flores, Yuridia

    2016-01-01

    The aspartic proteases, also called aspartyl and aspartate proteases or acid proteases (E.C.3.4.23), belong to the endopeptidase family and are characterized by the conserved sequence Asp-Gly-Thr at the active site. These enzymes are found in a wide variety of microorganisms in which they perform important functions related to nutrition and pathogenesis. In addition, their high activity and stability at acid pH make them attractive for industrial application in the food industry; specifically, they are used as milk-coagulating agents in cheese production or serve to improve the taste of some foods. This review presents an analysis of the characteristics and properties of secreted microbial aspartic proteases and their potential for commercial application.

  6. Vanadium inhibition of serine and cysteine proteases.

    PubMed

    Guerrieri, N; Cerletti, P; De Vincentiis, M; Salvati, A; Scippa, S

    1999-03-01

    A study was made on the effect of vanadium, in both the tetravalent state in vanadyl sulphate and in the pentavalent state in sodium meta-vanadate, and ortho-vanadate, on the proteolysis of azocasein by two serine proteases, trypsin and subtilisin and two cysteine proteases bromelain and papain. Also the proteolysis of bovine azoalbumin by serine proteases was considered. An inhibitory effect was present in all cases, except meta-vanadate with subtilisin. The oxidation level of vanadium by itself did not determine the inhibition kinetics, which also depended on the type and composition of the vanadium containing molecule and on the enzyme assayed. The pattern of inhibition was similar for proteases belonging to the same class. The highest inhibition was obtained with meta-vanadate on papain and with vanadyl sulphate on bromelain.

  7. Bioinformatics of proteases in the MEROPS database.

    PubMed

    Barrett, Alan J

    2004-05-01

    Proteolytic enzymes represent approximately approximately 2% of the total number of proteins present in all types of organisms. Many of these enzymes are of medical importance, and those that are of potential interest as drug targets can be divided into the endogenous enzymes encoded in the human genome, and the exogenous proteases encoded in the genomes of disease-causing organisms. There are also naturally occurring inhibitors of proteases, some of which have pharmaceutical relevance. The MEROPS database provides a rich source of information on proteases and their inhibitors. Storage and retrieval of this information is facilitated by the use of a hierarchical classification system (which was pioneered by the compilers of the database) in which homologous proteases and their inhibitors are divided into clans and families. PMID:15216937

  8. Electrically sensing protease activity with nanopores

    NASA Astrophysics Data System (ADS)

    Kukwikila, Mikiembo; Howorka, Stefan

    2010-11-01

    The enzymatic activity of a protease was electrically detected using nanopore recordings. A peptide substrate was tethered to microscale beads, and cleavage by the enzyme trypsin released a soluble fragment that was electrophoretically driven through the α-hemolysin protein pore, leading to detectable blockades in the ionic current. Owing to its simplicity, this approach to sense enzymatic activity may be applied to other proteases.

  9. Changing Patterns in the Neuropathogenesis of HIV During the HAART Era

    PubMed Central

    Langford, T. D.; Letendre, S. L.; Larrea, G. J.; Masliah, E.

    2015-01-01

    Rapid progress in the development of highly active antiretroviral therapy has changed the observed patterns in HIV encephalitis and AIDS-related CNS opportunistic infections. Early in the AIDS epidemic, autopsy studies pointed to a high prevalence of these conditions. With the advent of nucleoside reverse transcriptase inhibitors, the prevalence at autopsy of opportunistic infections, such as toxoplasmosis and progressive multifocal leukoencephalopathy, declined while that of HIV encephalitis increased. After the introduction of protease inhibitors, a decline in both HIV encephalitis and CNS opportunistic infections was observed. However, with the increasing resistance of HIV strains to anti-retrovirals, there has been a resurgence in the frequency of HIV encephalitis and HIV leukoencephalopathy. HIV leukoencephalopathy in AIDS patients failing highly active antiretroviral therapy is characterized by massive infiltration of HIV infected monocytes/macrophages into the brain and extensive white matter destruction. This condition may be attributable to interactions of anti-retrovirals with cerebrovascular endothelium, astroglial cells and white matter of the brain. These interactions may lead to cerebral ischemia, increased blood-brain barrier permeability and demyelination. Potential mechanisms of such interactions include alterations in host cell signaling that may result in trophic factor dysregulation and mitochondrial injury. We conclude that despite the initial success of combined anti-retroviral therapy, more severe forms of HIV encephalitis appear to be emerging as the epidemic matures. Factors that may contribute to this worsening include the prolonged survival of HIV-infected patients, thereby prolonging the brain’s exposure to HIV virions and proteins, the use of increasingly toxic combinations of poorly penetrating drugs in highly antiretroviral-experienced AIDS patients, and selection of more virulent HIV strains with higher replication rates and

  10. HIV Protease Inhibitors and Obesity

    PubMed Central

    Anuurad, Erdembileg; Bremer, Andrew; Berglund, Lars

    2011-01-01

    Purpose of review To review the current scientific literature and recent clinical trials on HIV protease inhibitors (PIs) and their potential role in the pathogenesis of lipodystrophy and metabolic disorders. Recent findings HIV PI treatment may affect the normal stimulatory effect of insulin on glucose and fat storage. Further, chronic inflammation from HIV infection and PI treatment trigger cellular homeostatic stress responses with adverse effects on intermediary metabolism. The physiologic outcome is such that total adipocyte storage capacity is decreased, and the remaining adipocytes resist further fat storage. This process leads to a pathologic cycle of lipodystrophy and lipotoxicity, a pro-atherogenic lipid profile, and a clinical phenotype of increased central body fat distribution similar to the metabolic syndrome. Summary PIs are a key component of antiretroviral therapy and have dramatically improved the life expectancy of HIV-infected individuals. However, they are also associated with abnormalities in glucose/lipid metabolism and body fat distribution. Further studies are needed to better define the pathogenesis of PI-associated metabolic and body fat changes and their potential treatment. PMID:20717021

  11. ADAM Proteases and Gastrointestinal Function.

    PubMed

    Jones, Jennifer C; Rustagi, Shelly; Dempsey, Peter J

    2016-01-01

    A disintegrin and metalloproteinases (ADAMs) are a family of cell surface proteases that regulate diverse cellular functions, including cell adhesion, migration, cellular signaling, and proteolysis. Proteolytically active ADAMs are responsible for ectodomain shedding of membrane-associated proteins. ADAMs rapidly modulate key cell signaling pathways in response to changes in the extracellular environment (e.g., inflammation) and play a central role in coordinating intercellular communication within the local microenvironment. ADAM10 and ADAM17 are the most studied members of the ADAM family in the gastrointestinal tract. ADAMs regulate many cellular processes associated with intestinal development, cell fate specification, and the maintenance of intestinal stem cell/progenitor populations. Several signaling pathway molecules that undergo ectodomain shedding by ADAMs [e.g., ligands and receptors from epidermal growth factor receptor (EGFR)/ErbB and tumor necrosis factor α (TNFα) receptor (TNFR) families] help drive and control intestinal inflammation and injury/repair responses. Dysregulation of these processes through aberrant ADAM expression or sustained ADAM activity is linked to chronic inflammation, inflammation-associated cancer, and tumorigenesis.

  12. Neutral serine proteases of neutrophils.

    PubMed

    Kettritz, Ralph

    2016-09-01

    Neutrophil serine proteases (NSPs) exercise tissue-degrading and microbial-killing effects. The spectrum of NSP-mediated functions grows continuously, not least because of methodological progress. Sensitive and specific FRET substrates were developed to study the proteolytic activity of each NSP member. Advanced biochemical methods are beginning to characterize common and specific NSP substrates. The resulting novel information indicates that NSPs contribute not only to genuine inflammatory neutrophil functions but also to autoimmunity, metabolic conditions, and cancer. Tight regulatory mechanisms control the proteolytic potential of NSPs. However, not all NSP functions depend on their enzymatic activity. Proteinase-3 (PR3) is somewhat unique among the NSPs for PR3 functions as an autoantigen. Patients with small-vessel vasculitis develop autoantibodies to PR3 that bind their target antigens on the neutrophil surface and trigger neutrophil activation. These activated cells subsequently contribute to vascular necrosis with life-threatening multiorgan failure. This article discusses various aspects of NSP biology and highlights translational aspects with strong clinical implications. PMID:27558338

  13. ADAM Proteases and Gastrointestinal Function

    PubMed Central

    Jones, Jennifer C.; Rustagi, Shelly; Dempsey, Peter J.

    2016-01-01

    A disintegrin and metalloproteinases (ADAMs) are a family of cell surface proteases that regulate diverse cellular functions, including cell adhesion, migration, cellular signaling, and proteolysis. Proteolytically active ADAMs are responsible for ectodomain shedding of membrane-associated proteins. ADAMs rapidly modulate key cell signaling pathways in response to changes in the extracellular environment (e.g., inflammation) and play a central role in coordinating intercellular communication within the local microenvironment. ADAM10 and ADAM17 are the most studied members of the ADAM family in the gastrointestinal tract. ADAMs regulate many cellular processes associated with intestinal development, cell fate specification, and the maintenance of intestinal stem cell/progenitor populations. Several signaling pathway molecules that undergo ectodomain shedding by ADAMs [e.g., ligands and receptors from epidermal growth factor receptor (EGFR)/ErbB and tumor necrosis factor α (TNFα) receptor (TNFR) families] help drive and control intestinal inflammation and injury/repair responses. Dysregulation of these processes through aberrant ADAM expression or sustained ADAM activity is linked to chronic inflammation, inflammation-associated cancer, and tumorigenesis. PMID:26667078

  14. Carbohydrate protease conjugates: Stabilized proteases for peptide synthesis

    SciTech Connect

    Wartchow, C.A.; Wang, Peng; Bednarski, M.D.; Callstrom, M.R. |

    1995-12-31

    The synthesis of oligopeptides using stable carbohydrate protease conjugates (CPCs) was examined in acetonitrile solvent systems. CPC[{alpha}-chymotrypsin] was used for the preparation of peptides containing histidine, phenylalanine, tryptophan in the P{sub 1} position in 60-93% yield. The CPC[{alpha}-chymotrypsin]-catalyzed synthesis of octamer Z-Gly-Gly-Phe-Gly-Gly-Phe-Gly-Gly-OEt from Z-Gly-Gly-Phe-Gly-Gly-Phe-OMe was achieved in 71% yield demonstrating that synthesis peptides containing both hydrophylic and hydrophobic amino acids. The P{sub 2} specificity of papain for aromatic residues was utilized for the 2 + 3 coupling of Z-Tyr-Gly-OMe to H{sub 2}N-Gly-Phe-Leu-OH to generate the leucine enkephalin derivative in 79% yield. Although papain is nonspecific for the hydrolysis of N-benzyloxycarbonyl amino acid methyl esters in aqueous solution, the rates of synthesis for these derivitives with nucleophile leucine tert-butyl ester differed by nearly 2 orders of magnitude. CPC[thermolysin] was used to prepare the aspartame precursor Z-Asp-Phe-OMe in 90% yield. The increased stability of CPCs prepared from periodate-modified poly(2-methacryl- amido-2-deoxy-D-glucose), poly(2-methacrylamido-2-deoxy-D-galactose), and poly(5-methacryl-amido-5-deoxy-D-ribose), carbohydrate materials designed to increase the aldehyde concentration in aqueous solution, suggests that the stability of CPCs is directly related to the aldehyde concentration of the carbohydrate material. Periodate oxidation of poly(2-methacrylamido-2-deoxy-D-glucose) followed by covalent attachment to {alpha}-chymotrypsin gave a CPC with catalytic activity in potassium phosphate buffer at 90{degrees}C for 2 h. 1 fig., 1 tab., 40 refs.

  15. The impact of integrating food supplementation, nutritional education and HAART (Highly Active Antiretroviral Therapy) on the nutritional status of patients living with HIV/AIDS in Mozambique: results from the DREAM Programme.

    PubMed

    Scarcella, P; Buonomo, E; Zimba, I; Doro Altan, A M; Germano, P; Palombi, L; Marazzi, M C

    2011-01-01

    DREAM (Drug Resources Enhancement against AIDS and Malnutrition) is a multiregional health program active in Mozambique since 2002 and provides free of charge an integrating package of care consisting of peer to peer nutritional and health education, food supplementation, voluntary counseling and testing, immunological, virological, clinical assessment and HAART (Highly Active AntiRetroviral Treatment). The main goals of this paper are to describe the state of health and nutrition and the adequacy of the diet of a sample of HIV/AIDS patients in Mozambique on HAART and not. A single-arm retrospective cohort study was conducted. 106 HIV/AIDS adult patients (84 in HAART), all receiving food supplementation and peer-to-peer nutritional education, were randomly recruited in Mozambique in two public health centres where DREAM is running. The programme is characterized by: provision of HAART, clinical and laboratory monitoring, peer to peer health and nutritional education and food supplementation. We measured BMI, haemoglobin, viral load, CD4 count at baseline (T0) and after at least 1 year (T1). Dietary intake was estimated using 24h food recall and dietary diversity was assessed by using the Dietary Diversity Score (DDS) at T1. Overall, the patients'diet appeared to be quite balanced in nutrients. In the cohort not in HAART the mean BMI values showed an increases but not significant (initial value: 21.9 ± 2.9; final value: 22.5 ± 3.3 ) and the mean haemoglobin values (g/dl) showed a significant increases (initial value: 10.5+ 2.1; final value: 11.5 ± 1.7 p< 0.024) . In the cohort in HAART, both the mean of BMI value (initial value: 20.7 ± 3.9; final value: 21.9 ± 3.3 p< 0.001) and of haemoglobin (initial value: 9.9 ± 2.2; final value: 10.8 ± 1.7 p< 0.001) showed a higher significant increase. The increase in BMI was statistically associated with the DDS in HAART patients. In conclusion nutritional status improvement was observed in both cohorts. The improvement

  16. Protease Inhibitors Targeting Coronavirus and Filovirus Entry

    PubMed Central

    Zhou, Yanchen; Vedantham, Punitha; Lu, Kai; Agudelo, Juliet; Carrion, Ricardo; Nunneley, Jerritt W.; Barnard, Dale; Pöhlmann, Stefan; McKerrow, James H.; Renslo, Adam R.; Simmons, Graham

    2016-01-01

    In order to gain entry into cells, diverse viruses, including Ebola virus, SARS-coronavirus and the emerging MERS-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. The respective enzymes are thus excellent targets for antiviral intervention. In cell culture, activation of Ebola virus, as well as SARS- and MERS-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin L (CTSL) and cathepsin B (CTSB). In addition, SARS- and MERS-coronavirus can use serine proteases localized at the cell surface, for their activation. However, it is currently unclear which protease(s) facilitate viral spread in the infected host. We report here that the cysteine protease inhibitor K11777, ((2S)-N-[(1E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2-{[(E)-4-methylpiperazine-1-carbonyl]amino}-3-phenylpropanamide) and closely-related vinylsulfones act as broad-spectrum antivirals by targeting cathepsin-mediated cell entry. K11777 is already in advanced stages of development for a number of parasitic diseases, such as Chagas disease, and has proven to be safe and effective in a range of animal models. K11777 inhibition of SARS-CoV and Ebola virus entry was observed in the sub-nanomolar range. In order to assess, whether cysteine or serine proteases promote viral spread in the host, we compared the antiviral activity of an optimized K11777-derivative with that of camostat, an inhibitor of TMPRSS2 and related serine proteases. Employing a pathogenic animal model of SARS-CoV infection, we demonstrated that viral spread and pathogenesis of SARS-CoV is driven by serine rather than cysteine proteases and can be effectively prevented by camostat. Camostat has been clinically used to treat chronic pancreatitis, and thus represents an exciting potential therapeutic for respiratory coronavirus infections. Our results indicate that camostat, or similar serine protease inhibitors, might be an effective option for treatment of SARS and

  17. New soluble ATP-dependent protease, Ti, in Escherichia coli that is distinct from protease La

    SciTech Connect

    Chung, C.H.; Hwang, B.J.; Park, W.J.; Goldberg, A.L.

    1987-05-01

    E. coli must contain other ATP-requiring proteolytic systems in addition to protease La (the lon gene product). A new ATP-dependent protease was purified from lon cells which lack protease La, as shown by immuno-blotting. This enzyme hydrolyzes (TH)casein to acid-soluble products in the presence of ATP (or dATP) and MgS . Nonhydrolyzable ATP analogs, other nucleoside triphosphates and AMP can not replace ATP. Therefore, ATP hydrolysis appears necessary for proteolysis. The enzyme appears to be a serine protease, but also contains essential thiol residues. Unlike protease La, it is not inhibited by vanadate, heparin, or the defective R9 subunit of protease La. On gel filtration, this enzyme has an apparent Mr of 340,000 and is comprised of two components of 190,000D and 130,000D, which can be separated by phosphocellulose chromatography. By themselves, these components do not show ATP-dependent proteolysis, but when mixed, full activity is restored. These finding and similar ones of Maurizi and Gottesman indicate that E. coli contain two soluble ATP-dependent proteases, which function by different mechanisms. This new enzyme may contribute to the rapid breakdown of abnormal polypeptides or of normal proteins during starvation. The authors propose to name it protease Ti.

  18. Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La

    SciTech Connect

    Hwang, B.J.; Park, W.J.; Chung, C.H.; Goldberg, A.L.

    1987-08-01

    The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon/sup -/ cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes (/sup 3/H)methyl-casein to acid-soluble products in the presence of ATP and Mg/sup 2 +/. ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. They propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show preteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles.

  19. Regulatory T cells generated during cytomegalovirus in vitro stimulation of mononuclear cells from HIV-infected individuals on HAART correlate with decreased lymphocyte proliferation

    SciTech Connect

    Jesser, Renee D.; Li, Shaobing; Weinberg, Adriana . E-mail: Adriana.Weinberg@uchsc.edu

    2006-09-01

    HIV-infected patients fail to fully recover cell-mediated immunity despite HAART. To identify regulatory factors, we studied the phenotype and function of in vitro cytomegalovirus (CMV)-stimulated T cells from HAART recipients. CFSE-measured proliferation showed CD4{sup +} and CD8{sup +} cells dividing in CMV-stimulated cultures. Compared with healthy controls, CMV-stimulated lymphocytes from HAART recipients had lower {sup 3}H-thymidine incorporation; lower IFN{gamma} and TNF{alpha} production; higher CD4{sup +}CD27{sup -}CD28{sup -} and CD8{sup +}CD27{sup -}CD28{sup -} frequencies; lower CD4{sup +}CD25{sup hi}; and higher FoxP3 expression in CD8{sup +}CD25{sup hi} cells. CMV-specific proliferation correlated with higher IFN{gamma}, TNF{alpha} and IL10 levels and higher CD4{sup +}perforin{sup +} and CD8{sup +}perforin{sup +} frequencies. Decreased proliferation correlated with higher CD4{sup +}CD27{sup -}CD28{sup -} frequencies and TGF{beta}1 production, which also correlated with each other. Anti-TGF{beta}1 neutralizing antibodies restored CMV-specific proliferation in a dose-dependent fashion. In HIV-infected subjects, decreased proliferation correlated with higher CMV-stimulated CD8{sup +}CD25{sup hi} frequencies and their FoxP3 expression. These data indicate that FoxP3- and TGF{beta}1-expressing regulatory T cells contribute to decreased immunity in HAART recipients.

  20. Risk factors, CD4 long-term evolution and mortality of HIV-infected patients who persistently maintain low CD4 counts, despite virological response to HAART.

    PubMed

    Pacheco, Yolanda M; Jarrín, Inmaculada; Del Amo, Julia; Moreno, Santiago; Iribarren, José A; Viciana, Pompeyo; Parra, Jorge; Gomez-Sirvent, Juan L; Gutierrez, Félix; Blanco, José R; Vidal, Francesc; Leal, Manuel

    2009-11-01

    A proportion of HIV-patients does not normally restore their CD4 counts despite virological response to HAART. Those whose CD4 counts persistently remain closed to the critical threshold for opportunistic infections deserve special interest. To study the risk factors, the long-term CD4 counts evolution, and the risk of death of patients who persistently maintain low CD4 counts, despite virological response to HAART, within a multicenter, hospital-based cohort study. A total of 147 patients were selected from CoRIS-MD and classified into a "Low-Group" or a "High-Group", depending on their CD4 counts after two-years of effective HAART (threshold 250 cells/microL). Associated risk factors were analysed by logistic regression, the CD4 dynamics were evaluated over a total period of 7.70 years (IQR, 6.70-9.00), and mortality was estimated by Cox proportional hazard. A total of 40 patients (27%) were classified into the "Low-Group". The odds ratio for this group increased with age, being 4.56 (2.23-9.33) for over 40, and was also higher among IDU, 3.63 (1.04-12.68). Six years thereafter, among these patients, only a 30% exceeded 350 CD4 cells/microL and a 12% exceeded 500 CD4 cells/microL. Furthermore, the "Low-Group" had a death rate of 2.42 per 100 persons/year (95%CI, 1.01-5.81), although once adjusted by age the estimates were no longer significant [4.14 (0.87-19.72)]. Our results suggest that those HIV patients who have not overcome the critical threshold of 250 CD4 cells/microL after a two years period of virologically effective HAART do persist with the aforementioned failure of CD4 restoration for a much longer time.

  1. Impact of genotypic drug resistance mutations on clinical and immunological outcomes in HIV-infected adults on HAART in West Africa

    PubMed Central

    Seyler, Catherine; Adjé-Touré, Christiane; Messou, Eugène; Dakoury-Dogbo, Nicole; Rouet, François; Gabillard, Delphine; Nolan, Monica; Toure, Siaka; Anglaret, Xavier

    2007-01-01

    Background The impact of HIV-1 drug resistance mutations in African adults on HAART has never been reported. Methods In 2004 in Abidjan, 106 adults on HAART had plasma viral load (VL) measurements. Patients with detectable VL had resistance genotypic tests. Patients were followed-up until 2006. Main outcomes were serious morbidity and immunological failure (CD4 count < 200/mm3). Results At study entry, the median previous time on HAART was 37 months and the median CD4 count 266/mm3; 58% of patients had undetectable VL, 20% detectable VL with no major resistance mutations, and 22% detectable VL with ≥1 major mutations. At study termination, 20% of patients had <200 CD4/mm3. Factors associated with immunological failure were a low baseline CD4 count (p=0.007) and ≥1 resistance mutations at baseline (p=0.04). Compared with patients with undetectable VL, those with detectable VL without mutations and those with ≥1 mutations had adjusted hazard ratios of immunological failure of 2.56 (95%CI 0.76–8.54) and 4.32 (1.38–13.57), respectively. In patients with undetectable VL and detectable VL without and with mutations, the median change in CD4 count between study entry and termination was +129/mm3, +51/mm3 and +3/mm3, respectively. One patient died. The 18-months probability of remaining free of morbidity was 0.79 in patients with undetectable VL and 0.69 in those with resistance mutations (p=0.19). Conclusion In this setting with restricted access to second-line HAART regimens, patients with major resistance mutations had higher rates of immunological failure, but most of them maintained stable CD4 count and stayed alive during 20 months. PMID:17502726

  2. Undernutrition and anaemia among HAART-naïve HIV infected children in Ile-Ife, Nigeria: a case-controlled, hospital based study

    PubMed Central

    Anyabolu, Henry Chineme; Adejuyigbe, Ebunoluwa Aderonke; Adeodu, Oluwagbemiga Oyewole

    2014-01-01

    Introduction Case control studies that assess the burden and factors associated with undernutrition and anaemia among HAART naïve HIV infected children in Nigeria is very sparse. This will help to formulate nutritional programs among these children. Methods Seventy HAART naive HIV infected children aged 18 months and above were as well as seventy age and sex matched HIV negative children were recruited from August 2007 to January 2009 at Paediatric Clinic of Obafemi Awolowo University Teaching Hospital Complex, Ile-Ife, Nigeria. Their bio data, WHO clinical stage, anthropometric measurements, haematocrit, serum albumin and CD4 counts were taken with other parameters according to a study proforma. Results The prevalence of stunting, underweight and wasting among the HIV infected subjects were 48. 6%,58. 6% and 31. 4% respectively which as significantly higher than 28. 1%, 7. 1% and 28. 1% among the HIV negative controls. 20. 1% of the HIV infected children were marasmic compared to 2. 3% of the controls. Triple anthropometric failure was found in 7. 1% of the subjects as compared to none among the controls. Anaemia is significantly more prevalent among the subjects than the controls (70. 0% vs 31. 4%; p<0. 001). The prevalence of anaemia was higher in the HIV infected subjects with undernutrition. Low socioeconomic status, hypoalbuminemia and severe immunosuppression are significantly associated with higher undernutrition prevalence. Conclusion Several years after availability of HAART, undernutrition and anaemia remain widely prevalent among newly presenting HAART naïve HIV infected Nigerian children. Nutritional supplementation and evaluation for anaemia still need close attention in the management of these children. PMID:25400844

  3. Stimulated proliferative responses in vertically HIV-infected children on HAART correlate with clinical and immunological markers

    PubMed Central

    RESINO, S; ABAD, M L; NAVARRO, J; BELLÓN, J M; SÁNCHEZ-RAMÓN, S; ÁNGELES MUÑOZ-FERNÁNDEZ, M

    2003-01-01

    The objective of the study was to investigate the relationship between various CD4+ T cell subsets and the ability of peripheral blood mononuclear cells (PBMC) to proliferate to several stimuli in vertically human immunodeficiency virus type 1 (HIV-1)-infected children. We studied 29 HIV-1-infected children on highly active antiretroviral therapy (HAART) (median duration: 12·3 months). T cell subsets were determined by flow cytometry. Plasma viral load (VL) was quantified using a standardized molecular method. Proliferative responses were evaluated by [3H]-thymidine incorporation. Decreased proliferative responses of PBMC to pokeweed mitogen (PWM) were found for HIV-1-infected children in Centers for Disease Control (CDC) clinical categories B and C when compared to the control group (P < 0·05). Similarly, children with ≤ 15% CD4+ T cells showed a decrease in proliferative responses to PWM (P < 0·01), anti-CD3 + anti-CD28 (P < 0·01) and phytohaemagglutinin (PHA) (P < 0·05) with respect to the control group and to children with CD4+ T cells ≥ 25%. Proliferative responses to PWM, anti-CD3+, anti-CD28 and PHA had a statistically significant positive correlation with CD3+/mm3, CD4+/mm3, % CD4 T cells, CD4/CD8 ratio and the percentage of naive T cell subsets (CD4+CD45RO−HLA-DR−, CD4+ CD45RA+ CD62L+, CD4+ CD45RA+), CD4+ CD62L+ and CD4+ T cells co-expressing CD38+ (CD4+ HLA-DR−CD38+, CD4+ CD38+). Moreover, we found a negative correlation between PBMC proliferative responses and % CD8 T cells, memory, memory-activated and activated CD4+ T cell subsets. Lower proliferative responses to PWM (P < 0·01) and PHA (P < 0·01) were associated with higher VL. Our data show that higher proliferative responses to PWM, anti-CD3 + anti-CD28 and PHA are associated with both non-activated and naive CD4+ T cell subsets in HIV-1-infected children on HAART. PMID:12519396

  4. Failure to Restore the Vγ2-Jγ1.2 Repertoire in HIV-infected Men Receiving Highly Active Antiretroviral Therapy (HAART)

    PubMed Central

    Hebbeler, Andrew M.; Propp, Nadia; Cairo, Cristiana; Li, Haishan; Cummings, Jean Saville; Jacobson, Lisa P.; Margolick, Joseph B.; Pauza, C. David

    2008-01-01

    Gammadelta (γδ) T cells expressing the Vγ2-Jγ1.2Vδ2 (Vγ9-JPVδ2, alternate nomenclature) T cell receptor (TCR) constitute the major peripheral blood population of γδ T cells in adult humans and are specifically depleted during human immunodeficiency virus (HIV) disease. Vγ2-Jγ1.2Vδ2 T cells provide a convenient model for assessing the impact of antiretroviral therapy on cell populations that are not susceptible to direct infection because they do not express CD4 and depletion occurs by indirect mechanisms. We obtained longitudinal PBMC samples from 16 HIV-infected individuals who were enrolled in the Multicenter AIDS Cohort Study (MACS) and starting highly active antiretroviral therapy (HAART). Vγ2-Jγ1.2Vδ2 T cells were depleted in these individuals as a result of HIV infection. Despite evidence for clinical benefits of HAART, the Vγ2-Jγ1.2Vδ2 T cell repertoire did not recover after HAART initiation irrespective of treatment duration. These studies highlight important defects among cell subsets lost due to indirect effects of HIV. PMID:18606571

  5. Production of alkaline protease from Cellulosimicrobium cellulans

    PubMed Central

    Ferracini-Santos, Luciana; Sato, Hélia H

    2009-01-01

    Cellulosimicrobium cellulans is one of the microorganisms that produces a wide variety of yeast cell wall-degrading enzymes, β-1,3-glucanase, protease and chitinase. Dried cells of Saccharomyces cerevisiae were used as carbon and nitrogen source for cell growth and protease production. The medium components KH2PO4, KOH and dried yeast cells showed a significant effect (p<0.05) on the factorial fractional design. A second design was prepared using two factors: pH and percentage of dried yeast cells. The results showed that the culture medium for the maximum production of protease was 0.2 g/l of MgSO4.7H2O, 2.0 g/l of (NH4)2SO4 and 8% of dried yeast cells in 0.15M phosphate buffer at pH 8.0. The maximum alkaline protease production was 7.0 ± 0.27 U/ml over the center point. Crude protease showed best activity at 50ºC and pH 7.0-8.0, and was stable at 50ºC. PMID:24031317

  6. Protease Inhibitors from Plants with Antimicrobial Activity

    PubMed Central

    Kim, Jin-Young; Park, Seong-Cheol; Hwang, Indeok; Cheong, Hyeonsook; Nah, Jae-Woon; Hahm, Kyung-Soo; Park, Yoonkyung

    2009-01-01

    Antimicrobial proteins (peptides) are known to play important roles in the innate host defense mechanisms of most living organisms, including plants, insects, amphibians and mammals. They are also known to possess potent antibiotic activity against bacteria, fungi, and even certain viruses. Recently, the rapid emergence of microbial pathogens that are resistant to currently available antibiotics has triggered considerable interest in the isolation and investigation of the mode of action of antimicrobial proteins (peptides). Plants produce a variety of proteins (peptides) that are involved in the defense against pathogens and invading organisms, including ribosome-inactivating proteins, lectins, protease inhibitors and antifungal peptides (proteins). Specially, the protease inhibitors can inhibit aspartic, serine and cysteine proteinases. Increased levels of trypsin and chymotrypsin inhibitors correlated with the plants resistance to the pathogen. Usually, the purification of antimicrobial proteins (peptides) with protease inhibitor activity was accomplished by salt-extraction, ultrafiltration and C18 reverse phase chromatography, successfully. We discuss the relation between antimicrobial and anti-protease activity in this review. Protease inhibitors from plants potently inhibited the growth of a variety of pathogenic bacterial and fungal strains and are therefore excellent candidates for use as the lead compounds for the development of novel antimicrobial agents. PMID:19582234

  7. High Viral Load and Elevated Angiogenic Markers Associated with Increased Risk of Preeclampsia among Women Initiating Highly Active Antiretroviral Therapy (HAART) in Pregnancy in the Mma Bana Study, Botswana

    PubMed Central

    Powis, Kathleen M; McElrath, Thomas F; Hughes, Michael D; Ogwu, Anthony; Souda, Sajini; Datwyler, Saul A; von Widenfelt, Erik; Moyo, Sikhulile; Nádas, Marisa; Makhema, Joseph; Machakaire, Esther; Lockman, Shahin; Essex, Max; Shapiro, Roger L

    2013-01-01

    Background Risk factors associated with preeclampsia in HIV-infected women remain largely unknown. Systemic angiogenic imbalance contributes to preeclampsia in HIV-uninfected women, but changes in angiogenic markers after HAART initiation have not been studied. Methods The Mma Bana study randomized 560 HIV-infected, HAART-naive pregnant women with CD4 counts ≥ 200 cells/mm3 between 26–34 weeks gestation to lopinavir/ritonavir/zidovudine/lamivudine or abacavir/zidovudine/lamivudine. Another 170 participants with CD4 counts < 200 cells/mm3 initiated nevirapine/zidovudine/lamivudine between 18–34 weeks gestation. Characteristics of 11 women who developed preeclampsia were compared with the remaining722 Mma Bana participants who delivered, using logistic regression. Plasma samples drawn at HAART initiation and one month later from 60 women without preeclampsia and at HAART initiation for all11 preeclamptic women were assayed for placental growth factor (PlGF) and soluble FMS toll-like tyrosine kinase-1 (sFlt-1), Results Pre-HAART viral load > 100,000 copies/ml was associated with preeclampsia (OR 5.8; 95% CI 1.8, 19.4; p = 0.004). Median pre-HAART PlGF level was lower and sFLT-1 was higher in women who developed preeclampsia versus those who did not (130 vs 992 pg/ml, p=0.001; 17.5 vs 9.4 pg/ml, p=0.03, respectively). In multivariate analysis, PlGF and viral load remained significantly associated with preeclampsia. No significant changes in angiogenic factors were noted after 1 month of HAART treatment among non-preeclamptic women. Conclusions Pre-HAART viral load > 100,000 copies/ml and PlGF predicted preeclampsia among women starting HAART in pregnancy. Among non-preeclamptic women, HAART treatment did not significantly alter levels of PlGF or sFlt-1 one month into treatment. PMID:23344545

  8. Hepatitis C Virus Quasispecies in HIV-Infected Women: Role of Injecting Drug Use and Highly Active Antiretroviral Therapy (HAART)

    PubMed Central

    Laskus, Tomasz; Wilkinson, Jeffrey; Karim, Roksana; Mack, Wendy; Radkowski, Marek; deGiacomo, Marina; Nasseri, Jonathan; Chen, Zhi; Xu, Jiaao; Kovacs, Andrea

    2012-01-01

    Despite the high frequency of HCV and HIV coinfection, little is known about HCV quasispecies in HIV-positive patients. The current analysis included 236 HIV+/anti-HCV+ women enrolled in the Women’s Interagency HIV Study (WIHS). Hypervariable region 1 of the second envelope gene was analyzed by single-strand conformation polymorphism (SSCP). The relationship between the HCV quasispecies and clinical and demographic features were analyzed in multivariate models. Age over 40 years and high HCV RNA load were the only factors significantly associated with quasispecies complexity, assessed as the number of SSCP bands. High HIV and HCV plasma loads were associated with quasispecies stability over time, as reflected by stable SSCP band patterns. However, women who were actively injecting drugs were 3 times more likely to experience quasispecies changes than their noninjecting counterparts. No affect on HCV quasi-species dynamics was noted in relation to CD4 count or highly active antiretroviral therapy (HAART). Conclusion: among HIV/HCV coinfected patients, HCV quasispecies complexity and dynamics correlate more closely with HIV and HCV plasma loads than with CD4+ cell counts. Active drug use is associated with quasispecies changes probably due to repeated superinfections with new HCV strains. This needs to be considered when planning treatment and prevention strategies for HCV in coinfected individuals. PMID:17659581

  9. HIV-1/HAART-Related Lipodystrophy Syndrome (HALS) Is Associated with Decreased Circulating sTWEAK Levels

    PubMed Central

    López-Dupla, Miguel; Maymó-Masip, Elsa; Martínez, Esteban; Domingo, Pere; Leal, Manuel; Peraire, Joaquim; Viladés, Consuelo; Veloso, Sergi; Arnedo, Mireia; Ferrando-Martínez, Sara; Beltrán-Debón, Raúl; Alba, Verónica; Gatell, Josep Mª; Vendrell, Joan; Vidal, Francesc; Chacón, Matilde R.

    2015-01-01

    Background and Objectives Obesity and HIV-1/HAART–associated lipodystrophy syndrome (HALS) share clinical, pathological and mechanistic features. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine that plays an important role in obesity and related diseases. We sought to explore the relationship between HALS and circulating levels of soluble (s) TWEAK and its scavenger receptor sCD163. Methods This was a cross-sectional multicenter study of 120 HIV-1-infected patients treated with a stable HAART regimen; 56 with overt HALS and 64 without HALS. Epidemiological and clinical variables were determined. Serum levels of sTWEAK and sCD163 levels were measured by ELISA. Results were analyzed with Student’s t-test, Mann-Whitney U and χ2 test. Pearson and Spearman correlation were used to estimate the strength of association between variables. Results Circulating sTWEAK was significantly decreased in HALS patients compared with non-HALS patients (2.81±0.2 vs. 2.94±0.28 pg/mL, p = 0.018). No changes were observed in sCD163 levels in the studied cohorts. On multivariate analysis, a lower log sTWEAK concentration was independently associated with the presence of HALS (OR 0.027, 95% CI 0.001–0.521, p = 0.027). Conclusions HALS is associated with decreased sTWEAK levels. PMID:26658801

  10. Socio-economic impact of antiretroviral treatment in HIV patients. An economic review of cost savings after introduction of HAART.

    PubMed

    Gonzalo, Teresa; García Goñi, Manuel; Muñoz-Fernández, María Angeles

    2009-01-01

    Star celebrities such as Rock Hudson, Freddie Mercury, Magic Johnson, and Isaac Asimov have unfortunately something in common: they were all victims of the HIV global pandemic. Since then HIV infection has become considered a pandemic disease, and it is regarded as a priority in healthcare worldwide. It is ranked as the first cause of death among young people in industrialized countries, and it is recognized as a public healthcare problem due to its human, social, mass media, and economic impact. Incorporation of new and highly active antiretroviral treatment, available since 1996 for HIV/AIDS treatment, has provoked a radical change in the disease pattern, as well as in the impact on patient survival and quality of life. The pharmaceutical industry's contribution, based on the research for more active new drugs, has been pivotal. Mortality rates have decreased significantly in 20 years by 50% and now AIDS is considered a chronic and controlled disease. In this review we have studied the impact of HAART treatment on infected patients, allowing them to maintain their status as active workers and the decreased absenteeism from work derived from this, contributing ultimately to overall social wealth and, thus, to economic growth. Furthermore, an analysis of the impact on healthcare costs, quality of life per year, life per year gained, cost economic savings and cost opportunity among other parameters has shown that society and governments are gaining major benefits from the inclusion of antiretroviral therapies in HIV/AIDS patients.

  11. Serine protease inhibitors of parasitic helminths.

    PubMed

    Molehin, Adebayo J; Gobert, Geoffrey N; McManus, Donald P

    2012-05-01

    Serine protease inhibitors (serpins) are a superfamily of structurally conserved proteins that inhibit serine proteases and play key physiological roles in numerous biological systems such as blood coagulation, complement activation and inflammation. A number of serpins have now been identified in parasitic helminths with putative involvement in immune regulation and in parasite survival through interference with the host immune response. This review describes the serpins and smapins (small serine protease inhibitors) that have been identified in Ascaris spp., Brugia malayi, Ancylostoma caninum Onchocerca volvulus, Haemonchus contortus, Trichinella spiralis, Trichostrongylus vitrinus, Anisakis simplex, Trichuris suis, Schistosoma spp., Clonorchis sinensis, Paragonimus westermani and Echinococcus spp. and discusses their possible biological functions, including roles in host-parasite interplay and their evolutionary relationships. PMID:22310379

  12. Coagulation, Protease Activated Receptors and Viral Myocarditis

    PubMed Central

    Antoniak, Silvio; Mackman, Nigel

    2013-01-01

    The coagulation protease cascade plays an essential role in hemostasis. In addition, a clot contributes to host defense by limiting the spread of pathogens. Coagulation proteases induce intracellular signaling by cleavage of cell surface receptors called protease-activated receptors (PARs). These receptors allow cells to sense changes in the extracellular environment, such as infection. Viruses activate the coagulation cascade by inducing tissue factor expression and by disrupting the endothelium. Virus infection of the heart can cause myocarditis, cardiac remodeling and heart failure. Recent studies using a mouse model have shown that tissue factor, thrombin and PAR-1 signaling all positively regulate the innate immune during viral myocarditis. In contrast, PAR-2 signaling was found to inhibit interferon-β expression and the innate immune response. These observations suggest that anticoagulants may impair the innate immune response to viral infection and that inhibition of PAR-2 may be a new target to reduce viral myocarditis.. PMID:24203054

  13. Subtilisin-like proteases in nematodes.

    PubMed

    Poole, Catherine B; Jin, Jingmin; McReynolds, Larry A

    2007-09-01

    Cleavage by subtilisin-like proteases (subtilases) is an essential step in post-translational processing of proteins found in organisms ranging from yeast to mammals. Our knowledge of the diversity of this protease family in nematodes is aided by the rapid increase in sequence information, especially from the Brugia malayi genome project. Genetic studies of the subtilases in Caenorhabitis elegans give valuable insight into the biological function of these proteases in other nematode species. In this review, we focus on the subtilases in filarial nematodes as well as other parasitic and free-living nematodes in comparison to what is known in C. elegans. Topics to be addressed include expansion and diversity of the subtilase gene family during evolution, enhanced complexity created by alternative RNA splicing, molecular and biochemical characterization of the different subtilases and the challenges of designing subtilase-specific inhibitors for parasitic nematodes. PMID:17570539

  14. Conformational selection in trypsin-like proteases

    PubMed Central

    Pozzi, Nicola; Vogt, Austin D.; Gohara, David W.; Di Cera, Enrico

    2012-01-01

    For over four decades, two competing mechanisms of ligand recognition – conformational selection and induced-fit - have dominated our interpretation of protein allostery. Defining the mechanism broadens our understanding of the system and impacts our ability to design effective drugs and new therapeutics. Recent kinetics studies demonstrate that trypsin-like proteases exist in equilibrium between two forms: one fully accessible to substrate (E) and the other with the active site occluded (E*). Analysis of the structural database confirms existence of the E* and E forms and vouches for the allosteric nature of the trypsin fold. Allostery in terms of conformational selection establishes an important paradigm in the protease field and enables protein engineers to expand the repertoire of proteases as therapeutics. PMID:22664096

  15. Dataset of cocoa aspartic protease cleavage sites.

    PubMed

    Janek, Katharina; Niewienda, Agathe; Wöstemeyer, Johannes; Voigt, Jürgen

    2016-09-01

    The data provide information in support of the research article, "The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors" (Janek et al., 2016) [1]. Three different protein substrates were partially digested with the aspartic protease isolated from cocoa beans and commercial pepsin, respectively. The obtained peptide fragments were analyzed by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS/MS) and identified using the MASCOT server. The N- and C-terminal ends of the peptide fragments were used to identify the corresponding in-vitro cleavage sites by comparison with the amino acid sequences of the substrate proteins. The same procedure was applied to identify the cleavage sites used by the cocoa aspartic protease during cocoa fermentation starting from the published amino acid sequences of oligopeptides isolated from fermented cocoa beans. PMID:27508221

  16. Nidovirus papain-like proteases: multifunctional enzymes with protease, deubiquitinating and deISGylating activities

    PubMed Central

    Mielech, Anna M.; Chen, Yafang; Mesecar, Andrew D.; Baker, Susan C.

    2014-01-01

    Coronaviruses and arteriviruses, members of the order Nidovirales, are positive strand RNA viruses that encode large replicase polyproteins that are processed by viral proteases to generate the nonstructural proteins which mediate viral RNA synthesis. The viral papain-like proteases (PLPs) are critical for processing the amino-terminal end of the replicase and are attractive targets for antiviral therapies. With the analysis of the papain-like protease of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), came the realization of the multifunctional nature of these enzymes. Structural and enzymatic studies revealed that SARS-CoV PLpro can act as both a protease to cleave peptide bonds and also as a deubiquitinating (DUB) enzyme to cleave the isopeptide bonds found in polyubiquitin chains. Furthermore, viral DUBs can also remove the protective effect of conjugated ubiquitin-like molecules such as interferon stimulated gene 15 (ISG15). Extension of these studies to other coronaviruses and arteriviruses led to the realization that viral protease/DUB activity is conserved in many family members. Overexpression studies revealed that viral protease/DUB activity can modulate or block activation of the innate immune response pathway. Importantly, mutations that alter DUB activity but not viral protease activity have been identified and arteriviruses expressing DUB mutants stimulated higher levels of acute inflammatory cytokines after infection. Further understanding of the multifunctional nature of the Nidovirus PLP/DUBs may facilitate vaccine development. Here, we review studies describing the PLPs’ enzymatic activity and their role in virus pathogenesis. PMID:24512893

  17. Therapeutic Immunization with HIV-1 Tat Reduces Immune Activation and Loss of Regulatory T-Cells and Improves Immune Function in Subjects on HAART

    PubMed Central

    Ensoli, Barbara; Bellino, Stefania; Tripiciano, Antonella; Longo, Olimpia; Francavilla, Vittorio; Marcotullio, Simone; Cafaro, Aurelio; Picconi, Orietta; Paniccia, Giovanni; Scoglio, Arianna; Arancio, Angela; Ariola, Cristina; Ruiz Alvarez, Maria J.; Campagna, Massimo; Scaramuzzi, Donato; Iori, Cristina; Esposito, Roberto; Mussini, Cristina; Ghinelli, Florio; Sighinolfi, Laura; Palamara, Guido; Latini, Alessandra; Angarano, Gioacchino; Ladisa, Nicoletta; Soscia, Fabrizio; Mercurio, Vito S.; Lazzarin, Adriano; Tambussi, Giuseppe; Visintini, Raffaele; Mazzotta, Francesco; Di Pietro, Massimo; Galli, Massimo; Rusconi, Stefano; Carosi, Giampiero; Torti, Carlo; Di Perri, Giovanni; Bonora, Stefano; Ensoli, Fabrizio; Garaci, Enrico

    2010-01-01

    Although HAART suppresses HIV replication, it is often unable to restore immune homeostasis. Consequently, non-AIDS-defining diseases are increasingly seen in treated individuals. This is attributed to persistent virus expression in reservoirs and to cell activation. Of note, in CD4+ T cells and monocyte-macrophages of virologically-suppressed individuals, there is continued expression of multi-spliced transcripts encoding HIV regulatory proteins. Among them, Tat is essential for virus gene expression and replication, either in primary infection or for virus reactivation during HAART, when Tat is expressed, released extracellularly and exerts, on both the virus and the immune system, effects that contribute to disease maintenance. Here we report results of an ad hoc exploratory interim analysis (up to 48 weeks) on 87 virologically-suppressed HAART-treated individuals enrolled in a phase II randomized open-label multicentric clinical trial of therapeutic immunization with Tat (ISS T-002). Eighty-eight virologically-suppressed HAART-treated individuals, enrolled in a parallel prospective observational study at the same sites (ISS OBS T-002), served for intergroup comparison. Immunization with Tat was safe, induced durable immune responses, and modified the pattern of CD4+ and CD8+ cellular activation (CD38 and HLA-DR) together with reduction of biochemical activation markers and persistent increases of regulatory T cells. This was accompanied by a progressive increment of CD4+ T cells and B cells with reduction of CD8+ T cells and NK cells, which were independent from the type of antiretroviral regimen. Increase in central and effector memory and reduction in terminally-differentiated effector memory CD4+ and CD8+ T cells were accompanied by increases of CD4+ and CD8+ T cell responses against Env and recall antigens. Of note, more immune-compromised individuals experienced greater therapeutic effects. In contrast, these changes were opposite, absent or partial in the

  18. Exercise Training Reduces Central Adiposity and Improves Metabolic Indices in HAART-Treated HIV-Positive Subjects in Rwanda: A Randomized Controlled Trial

    PubMed Central

    MUTIMURA, EUGENE; CROWTHER, NIGEL J.; CADE, TODD W.; YARASHESKI, KEVIN E.; STEWART, AIMEE

    2014-01-01

    As HAART becomes more accessible in sub-Saharan Africa, metabolic syndromes, body fat redistribution (BFR), and cardiovascular disease may become more prevalent. We conducted a 6-month, randomized controlled trial to test whether cardiorespiratory exercise training (CET), improves metabolic, body composition and cardiorespiratory fitness parameters in HAART-treated HIV+ African subjects with BFR. Six months of CET reduced waist circumference (−7.13 ± 4.4 cm, p < 0.0001), WHR (−0.10 ± 0.1, p < 0.0001), sum skinfold thickness (−6.15 ± 8.2 mm, p < 0.0001) and % body fat mass (−1.5 ± 3.3, p < 0.0001) in HIV+BFR+EXS. Hip circumference was unchanged in non-exercise control groups. CET reduced fasting total cholesterol (−0.03 ± 1.11 mM, p < 0.05), triglycerides (−0.22 ± 0.48 mM, p < 0.05) and glucose levels (−0.21 ± 0.71 mM, p < 0.05) (p < 0.0001). HDL-, LDL-cholesterol and HOMA values were unchanged after CET. Interestingly, HIV+ subjects randomized to non-exercising groups experienced increases in fasting plasma glucose levels, whereas HIV seronegative controls did not (p < 0.001). Predicted VO2 peak increased more in the HIV+BFR+EXS than in all other groups (4.7 ± 3.9 ml/kg/min, p < 0.0001). Exercise training positively modulated body composition and metabolic profiles, and improved cardiorespiratory fitness in HAART-treated HIV+ Africans. These beneficial adaptations imply that exercise training is a safe, inexpensive, practical, and effective treatment for evolving metabolic and cardiovascular syndromes associated with HIV and HAART exposure in resource-limited sub-Saharan countries, where treatment is improving, morbidity and mortality rates are declining, but where minimal resources are available to manage HIV- and HAART-associated cardiovascular and metabolic syndromes. PMID:18275343

  19. Residual Viremia in an RT-SHIV Rhesus Macaque HAART Model Marked by the Presence of a Predominant Plasma Clone and a Lack of Viral Evolution

    PubMed Central

    Kauffman, Robert C.; Villalobos, Andradi; Bowen, Joanne H.; Adamson, Lourdes; Schinazi, Raymond F.

    2014-01-01

    Highly active antiretroviral therapy (HAART) significantly reduces HIV-1 replication and prevents progression to AIDS. However, residual low-level viremia (LLV) persists and long-lived viral reservoirs are maintained in anatomical sites. These reservoirs permit a recrudescence of viremia upon cessation of therapy and thus HAART must be maintained indefinitely. HIV-1 reservoirs include latently infected resting memory CD4+ T-cells and macrophages which may contribute to residual viremia. It has not been conclusively determined if a component of LLV may also be due to residual replication in cells with sub-therapeutic drug levels and/or long-lived chronically infected cells. In this study, RT-SHIVmac239 diversity was characterized in five rhesus macaques that received a five-drug HAART regimen [tenofovir, emtricitabine, zidovudine, amdoxovir, (A, C, T, G nucleoside analogs) and the non-nucleoside reverse transcriptase (RT) inhibitor efavirenz]. Before maximal viral load suppression, longitudinal plasma viral RNA RT diversity was analyzed using a 454 sequencer. After suppression, LLV RT diversity (amino acids 65-210) was also assessed. LLV samples had viral levels less than our standard detection limit (50 viral RNA copies/mL) and few transient blips <200 RNA copies/mL. HAART was discontinued in three macaques after 42 weeks of therapy resulting in viral rebound. The level of viral divergence and the prevalence of specific alleles in LLV was similar to pre-suppression viremia. While some LLV sequences contained mutations not observed in the pre-suppression profile, LLV was not characterized by temporal viral evolution or apparent selection of drug resistance mutations. Similarly, resistance mutations were not detected in the viral rebound population. Interestingly, one macaque maintained a putative LLV predominant plasma clone sequence. Together, these results suggest that residual replication did not markedly contribute to LLV and that this model mimics the

  20. Serine Protease(s) Secreted by the Nematode Trichuris muris Degrade the Mucus Barrier

    PubMed Central

    Hasnain, Sumaira Z.; McGuckin, Michael A.; Grencis, Richard K.; Thornton, David J.

    2012-01-01

    The polymeric mucin component of the intestinal mucus barrier changes during nematode infection to provide not only physical protection but also to directly affect pathogenic nematodes and aid expulsion. Despite this, the direct interaction of the nematodes with the mucins and the mucus barrier has not previously been addressed. We used the well-established Trichuris muris nematode model to investigate the effect on mucins of the complex mixture of immunogenic proteins secreted by the nematode called excretory/secretory products (ESPs). Different regimes of T. muris infection were used to simulate chronic (low dose) or acute (high dose) infection. Mucus/mucins isolated from mice and from the human intestinal cell line, LS174T, were treated with ESPs. We demonstrate that serine protease(s) secreted by the nematode have the ability to change the properties of the mucus barrier, making it more porous by degrading the mucin component of the mucus gel. Specifically, the serine protease(s) acted on the N-terminal polymerising domain of the major intestinal mucin Muc2, resulting in depolymerisation of Muc2 polymers. Importantly, the respiratory/gastric mucin Muc5ac, which is induced in the intestine and is critical for worm expulsion, was protected from the depolymerising effect exerted by ESPs. Furthermore, serine protease inhibitors (Serpins) which may protect the mucins, in particular Muc2, from depolymerisation, were highly expressed in mice resistant to chronic infection. Thus, we demonstrate that nematodes secrete serine protease(s) to degrade mucins within the mucus barrier, which may modify the niche of the parasite to prevent clearance from the host or facilitate efficient mating and egg laying from the posterior end of the parasite that is in intimate contact with the mucus barrier. However, during a TH2-mediated worm expulsion response, serpins, Muc5ac and increased levels of Muc2 protect the barrier from degradation by the nematode secreted protease(s). PMID

  1. Current and Novel Inhibitors of HIV Protease

    PubMed Central

    Pokorná, Jana; Machala, Ladislav; Řezáčová, Pavlína; Konvalinka, Jan

    2009-01-01

    The design, development and clinical success of HIV protease inhibitors represent one of the most remarkable achievements of molecular medicine. This review describes all nine currently available FDA-approved protease inhibitors, discusses their pharmacokinetic properties, off-target activities, side-effects, and resistance profiles. The compounds in the various stages of clinical development are also introduced, as well as alternative approaches, aiming at other functional domains of HIV PR. The potential of these novel compounds to open new way to the rational drug design of human viruses is critically assessed. PMID:21994591

  2. Using specificity to strategically target proteases

    PubMed Central

    Lim, Mark D.; Craik, Charles S.

    2009-01-01

    Proteases are a family of naturally occurring enzymes in the body whose dysregulation has been implicated in numerous diseases and cancers. Their ability to selectively and catalytically turnover substrate adds both signal amplification and functionality as parameters for the detection of disease. This review will focus on the development of activity-based methodologies to characterize proteases, and in particular, the use of positional scanning, synthetic combinatorial libraries (PS-SCL’s), and substrate activity screening (SAS) assays. The use of these approaches to better understand a protease’s natural substrate will be discussed as well as the technologies that emerged. PMID:18434168

  3. Detection of protease and protease activity using a single nanoscrescent SERS probe

    DOEpatents

    Liu, Gang L.; Ellman, Jonathan A.; Lee, Luke P.; Chen, Fanqing Frank

    2013-01-29

    This invention pertains to the in vitro detection of proteases using a single peptide-conjugate nanocrescent surface enhanced Raman scattering (SERS) probes with at least nanomolar sensitivity. The probe enables detection of proteolytic activity in extremely small volume and at low concentration. In certain embodiments the probes comprise an indicator for the detection of an active protease, where the indicator comprises a nanocrescent attached to a peptide, where said peptide comprises a recognition site for the protease and a Raman tag attached to the peptide.

  4. HIV protease inhibitors and onset of cardiovascular diseases: a central role for oxidative stress and dysregulation of the ubiquitin-proteasome system.

    PubMed

    Reyskens, Kathleen M S E; Essop, M Faadiel

    2014-02-01

    The successful roll-out of highly active antiretroviral therapy (HAART) has extended life expectancy and enhanced the overall well-being of HIV-positive individuals. There are, however, increased concerns regarding HAART-mediated metabolic derangements and its potential risk for cardiovascular diseases (CVD) in the long-term. Here certain classes of antiretroviral drugs such as the HIV protease inhibitors (PIs) are strongly implicated in this process. This article largely focuses on the direct PI-linked development of cardio-metabolic complications, and reviews the inter-linked roles of oxidative stress and the ubiquitin-proteasome system (UPS) as key mediators driving this process. It is proposed that PIs trigger reactive oxygen species (ROS) production that leads to serious downstream consequences such as cell death, impaired mitochondrial function, and UPS dysregulation. Moreover, we advocate that HIV PIs may also directly lower myocardial UPS function. The attenuation of cardiac UPS can initiate transcriptional changes that contribute to perturbed lipid metabolism, thereby fueling a pro-atherogenic milieu. It may also directly alter ionic channels and interfere with electrical signaling in the myocardium. Therefore HIV PI-induced ROS together with a dysfunctional UPS elicit detrimental effects on the cardiovascular system that will eventually result in the onset of heart diseases. Thus while HIV PIs substantially improve life expectancy and quality of life in HIV-positive patients, its longer-term side-effects on the cardiovascular system should lead to a) greater clinical awareness regarding its benefit-harm paradigm, and b) the development and evaluation of novel co-treatment strategies.

  5. Transient ECM protease activity promotes synaptic plasticity

    PubMed Central

    Magnowska, Marta; Gorkiewicz, Tomasz; Suska, Anna; Wawrzyniak, Marcin; Rutkowska-Wlodarczyk, Izabela; Kaczmarek, Leszek; Wlodarczyk, Jakub

    2016-01-01

    Activity-dependent proteolysis at a synapse has been recognized as a pivotal factor in controlling dynamic changes in dendritic spine shape and function; however, excessive proteolytic activity is detrimental to the cells. The exact mechanism of control of these seemingly contradictory outcomes of protease activity remains unknown. Here, we reveal that dendritic spine maturation is strictly controlled by the proteolytic activity, and its inhibition by the endogenous inhibitor (Tissue inhibitor of matrix metalloproteinases-1 – TIMP-1). Excessive proteolytic activity impairs long-term potentiation of the synaptic efficacy (LTP), and this impairment could be rescued by inhibition of protease activity. Moreover LTP is altered persistently when the ability of TIMP-1 to inhibit protease activity is abrogated, further demonstrating the role of such inhibition in the promotion of synaptic plasticity under well-defined conditions. We also show that dendritic spine maturation involves an intermediate formation of elongated spines, followed by their conversion into mushroom shape. The formation of mushroom-shaped spines is accompanied by increase in AMPA/NMDA ratio of glutamate receptors. Altogether, our results identify inhibition of protease activity as a critical regulatory mechanism for dendritic spines maturation. PMID:27282248

  6. Proteases and Peptidases of Castor Bean Endosperm

    PubMed Central

    Tully, Raymond E.; Beevers, Harry

    1978-01-01

    The endosperm of castor bean seeds (Ricinus communis L.) contains two —SH-dependent aminopeptidases, one hydrolyzing l-leucine-β-naphthylamide optimally at pH 7.0, and the other hydrolyzing l-proline-β-naphthylamide optimally at pH 7.5. After germination the endosperm contains in addition an —SH-dependent hemoglobin protease, a serine-dependent carboxypeptidase, and at least two —SH-dependent enzymes hydrolyzing the model substrate α-N-benzoyl-dl-arginine-β-naphthylamide (BANA). The carboxypeptidase is active on a variety of N-carbobenzoxy dipeptides, especially N-carbobenzoxy-L-phenylalanine-l-alanine and N-carbobenzoxy-l-tyrosine-l-leucine. The pH optima for the protease, carboxypeptidase, and BANAase acivities are 3.5 to 4.0, 5.0 to 5.5, and 6 to 8, respectively. The two aminopeptidases increased about 4-fold in activity during the first 4 days of growth, concurrent with the period of rapid depletion of storage protein. Activities then declined as the endosperm senesced, but were still evident after 6 days. Senescence was complete by day 7 to 8. Hemoglobin protease, carboxypeptidase, and BANAase activities appeared in the endosperm at day 2 to 3, and reached peak activity at day 5 to 6. The data indicate that the aminopeptidases are involved in the early mobilization of endosperm storage protein, whereas protease, carboxypeptidase, and BANAase may take part in later turnover and/or senescence. PMID:16660598

  7. Proteases and Protease Inhibitors of Urinary Extracellular Vesicles in Diabetic Nephropathy

    PubMed Central

    Tataruch, Dorota; Gu, Dongfeng; Liu, Xinyu; Forsblom, Carol; Groop, Per-Henrik; Holthofer, Harry

    2015-01-01

    Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus (DM), leads to chronic kidney disease (CKD), and, ultimately, is the main cause for end-stage kidney disease (ESKD). Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs) have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profile urines of type 1 diabetes patients at different stages of kidney involvement. Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively. Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups. Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL) in the normoalbuminuric group. This study shows for the first time the distinctive alterations in comprehensive protease profiles of UEVs in diabetic nephropathy and uncovers intriguing mechanistic, prognostic, and diagnostic features of kidney damage in diabetes. PMID:25874235

  8. Protease IV, a quorum sensing-dependent protease of Pseudomonas aeruginosa modulates insect innate immunity.

    PubMed

    Park, Su-Jin; Kim, Soo-Kyoung; So, Yong-In; Park, Ha-Young; Li, Xi-Hui; Yeom, Doo Hwan; Lee, Mi-Nan; Lee, Bok-Luel; Lee, Joon-Hee

    2014-12-01

    In Pseudomonas aeruginosa, quorum sensing (QS) plays an essential role in pathogenesis and the QS response controls many virulence factors. Using a mealworm, Tenebrio molitor as a host model, we found that Protease IV, a QS-regulated exoprotease of P. aeruginosa functions as a key virulence effector causing the melanization and death of T. molitor larvae. Protease IV was able to degrade zymogens of spätzle processing enzyme (SPE) and SPE-activating enzyme (SAE) without the activation of the antimicrobial peptide (AMP) production. Since SPE and SAE function to activate spätzle, a ligand of Toll receptor in the innate immune system of T. molitor, we suggest that Protease IV may interfere with the activation of the Toll signaling. Independently of the Toll pathway, the melanization response, another innate immunity was still generated, since Protease IV directly converted Tenebrio prophenoloxidase into active phenoloxidase. Protease IV also worked as an important factor in the virulence to brine shrimp and nematode. These results suggest that Protease IV provides P. aeruginosa with a sophisticated way to escape the immune attack of host by interfering with the production of AMPs. PMID:25315216

  9. A novel protease activity assay using a protease-responsive chaperone protein

    SciTech Connect

    Sao, Kentaro; Murata, Masaharu; Fujisaki, Yuri; Umezaki, Kaori; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki; Hashizume, Makoto

    2009-06-05

    Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

  10. Coagulation factor XII protease domain crystal structure

    PubMed Central

    Pathak, M; Wilmann, P; Awford, J; Li, C; Hamad, BK; Fischer, PM; Dreveny, I; Dekker, LV; Emsley, J

    2015-01-01

    Background Coagulation factor XII is a serine protease that is important for kinin generation and blood coagulation, cleaving the substrates plasma kallikrein and FXI. Objective To investigate FXII zymogen activation and substrate recognition by determining the crystal structure of the FXII protease domain. Methods and results A series of recombinant FXII protease constructs were characterized by measurement of cleavage of chromogenic peptide and plasma kallikrein protein substrates. This revealed that the FXII protease construct spanning the light chain has unexpectedly weak proteolytic activity compared to β-FXIIa, which has an additional nine amino acid remnant of the heavy chain present. Consistent with these data, the crystal structure of the light chain protease reveals a zymogen conformation for active site residues Gly193 and Ser195, where the oxyanion hole is absent. The Asp194 side chain salt bridge to Arg73 constitutes an atypical conformation of the 70-loop. In one crystal form, the S1 pocket loops are partially flexible, which is typical of a zymogen. In a second crystal form of the deglycosylated light chain, the S1 pocket loops are ordered, and a short α-helix in the 180-loop of the structure results in an enlarged and distorted S1 pocket with a buried conformation of Asp189, which is critical for P1 Arg substrate recognition. The FXII structures define patches of negative charge surrounding the active site cleft that may be critical for interactions with inhibitors and substrates. Conclusions These data provide the first structural basis for understanding FXII substrate recognition and zymogen activation. PMID:25604127

  11. Serine Protease Autotransporters of Enterobacteriaceae (SPATEs): Biogenesis and Function

    PubMed Central

    Dautin, Nathalie

    2010-01-01

    Serine Protease Autotransporters of Enterobacteriaceae (SPATEs) constitute a large family of proteases secreted by Escherichia coli and Shigella. SPATEs exhibit two distinct proteolytic activities. First, a C-terminal catalytic site triggers an intra-molecular cleavage that releases the N-terminal portion of these proteins in the extracellular medium. Second, the secreted N-terminal domains of SPATEs are themselves proteases; each contains a canonical serine-protease catalytic site. Some of these secreted proteases are toxins, eliciting various effects on mammalian cells. Here, we discuss the biogenesis of SPATEs and their function as toxins. PMID:22069633

  12. Six-transmembrane Topology for Golgi Anti-apoptotic Protein (GAAP) and Bax Inhibitor 1 (BI-1) Provides Model for the Transmembrane Bax Inhibitor-containing Motif (TMBIM) Family*

    PubMed Central

    Carrara, Guia; Saraiva, Nuno; Gubser, Caroline; Johnson, Benjamin F.; Smith, Geoffrey L.

    2012-01-01

    The Golgi anti-apoptotic protein (GAAP) is a hydrophobic Golgi protein that regulates intracellular calcium fluxes and apoptosis. GAAP is highly conserved throughout eukaryotes and some strains of vaccinia virus (VACV) and camelpox virus. Based on sequence, phylogeny, and hydrophobicity, GAAPs were classified within the transmembrane Bax inhibitor-containing motif (TMBIM) family. TMBIM members are anti-apoptotic and were predicted to have seven-transmembrane domains (TMDs). However, topology prediction programs are inconsistent and predicted that GAAP and other TMBIM members have six or seven TMDs. To address this discrepancy, we mapped the transmembrane topology of viral (vGAAP) and human (hGAAP), as well as Bax inhibitor (BI-1). Data presented show a six-, not seven-, transmembrane topology for vGAAP with a putative reentrant loop at the C terminus and both termini located in the cytosol. We find that this topology is also conserved in hGAAP and BI-1. This places the charged C terminus in the cytosol, and mutation of these charged residues in hGAAP ablated its anti-apoptotic function. Given the highly conserved hydrophobicity profile within the TMBIM family and recent phylogenetic data indicating that a GAAP-like protein may have been the ancestral progenitor of a subset of the TMBIM family, we propose that this vGAAP topology may be used as a model for the remainder of the TMBIM family of proteins. The topology described provides valuable information on the structure and function of an important but poorly understood family of proteins. PMID:22418439

  13. Proteases from the Regenerating Gut of the Holothurian Eupentacta fraudatrix

    PubMed Central

    Lamash, Nina E.; Dolmatov, Igor Yu

    2013-01-01

    Four proteases with molecular masses of 132, 58, 53, and 47 kDa were detected in the digestive system of the holothurian Eupentacta fraudatrix. These proteases displayed the gelatinase activity and characteristics of zinc metalloproteinases. The 58 kDa protease had similar protease inhibitor sensitivity to that of mammalian matrix metalloproteinases. Zymographic assay revealed different lytic activities of all four proteases during intestine regeneration in the holothurian. The 132 kDa protease showed the highest activity at the first stage. During morphogenesis (stages 2–4 of regeneration), the highest activity was measured for the 53 and 58 kDa proteases. Inhibition of protease activity exerts a marked effect on regeneration, which was dependent on the time when 1,10-phenanthroline injections commenced. When metalloproteinases were inhibited at the second stage of regeneration, the restoration rates were decreased. However, such an effect proved to be reversible, and when inhibition ceased, the previous rate of regeneration was recovered. When protease activity is inhibited at the first stage, regeneration is completely abolished, and the animals die, suggesting that early activation of the proteases is crucial for triggering the regenerative process in holothurians. The role of the detected proteases in the regeneration processes of holothurians is discussed. PMID:23505505

  14. New directions for protease inhibitors directed drug discovery.

    PubMed

    Hamada, Yoshio; Kiso, Yoshiaki

    2016-11-01

    Proteases play crucial roles in various biological processes, and their activities are essential for all living organisms-from viruses to humans. Since their functions are closely associated with many pathogenic mechanisms, their inhibitors or activators are important molecular targets for developing treatments for various diseases. Here, we describe drugs/drug candidates that target proteases, such as malarial plasmepsins, β-secretase, virus proteases, and dipeptidyl peptidase-4. Previously, we reported inhibitors of aspartic proteases, such as renin, human immunodeficiency virus type 1 protease, human T-lymphotropic virus type I protease, plasmepsins, and β-secretase, as drug candidates for hypertension, adult T-cell leukaemia, human T-lymphotropic virus type I-associated myelopathy, malaria, and Alzheimer's disease. Our inhibitors are also described in this review article as examples of drugs that target proteases. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 563-579, 2016. PMID:26584340

  15. Evolution of Framingham cardiovascular risk score in HIV-infected patients initiating EFV- and LPV/r-based HAART in a Latin American cohort

    PubMed Central

    Cecchini, Diego; Ines Mattioli, Maria; Cassetti, Julia; Chan, Debora; Cassetti, Isabel

    2014-01-01

    Introduction Epidemiological studies suggest that some antiretroviral drugs may contribute to increase cardiovascular risk in HIV-infected patients. However, data from Latin American countries are limited, as impact of HAART on cardiovascular risk remains understudied. In this context, we aimed to evaluate if 10-year Framingham Cardiovascular Risk Score (FCRS) increases in patients following exposure to EFV- and LPV/r-based HAART in a Latin American cohort. Materials and Methods Retrospective 48-week cohort study. We reviewed clinical charts of randomly selected samples of patients initiating (according to national guidelines) EFV first-line HAART and LPV/r first- or second-line (but first PI-based) HAART assisted at a reference HIV centre in Buenos Aires, Argentina (period 2004–2012). Each patient could only be included in one arm. FCRS was calculated according to National Institutes of Health risk assessment tool (http://cvdrisk.nhlbi.nih.gov/). Results A total of 357 patients were included: 249 in EFV arm and 108 in LPV/r arm (80 as first line and 28 as second line, but first PI-based HAART). Baseline characteristics (median, interquartile range): age, 38 (33–45) years; male, 247 (69%); viral load, 98200 (20550–306000) copies/mL; CD4 T-cell count, 115 (60–175) cel/µL; total cholesterol, 159 (135–194) mg/dL; HDL: 39 (31–41) mg/dL; LDL: 94 (72–123) mg/dL; current smoker, 29%; on antihypertensive drugs: 14 (4%), diabetic: 4 (1%). Most frequent accompanying nucleoside reverse transcriptase inhibitors (NRTIs) were 3TC (92%) and zidovudine (AZT; 76%). Baseline FCRS was low, moderate and high for 93%, 7% and 0% of patients on EFV arm and 96.7%, 1.7% and 1.7% on LPV/r arm. On EFV arm, an increase in FCRS category (low to moderate or moderate to high) was observed in 1 patient (0.9%) at 24 weeks and 6 (5,6%) at 48 weeks; 5 (4.7%) decreased category. On LPV/r arm no one varied FCRS category at 24 weeks and 2 (3.4%) increased from low to moderate at 48 weeks

  16. Carotid artery intima–media thickness and HIV infection: traditional risk factors overshadow impact of protease inhibitor exposure

    PubMed Central

    Currier, Judith S.; Kendall, Michelle A.; Zackin, Robert; Henry, W. Keith; Alston-Smith, Beverly; Torriani, Francesca J.; Schouten, Jeff; Mickelberg, Keith; Li, Yanjie; Hodis, Howard N.

    2005-01-01

    Context The impact of HIV infection and exposure to antiretroviral therapy on the development of subclinical atherosclerosis is incompletely understood. Objective To compare intima–media thickness (IMT) of the carotid artery between HIV-infected subjects receiving protease inhibitor-containing regimens and subjects not receiving these regimens and to compare differences in the IMT of the carotid artery between HIV-infected subjects and HIV-uninfected subjects. Methods A prospective matched cohort study in university-based outpatient clinics. Groups of three individuals (triads) matched on the following characteristics were enrolled: age, sex, race/ethnicity, smoking status, blood pressure and menopausal status. Group 1, HIV-infected subjects with continuous use of protease inhibitor (PI) therapy for ≥ 2 years; group 2, HIV-infected subjects without prior PI use; and group 3: HIV-uninfected. Ultrasonographers at six sites sent standardized ultrasound images to a central reading site for carotid IMT measurements. Carotid IMT was compared within the HIV-infected groups (1 and 2) and between the HIV-infected and uninfected groups in a matched analysis. Results One hundred and thirty-four individuals were enrolled in 45 triads. The median IMT in groups 1, 2 and 3 was 0.690, 0.712 and 0.698 mm, respectively. There were no statistically significant differences in IMT between groups 1 and 2, or in the combined HIV groups compared with the HIV uninfected group. Significant predictors of carotid IMT in a multivariate model included high-density lipoprotein (HDL) cholesterol, the interaction of HDL cholesterol and triglycerides, age and body mass index. Conclusions We found no association between PI inhibitor exposure or HIV infection and carotid IMT. PMID:15905673

  17. Hepatitis C virus NS3 protease is activated by low concentrations of protease inhibitors.

    PubMed

    Dahl, Göran; Arenas, Omar Gutiérrez; Danielson, U Helena

    2009-12-01

    The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) is a bifunctional enzyme with a protease and a helicase functionality located in each of the two domains of the single peptide chain. There is little experimental evidence for a functional role of this unexpected arrangement since artificial single domain forms of both enzymes are catalytically competent. We have observed that low concentrations of certain protease inhibitors activate the protease of full-length NS3 from HCV genotype 1a with up to 100%, depending on the preincubation time and the inhibitor used. The activation was reduced, but not eliminated, by increased ionic strength, lowered glycerol concentration, or lowered pH. In all cases, it was at the expense of a significant loss of activity. Activation was not seen with the artificial protease domain of genotype 1b NS3 fused with a fragment of the NS4A cofactor. This truncated and covalently modified enzyme form was much less active and exhibited fundamentally different catalytic properties to the full-length NS3 protease without the fused cofactor. The most plausible explanation for the activation was found to involve a slow transition between two enzyme conformations, which differed in their catalytic ability and affinity for inhibitors. Equations derived based on this assumption resulted in better fits to the experimental data than the equation for simple competitive inhibition. The mechanism may involve an inhibitor-induced stabilization of the helicase domain in a conformation that enhances the protease activity, or an improved alignment of the catalytic triad in the protease. The proposed mnemonic mechanism and derived equations are viable for both these explanations and can serve as a basic framework for future studies of enzymes activated by inhibitors or other ligands.

  18. Three monoclonal antibodies against the serpin protease nexin-1 prevent protease translocation.

    PubMed

    Kousted, Tina M; Skjoedt, Karsten; Petersen, Steen V; Koch, Claus; Vitved, Lars; Sochalska, Maja; Lacroix, Céline; Andersen, Lisbeth M; Wind, Troels; Andreasen, Peter A; Jensen, Jan K

    2014-01-01

    Protease nexin-1 (PN-1) belongs to the serpin family and is an inhibitor of thrombin, plasmin, urokinase-type plasminogen activator, and matriptase. Recent studies have suggested PN-1 to play important roles in vascular-, neuro-, and tumour-biology. The serpin inhibitory mechanism consists of the serpin presenting its so-called reactive centre loop as a substrate to its target protease, resulting in a covalent complex with the inactivated enzyme. Previously, three mechanisms have been proposed for the inactivation of serpins by monoclonal antibodies: steric blockage of protease recognition, conversion to an inactive conformation or induction of serpin substrate behaviour. Until now, no inhibitory antibodies against PN-1 have been thoroughly characterised. Here we report the development of three monoclonal antibodies binding specifically and with high affinity to human PN-1. The antibodies all abolish the protease inhibitory activity of PN-1. In the presence of the antibodies, PN-1 does not form a complex with its target proteases, but is recovered in a reactive centre cleaved form. Using site-directed mutagenesis, we mapped the three overlapping epitopes to an area spanning the gap between the loop connecting α-helix F with β-strand 3A and the loop connecting α-helix A with β-strand 1B. We conclude that antibody binding causes a direct blockage of the final critical step of protease translocation, resulting in abortive inhibition and premature release of reactive centre cleaved PN-1. These new antibodies will provide a powerful tool to study the in vivo role of PN-1's protease inhibitory activity.

  19. Gender and other psychosocial factors as predictors of adherence to highly active antiretroviral therapy (HAART) in adults with comorbid HIV/AIDS, psychiatric and substance-related disorder.

    PubMed

    Applebaum, Allison J; Richardson, Mark A; Brady, Stephen M; Brief, Deborah J; Keane, Terence M

    2009-02-01

    This study assessed adherence to HAART among 67 HIV-infected adults, and the degree to which gender and psychological factors-including depression, drug and alcohol use, quality of life, and medication side effects-influenced adherence. Although overall adherence was greater than rates reported in similar studies, no significant difference in adherence was observed between men and women in the present sample. Medication side effects were a significant predictor of non-adherence in the sample at large and among women in particular, while alcohol dependence was a significant predictor of non-adherence only in women. Possible explanations are explored.

  20. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment.

    PubMed

    Fyfe, Cameron D; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W; Cogdell, Richard J; Wall, Daniel M; Burchmore, Richard J S; Byron, Olwyn; Walker, Daniel

    2015-07-01

    Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.

  1. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

    PubMed Central

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W.; Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel

    2015-01-01

    Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macro­globulin, this protease-activation mechanism is likely to operate across the diverse members of this group. PMID:26143919

  2. Dysregulation of protease and protease inhibitors in a mouse model of human pelvic organ prolapse.

    PubMed

    Budatha, Madhusudhan; Silva, Simone; Montoya, Teodoro Ignacio; Suzuki, Ayako; Shah-Simpson, Sheena; Wieslander, Cecilia Karin; Yanagisawa, Masashi; Word, Ruth Ann; Yanagisawa, Hiromi

    2013-01-01

    Mice deficient for the fibulin-5 gene (Fbln5(-/-)) develop pelvic organ prolapse (POP) due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP)-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5(-/-) mice, herein named V1 (25 kDa). V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS) 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5(-/-) mice. PRSS3 was (a) localized in epithelial secretions, (b) detected in media of vaginal organ culture from both Fbln5(-/-) and wild type mice, and (c) cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin) and Elafin] was dysregulated in Fbln5(-/-) epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice. PMID:23437119

  3. Structural determinants of tobacco vein mottling virus protease substrate specificity

    SciTech Connect

    Sun, Ping; Austin, Brian P.; Tozer, Jozsef; Waugh, David

    2010-10-28

    Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMV protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac-RETVRFQSD) at 1.7-{angstrom} resolution. As observed in several crystal structures of TEV protease, the C-terminus ({approx}20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by {approx}10-fold, an analogous truncation mutant of TVMV protease is significantly more active. Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1{prime} position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters k{sub cat} and K{sub m} for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease.

  4. Mitochondrial Proteases as Emerging Pharmacological Targets.

    PubMed

    Gibellini, Lara; De Biasi, Sara; Nasi, Milena; Iannone, Anna; Cossarizza, Andrea; Pinti, Marcello

    2016-01-01

    The preservation of mitochondrial function and integrity is critical for cell viability. Under stress conditions, unfolded, misfolded or damaged proteins accumulate in a certain compartment of the organelle, interfering with oxidative phosphorylation and normal mitochondrial functions. In stress conditions, several mechanisms, including mitochondrial unfolded protease response (UPRmt), fusion and fission, and mitophagy are engaged to restore normal proteostasis of the organelle. Mitochondrial proteases are a family of more than 20 enzymes that not only are involved in the UPRmt, but actively participate at multiple levels in the stress-response system. Alterations in their expression levels, or mutations that determine loss or gain of function of these proteases deeply impair mitochondrial functionality and can be associated with the onset of inherited diseases, with the development of neurodegenerative disorders and with the process of carcinogenesis. In this review, we focus our attention on six of them, namely CLPP, HTRA2 and LONP1, by analysing the current knowledge about their functions, their involvement in the pathogenesis of human diseases, and the compounds currently available for inhibiting their functions. PMID:26831646

  5. Proteases of human rhinovirus: role in infection.

    PubMed

    Jensen, Lora M; Walker, Erin J; Jans, David A; Ghildyal, Reena

    2015-01-01

    Human rhinoviruses (HRV) are the major etiological agents of the common cold and asthma exacerbations, with significant worldwide health and economic impact. Although large-scale population vaccination has proved successful in limiting or even eradicating many viruses, the more than 100 distinct serotypes mean that conventional vaccination is not a feasible strategy to combat HRV. An alternative strategy is to target conserved viral proteins such as the HRV proteases, 2A(pro) and 3C(pro), the focus of this review. Necessary for host cell shutoff, virus replication, and pathogenesis, 2A(pro) and 3C(pro) are clearly viable drug targets, and indeed, 3C(pro) has been successfully targeted for treating the common cold in experimental infection. 2A(pro) and 3C(pro) are crucial for virus replication due to their role in polyprotein processing as well as cleavage of key cellular proteins to inhibit cellular transcription and translation. Intriguingly, the action of the HRV proteases also disrupts nucleocytoplasmic trafficking, contributing to HRV cytopathic effects. Improved understanding of the protease-cell interactions should enable new therapeutic approaches to be identified for drug development. PMID:25261311

  6. Oligomeric state study of prokaryotic rhomboid proteases.

    PubMed

    Sampathkumar, Padmapriya; Mak, Michelle W; Fischer-Witholt, Sarah J; Guigard, Emmanuel; Kay, Cyril M; Lemieux, M Joanne

    2012-12-01

    Rhomboid peptidases (proteases) play key roles in signaling events at the membrane bilayer. Understanding the regulation of rhomboid function is crucial for insight into its mechanism of action. Here we examine the oligomeric state of three different rhomboid proteases. We subjected Haemophilus influenzae, (hiGlpG), Escherichia coli GlpG (ecGlpG) and Bacillus subtilis (YqgP) to sedimentation equilibrium analysis in detergent-solubilized dodecylmaltoside (DDM) solution. For hiGlpG and ecGlpG, rhomboids consisting of the core 6 transmembrane domains without and with soluble domains respectively, and YqgP, predicted to have 7 transmembrane domains with larger soluble domains at the termini, the predominant species was dimeric with low amounts of monomer and tetramers observed. To examine the effect of the membrane domain alone on oligomeric state of rhomboid, hiGlpG, the simplest form from the rhomboid class of intramembrane proteases representing the canonical rhomboid core of six transmembrane domains, was studied further. Using gel filtration and crosslinking we demonstrate that hiGlpG is dimeric and functional in DDM detergent solution. More importantly co-immunoprecipitation studies demonstrate that the dimer is present in the lipid bilayer suggesting a physiological dimer. Overall these results indicate that rhomboids form oligomers which are facilitated by the membrane domain. For hiGlpG we have shown that these oligomers exist in the lipid bilayer. This is the first detailed oligomeric state characterization of the rhomboid family of peptidases. PMID:22921757

  7. Proteases of human rhinovirus: role in infection.

    PubMed

    Jensen, Lora M; Walker, Erin J; Jans, David A; Ghildyal, Reena

    2015-01-01

    Human rhinoviruses (HRV) are the major etiological agents of the common cold and asthma exacerbations, with significant worldwide health and economic impact. Although large-scale population vaccination has proved successful in limiting or even eradicating many viruses, the more than 100 distinct serotypes mean that conventional vaccination is not a feasible strategy to combat HRV. An alternative strategy is to target conserved viral proteins such as the HRV proteases, 2A(pro) and 3C(pro), the focus of this review. Necessary for host cell shutoff, virus replication, and pathogenesis, 2A(pro) and 3C(pro) are clearly viable drug targets, and indeed, 3C(pro) has been successfully targeted for treating the common cold in experimental infection. 2A(pro) and 3C(pro) are crucial for virus replication due to their role in polyprotein processing as well as cleavage of key cellular proteins to inhibit cellular transcription and translation. Intriguingly, the action of the HRV proteases also disrupts nucleocytoplasmic trafficking, contributing to HRV cytopathic effects. Improved understanding of the protease-cell interactions should enable new therapeutic approaches to be identified for drug development.

  8. Corruption of innate immunity by bacterial proteases.

    PubMed

    Potempa, Jan; Pike, Robert N

    2009-01-01

    The innate immune system of the human body has developed numerous mechanisms to control endogenous and exogenous bacteria and thus prevent infections by these microorganisms. These mechanisms range from physical barriers such as the skin or mucosal epithelium to a sophisticated array of molecules and cells that function to suppress or prevent bacterial infection. Many bacteria express a variety of proteases, ranging from non-specific and powerful enzymes that degrade many proteins involved in innate immunity to proteases that are extremely precise and specific in their mode of action. Here we have assembled a comprehensive picture of how bacterial proteases affect the host's innate immune system to gain advantage and cause infection. This picture is far from being complete since the numbers of mechanisms utilized are as astonishing as they are diverse, ranging from degradation of molecules vital to innate immune mechanisms to subversion of the mechanisms to allow the bacterium to hide from the system or take advantage of it. It is vital that such mechanisms are elucidated to allow strategies to be developed to aid the innate immune system in controlling bacterial infections.

  9. Adherence to HAART: A Systematic Review of Developed and Developing Nation Patient-Reported Barriers and Facilitators

    PubMed Central

    Mills, Edward J; Nachega, Jean B; Bangsberg, David R; Singh, Sonal; Rachlis, Beth; Wu, Ping; Wilson, Kumanan; Buchan, Iain; Gill, Christopher J; Cooper, Curtis

    2006-01-01

    Background Adherence to highly active antiretroviral therapy (HAART) medication is the greatest patient-enabled predictor of treatment success and mortality for those who have access to drugs. We systematically reviewed the literature to determine patient-reported barriers and facilitators to adhering to antiretroviral therapy. Methods and Findings We examined both developed and developing nations. We searched the following databases: AMED (inception to June 2005), Campbell Collaboration (inception to June 2005), CinAhl (inception to June 2005), Cochrane Library (inception to June 2005), Embase (inception to June 2005), ERIC (inception to June 2005), MedLine (inception to June 2005), and NHS EED (inception to June 2005). We retrieved studies conducted in both developed and developing nation settings that examined barriers and facilitators addressing adherence. Both qualitative and quantitative studies were included. We independently, in duplicate, extracted data reported in qualitative studies addressing adherence. We then examined all quantitative studies addressing barriers and facilitators noted from the qualitative studies. In order to place the findings of the qualitative studies in a generalizable context, we meta-analyzed the surveys to determine a best estimate of the overall prevalence of issues. We included 37 qualitative studies and 47 studies using a quantitative methodology (surveys). Seventy-two studies (35 qualitative) were conducted in developed nations, while the remaining 12 (two qualitative) were conducted in developing nations. Important barriers reported in both economic settings included fear of disclosure, concomitant substance abuse, forgetfulness, suspicions of treatment, regimens that are too complicated, number of pills required, decreased quality of life, work and family responsibilities, falling asleep, and access to medication. Important facilitators reported by patients in developed nation settings included having a sense of self

  10. Four Amino Acid Changes in HIV-2 Protease Confer Class-Wide Sensitivity to Protease Inhibitors

    PubMed Central

    Smith, Robert A.; Gottlieb, Geoffrey S.

    2015-01-01

    ABSTRACT Protease is essential for retroviral replication, and protease inhibitors (PI) are important for treating HIV infection. HIV-2 exhibits intrinsic resistance to most FDA-approved HIV-1 PI, retaining clinically useful susceptibility only to lopinavir, darunavir, and saquinavir. The mechanisms for this resistance are unclear; although HIV-1 and HIV-2 proteases share just 38 to 49% sequence identity, all critical structural features of proteases are conserved. Structural studies have implicated four amino acids in the ligand-binding pocket (positions 32, 47, 76, and 82). We constructed HIV-2ROD9 molecular clones encoding the corresponding wild-type HIV-1 amino acids (I32V, V47I, M76L, and I82V) either individually or together (clone PRΔ4) and compared the phenotypic sensitivities (50% effective concentration [EC50]) of mutant and wild-type viruses to nine FDA-approved PI. Single amino acid replacements I32V, V47I, and M76L increased the susceptibility of HIV-2 to multiple PI, but no single change conferred class-wide sensitivity. In contrast, clone PRΔ4 showed PI susceptibility equivalent to or greater than that of HIV-1 for all PI. We also compared crystallographic structures of wild-type HIV-1 and HIV-2 proteases complexed with amprenavir and darunavir to models of the PRΔ4 enzyme. These models suggest that the amprenavir sensitivity of PRΔ4 is attributable to stabilizing enzyme-inhibitor interactions in the P2 and P2′ pockets of the protease dimer. Together, our results show that the combination of four amino acid changes in HIV-2 protease confer a pattern of PI susceptibility comparable to that of HIV-1, providing a structural rationale for intrinsic HIV-2 PI resistance and resolving long-standing questions regarding the determinants of differential PI susceptibility in HIV-1 and HIV-2. IMPORTANCE Proteases are essential for retroviral replication, and HIV-1 and HIV-2 proteases share a great deal of structural similarity. However, only three of nine

  11. Neuropathology of AIDS: An Autopsy Review of 284 Cases from Brazil Comparing the Findings Pre- and Post-HAART (Highly Active Antiretroviral Therapy) and Pre- and Postmortem Correlation

    PubMed Central

    Silva, Ana Cristina Araújo Lemos; Rodrigues, Blenda Sousa Carli; Micheletti, Adilha Misson Rua; Tostes, Sebastião; Meneses, Antonio Carlos Oliveira; Silva-Vergara, Mário Leon; Adad, Sheila Jorge

    2012-01-01

    A retrospective study of central nervous system (CNS) in 284 autopsy AIDS cases in Brazil (1989–2008) divided into 3 groups: A (without antiretroviral treatment: 163 cases); B (other antiretroviral therapies: 76 cases); C (HAART for 3 months or more: 45 cases). In 165 (58.1%) cases, relevant lesions were found, predominantly infections (54.2%); the most frequent was toxoplasmosis (29.9%) followed by cryptococcosis (15.8%), purulent bacterial infections (3.9%), and HIV encephalitis (2.8%); non-Hodgkin lymphomas occurred in 1.4% and vascular lesions in 1.1%. There was no difference when compared the frequency of lesion among the groups; however, toxoplasmosis was less common while HIV encephalitis was more frequent in group C related to A. CNS lesions remain a frequent cause of death in AIDS; however, the mean survival time was four times greater in group C than in A. In 91 (55.1%) of 165 cases with relevant brain lesions (or 32% of the total 284 cases), there was discordance between pre- and postmortem diagnosis; disagreement type 1 (important disease that if diagnosed in life could change the patient prognosis) occurred in 49 (53.8%) of 91 discordant cases (17.6% of the total 284) indicating the autopsy importance, even with HAART and advanced diagnostics technologies. PMID:22461978

  12. Recruitment in HIV/AIDS treatment naïve clinical trials in the HAART era - influence of gender, sexual orientation and race

    PubMed Central

    Menezes, P; Eron, JJ; Leone, PA; Adimora, AA; Wohl, DA; Miller, WC

    2010-01-01

    Background In the United States, women, racial/ethnic minorities and persons who acquire HIV infection through heterosexual intercourse represent an increasing proportion of HIV infected persons, yet are frequently underrepresented in clinical trials. We assessed the demographic predictors of trial participation in antiretroviral naïve patients. Methods Patients were characterized as trial participants if highly-active antiretroviral therapy (HAART) was initiated within a clinical trial. Prevalence ratios (PR) were obtained using binomial regression. Results Between 1996–2006, 30% of 738 treatment naïve patients initiated HAART in a clinical trial. Trial participation rates for MSM, heterosexual men, and women were respectively 36.5%, 29.6% and 24.3%. After adjustment for other factors, heterosexual men appeared less likely to participate in trials compared to MSM (PR: 0.79, 95%CI 0.57, 1.11) while women were as likely to participate as MSM (PR 0.97, 95%CI 0.68, 1.39). The participation rate in blacks (25.9%) was lower compared to non-blacks (37.5%) (adjusted PR 0.80, 95%CI 0.60, 1.06). Conclusions In our clinical setting gender did not appear to impact participation in HIV treatment trials but blacks were slightly less likely to participate in these trials. Considering the substantial proportion of HIV patients who are black, future trials need to consider strategies to incorporate underrepresented populations. PMID:20807254

  13. Distinctive in vitro effects of T-cell growth cytokines on cytomegalovirus-stimulated T-cell responses of HIV-infected HAART recipients

    SciTech Connect

    Patterson, Julie; Jesser, Renee; Weinberg, Adriana

    2008-08-15

    Functional immune reconstitution is limited after HAART, maintaining the interest in adjunctive immune-modulators. We compared in vitro the effects of the {gamma}-chain T-cell growth cytokines IL-2, IL-4, IL-7 and IL-15 on cytomegalovirus-stimulated cell-mediated immunity. IL-2 and IL-15 increased cytomegalovirus-specific lymphocyte proliferation in HAART recipients, whereas IL-4 and IL-7 did not. The boosting effect of IL-2 and IL-15 on proliferation correlated with their ability to prevent late apoptosis. However, IL-2 increased the frequency of cells in early apoptosis, whereas IL-15 increased the frequency of fully viable cells. Both IL-2 and IL-15 increased cytomegalovirus-induced CD4{sup +} and CD8{sup +} T-cell proliferation and the synthesis of Th1 and pro-inflammatory cytokines and chemokines. However, only IL-2 increased the frequency of regulatory T cells and Th2 cytokine production, both of which have the potential to attenuate antiviral immune responses. Overall, compared to other {gamma}-chain cytokines, IL-15 had the most favorable profile for boosting antiviral cell-mediated immunity.

  14. Preventive measures to prevent loss to follow-up in highly active antiretroviral therapy (HAART): implementing a strategy in Ziguinchor (Casamance, Senegal) in 2014.

    PubMed

    Randé, H; Rouffy, D

    2016-05-01

    Since 2010, the Pharmacie et Aide Humanitaire (PAH) in Casamance (Senegal) has been maintaining a software package (Tacojo) that allows monthly monitoring of the distribution of treatment to every patient with HIV infection receiving highly active antiretroviral therapy (HAART). We used this program to set up measures to prevent the loss to follow-up of patients receiving HAART. Our involvement focused on two main areas. First, each patient is routinely contacted after inclusion, to help us to understand the patient's experience of the disease and the treatment. This process aims to improve adherence to the treatment. Then, all patients who miss an appointment are routinely contacted by telephone within seven days of that appointment. The goal is to understand the reasons for the absence and to encourage patients to continue their treatment. Despite the lack of distance due to the relative newness of this program, these preventive measures have shown hopeful results (80% of the patients came back after a call). It would be interesting to apply it in a sustainable manner and in more medical facilities. PMID:27412981

  15. Diversity of Both the Cultivable Protease-Producing Bacteria and Bacterial Extracellular Proteases in the Coastal Sediments of King George Island, Antarctica

    PubMed Central

    Zhou, Ming-Yang; Wang, Guang-Long; Li, Dan; Zhao, Dian-Li; Qin, Qi-Long; Chen, Xiu-Lan; Chen, Bo; Zhou, Bai-Cheng; Zhang, Xi-Ying; Zhang, Yu-Zhong

    2013-01-01

    Protease-producing bacteria play a vital role in degrading sedimentary organic nitrogen. However, the diversity of these bacteria and their extracellular proteases in most regions remain unknown. In this paper, the diversity of the cultivable protease-producing bacteria and of bacterial extracellular proteases in the sediments of Maxwell Bay, King George Island, Antarctica was investigated. The cultivable protease-producing bacteria reached 105 cells/g in all 8 sediment samples. The cultivated protease-producing bacteria were mainly affiliated with the phyla Actinobacteria, Firmicutes, Bacteroidetes, and Proteobacteria, and the predominant genera were Bacillus (22.9%), Flavobacterium (21.0%) and Lacinutrix (16.2%). Among these strains, Pseudoalteromonas and Flavobacteria showed relatively high protease production. Inhibitor analysis showed that nearly all the extracellular proteases from the bacteria were serine proteases or metalloproteases. These results begin to address the diversity of protease-producing bacteria and bacterial extracellular proteases in the sediments of the Antarctic Sea. PMID:24223990

  16. Economic Methods of Ginger Protease'sextraction and Purification

    NASA Astrophysics Data System (ADS)

    Qiao, Yuanyuan; Tong, Junfeng; Wei, Siqing; Du, Xinyong; Tang, Xiaozhen

    This article reports the ginger protease extraction and purification methods from fresh ginger rhizome. As to ginger protease extraction, we adapt the steps of organic solvent dissolving, ammonium sulfate depositing and freeze-drying, and this method can attain crude enzyme powder 0.6% weight of fresh ginger rhizome. The purification part in this study includes two steps: cellulose ion exchange (DEAE-52) and SP-Sephadex 50 chromatography, which can purify crude ginger protease through ion and molecular weight differences respectively.

  17. A preliminary neutron diffraction analysis of Achromobacter protease I

    NASA Astrophysics Data System (ADS)

    Ohnishi, Yuki; Masaki, Takeharu; Yamada, Taro; Kurihara, Kazuo; Tanaka, Ichiro; Niimura, Nobuo

    2010-11-01

    Achromobacter protease I (API, E.C. 3.4.21.50) is one of the serine proteases produced by Achromobacter lyticus M497-1. API is distinct from the other tripsin type protease in its lysine specificity. The neutron structure analysis of catalytic triad with Trp169 and His210 was presented. His57 was double protonated and formed hydrogen bonds to Ser194Oγ and Asp113Oδ1, Oδ2.

  18. Role of Protease-Inhibitors in Ocular Diseases.

    PubMed

    Pescosolido, Nicola; Barbato, Andrea; Pascarella, Antonia; Giannotti, Rossella; Genzano, Martina; Nebbioso, Marcella

    2014-01-01

    It has been demonstrated that the balance between proteases and protease-inhibitors system plays a key role in maintaining cellular and tissue homeostasis. Indeed, its alteration has been involved in many ocular and systemic diseases. In particular, research has focused on keratoconus, corneal wounds and ulcers, keratitis, endophthalmitis, age-related macular degeneration, Sorsby fundus dystrophy, loss of nerve cells and photoreceptors during optic neuritis both in vivo and in vitro models. Protease-inhibitors have been extensively studied, rather than proteases, because they may represent a therapeutic approach for some ocular diseases. The protease-inhibitors mainly involved in the onset of the above-mentioned ocular pathologies are: α2-macroglobulin, α1-proteinase inhibitor (α1-PI), metalloproteinase inhibitor (TIMP), maspin, SERPINA3K, SERPINB13, secretory leukocyte protease inhibitor (SLPI), and calpeptin. This review is focused on the several characteristics of dysregulation of this system and, particularly, on a possible role of proteases and protease-inhibitors in molecular remodeling that may lead to some ocular diseases. Recently, researchers have even hypothesized a possible therapeutic effect of the protease-inhibitors in the treatment of injured eye in animal models. PMID:25493637

  19. Fibrin(ogen)olytic activity of bumblebee venom serine protease

    SciTech Connect

    Qiu Yuling; Choo, Young Moo; Yoon, Hyung Joo; Jia Jingming; Cui Zheng; Wang Dong; Kim, Doh Hoon; Sohn, Hung Dae; Jin, Byung Rae

    2011-09-01

    Bee venom is a rich source of pharmacologically active components; it has been used as an immunotherapy to treat bee venom hypersensitivity, and venom therapy has been applied as an alternative medicine. Here, we present evidence that the serine protease found in bumblebee venom exhibits fibrin(ogen)olytic activity. Compared to honeybee venom, bumblebee venom contains a higher content of serine protease, which is one of its major components. Venom serine proteases from bumblebees did not cross-react with antibodies against the honeybee venom serine protease. We provide functional evidence indicating that bumblebee (Bombus terrestris) venom serine protease (Bt-VSP) acts as a fibrin(ogen)olytic enzyme. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. However, Bt-VSP is not a plasminogen activator, and its fibrinolytic activity is less than that of plasmin. Taken together, our results define roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings offer significant insight into the allergic reaction sequence that is initiated by bee venom serine protease and its potential usefulness as a clinical agent in the field of hemostasis and thrombosis. - Graphical abstract: Display Omitted Highlights: > Bumblebee venom serine protease (Bt-VSP) is a fibrin(ogen)olytic enzyme. > Bt-VSP activates prothrombin. > Bt-VSP directly degrades fibrinogen into fibrin degradation products. > Bt-VSP is a hemostatically active protein that is a potent clinical agent.

  20. Extracellular Bacterial Proteases in Chronic Wounds: A Potential Therapeutic Target?

    PubMed Central

    Suleman, Louise

    2016-01-01

    Significance: Bacterial biofilms are considered to be responsible for over 80% of persistent infections, including chronic lung infections, osteomyelitis, periodontitis, endocarditis, and chronic wounds. Over 60% of chronic wounds are colonized with bacteria that reside within a biofilm. The exaggerated proteolytic environment of chronic wounds, more specifically elevated matrix metalloproteinases, is thought to be one of the possible reasons as to why chronic wounds fail to heal. However, the role of bacterial proteases within chronic wounds is not fully understood. Recent Advances: Recent research has shown that bacterial proteases can enable colonization and facilitate bacterial immune evasion. The inhibition of bacterial proteases such as Pseudomonas aeruginosa elastase B (LasB) has resulted in the disruption of the bacterial biofilm in vitro. P. aeruginosa is thought to be a key pathogen in chronic wound infection, and therefore, the disruption of these biofilms, potentially through the targeting of P. aeruginosa bacterial proteases, is an attractive therapeutic endeavor. Critical Issues: Disrupting biofilm formation through the inhibition of bacterial proteases may lead to the dissemination of bacteria from the biofilm, allowing planktonic cells to colonize new sites within the wound. Future Directions: Despite a plethora of evidence supporting the role of bacterial proteases as virulence factors in infection, there remains a distinct lack of research into the effect of bacterial proteases in chronic wounds. To assess the viability of targeting bacterial proteases, future research should aim to understand the role of these proteases in a variety of chronic wound subtypes. PMID:27785379

  1. Role of Protease-Inhibitors in Ocular Diseases.

    PubMed

    Pescosolido, Nicola; Barbato, Andrea; Pascarella, Antonia; Giannotti, Rossella; Genzano, Martina; Nebbioso, Marcella

    2014-01-01

    It has been demonstrated that the balance between proteases and protease-inhibitors system plays a key role in maintaining cellular and tissue homeostasis. Indeed, its alteration has been involved in many ocular and systemic diseases. In particular, research has focused on keratoconus, corneal wounds and ulcers, keratitis, endophthalmitis, age-related macular degeneration, Sorsby fundus dystrophy, loss of nerve cells and photoreceptors during optic neuritis both in vivo and in vitro models. Protease-inhibitors have been extensively studied, rather than proteases, because they may represent a therapeutic approach for some ocular diseases. The protease-inhibitors mainly involved in the onset of the above-mentioned ocular pathologies are: α2-macroglobulin, α1-proteinase inhibitor (α1-PI), metalloproteinase inhibitor (TIMP), maspin, SERPINA3K, SERPINB13, secretory leukocyte protease inhibitor (SLPI), and calpeptin. This review is focused on the several characteristics of dysregulation of this system and, particularly, on a possible role of proteases and protease-inhibitors in molecular remodeling that may lead to some ocular diseases. Recently, researchers have even hypothesized a possible therapeutic effect of the protease-inhibitors in the treatment of injured eye in animal models.

  2. Detergent alkaline proteases: enzymatic properties, genes, and crystal structures.

    PubMed

    Saeki, Katsuhisa; Ozaki, Katsuya; Kobayashi, Tohru; Ito, Susumu

    2007-06-01

    Subtilisin-like serine proteases from bacilli have been used in various industrial fields worldwide, particularly in the production of laundry and automatic dishwashing detergents. They belong to family A of the subtilase superfamily, which is composed of three clans, namely, true subtilisins, high-alkaline proteases, and intracellular proteases. We succeeded in the large-scale production of a high-alkaline protease (M-protease) from alkaliphilic Bacillus clausii KSM-K16, and the enzyme has been introduced into compact heavy-duty laundry detergents. We have also succeeded in the industrial-scale production of a new alkaline protease, KP-43, which was originally resistant to chemical oxidants and to surfactants, produced by alkaliphilic Bacillus sp. strain KSM-KP43 and have incorporated it into laundry detergents. KP-43 and related proteases form a new clan, oxidatively stable proteases, in subtilase family A. In this review, we describe the enzymatic properties, gene sequences, and crystal structures of M-protease, KP-43, and related enzymes. PMID:17630120

  3. Intracellular alkaline proteases produced by thermoacidophiles: detection of protease heterogeneity by gelatin zymography and polymerase chain reaction (PCR).

    PubMed

    Kocabiyik, Semra; Erdem, Bilge

    2002-08-01

    In this study 24 thermoacidophilic archeal and bacterial strains isolated from hot-springs and hot-soils were screened for their ability to produce intracellular alkaline proteases. The protease activities of the strains, based on azocasein hydrolysis, showed a variation from 0.6 to 5.1 U. The cell extracts of three most potent producers were further examined and it was found that their proteases exhibited maximum activity at 60-70 degrees C and showed a pH optimum over a range of pH 7.0-8.5. Gelatin zymography revealed that two of the selected archeal strains produced multiple active SDS-resistant proteases. On the other hand, PCR amplification of alkaline serine protease gene sequences of total DNA from all isolates yielded four distinct amplification fragments of 650, 450, 400 and 300 bp, which might have been derived from different serine protease genes.

  4. Origin and Diversification of Meprin Proteases.

    PubMed

    Marín, Ignacio

    2015-01-01

    Meprins are astacin metalloproteases with a characteristic, easily recognizable structure, given that they are the only proteases with both MAM and MATH domains plus a transmembrane region. So far assumed to be vertebrate-specific, it is shown here, using a combination of evolutionary and genomic analyses, that meprins originated before the urochordates/vertebrates split. In particular, three genes encoding structurally typical meprin proteins are arranged in tandem in the genome of the urochordate Ciona intestinalis. Phylogenetic analyses showed that the protease and MATH domains present in the meprin-like proteins encoded by the Ciona genes are very similar in sequence to the domains found in vertebrate meprins, which supports them having a common origin. While many vertebrates have the two canonical meprin-encoding genes orthologous to human MEP1A and MEP1B (which respectively encode for the proteins known as meprin α and meprin β), a single gene has been found so far in the genome of the chondrichthyan fish Callorhinchus milii, and additional meprin-encoding genes are present in some species. Particularly, a group of bony fish species have genes encoding highly divergent meprins, here named meprin-F. Genes encoding meprin-F proteins, derived from MEP1B genes, are abundant in some species, as the Amazon molly, Poecilia formosa, which has 7 of them. Finally, it is confirmed that the MATH domains of meprins are very similar to the ones in TRAF ubiquitin ligases, which suggests that meprins originated when protease and TRAF E3-encoding sequences were combined. PMID:26288188

  5. Purification of Pseudomonas aeruginosa proteases and microscopic characterization of pseudomonal protease-induced rabbit corneal damage.

    PubMed Central

    Kreger, A S; Gray, L D

    1978-01-01

    Extracellular proteases of three cornea-virulent strains of Pseudomonas aeruginosa were isolated by sequential ammonium sulfate precipitation, Ultrogel AcA 54 gel filtration, and flat-bed isoelectric focusing. The purity of the preparations was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis , thin-layer isoelectric focusing in polyacrylamide gel, immunodiffusion and immunoelectrophoretic procedures, and tests for the presence of other known pseudomonal products. Light and electron microscopic examination of rabbit corneal lesions observed 4 to 6 h after the intracorneal injection of submicrogram amounts of the proteases revealed: (i) degeneration and necrosis of epithelium, endothelium, and keratocytes, (ii) infiltration, degeneration, and necrosis of polymorphonuclear leukocytes, (iii) loss of the characteristic weblike pattern, colloidal iron staining, and ruthenium red staining of the stromal proteoglycan ground substance, (iv) dispersal of strucutrally normal appearing collagen fibrils, ground substance, (iv) dispersal of structurally normal appearing collagen fibrils, and (v) accumulation of plasma proteins and fibrin in the necrotic corneas. These structural alterations are very similar to those observed previously during experimental P. aeruginosa keratitis, and this similarity supports the idea that pseudomonal proteases are responsible, at least in part, for the rapid and extensive liquefaction necrosis characteristic of pseudomonal-induced keratitis. In addition, the results support the idea that pseudomonal proteases elicit severe corneal damage by causing the loss of the corneal proteoglycan ground substance, thus resulting in dispersal of undamaged collagen fibrils, weakening of the corneal stroma, and subsequent descemetocele formation and corneal perforation by the anterior chamber pressure. Images PMID:415981

  6. A bumblebee (Bombus ignitus) venom serine protease inhibitor that acts as a microbial serine protease inhibitor.

    PubMed

    Wan, Hu; Kim, Bo Yeon; Lee, Kwang Sik; Yoon, Hyung Joo; Lee, Kyung Yong; Jin, Byung Rae

    2014-01-01

    Serine protease inhibitors from bumblebee venom have been shown to block plasmin activity. In this study, we identified the protein BiVSPI from the venom of Bombus ignitus to be a serine protease inhibitor and an antimicrobial factor. BiVSPI is a 55-amino acid mature peptide with ten conserved cysteine residues and a P1 methionine residue. BiVSPI is expressed in the venom gland and also found in the venom as an 8-kDa peptide. Recombinant BiVSPI that was expressed in baculovirus-infected insect cells exhibited inhibitory activity against chymotrypsin but not trypsin. BiVSPI also inhibited microbial serine proteases, such as subtilisin A (Ki=6.57nM) and proteinase K (Ki=7.11nM). In addition, BiVSPI was shown to bind directly to Bacillus subtilis, Bacillus thuringiensis, and Beauveria bassiana but not to Escherichia coli. Consistent with these results, BiVSPI exhibited antimicrobial activity against Gram-positive bacteria and fungi. These findings provide evidence for a novel serine protease inhibitor in bumblebee venom that has antimicrobial functions.

  7. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

    SciTech Connect

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W.; Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel

    2015-06-30

    The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.

  8. Rigidity analysis of HIV-1 protease

    NASA Astrophysics Data System (ADS)

    Heal, J. W.; Wells, S. A.; Jimenez-Roldan, E.; Freedman, R. F.; Römer, R. A.

    2011-03-01

    We present a rigidity analysis on a large number of X-ray crystal structures of the enzyme HIV-1 protease using the 'pebble game' algorithm of the software FIRST. We find that although the rigidity profile remains similar across a comprehensive set of high resolution structures, the profile changes significantly in the presence of an inhibitor. Our study shows that the action of the inhibitors is to restrict the flexibility of the β-hairpin flaps which allow access to the active site. The results are discussed in the context of full molecular dynamics simulations as well as data from NMR experiments.

  9. Endogenous Protease Activation of ENaC

    PubMed Central

    Adebamiro, Adedotun; Cheng, Yi; Johnson, John P.; Bridges, Robert J.

    2005-01-01

    Endogenous serine proteases have been reported to control the reabsorption of Na+ by kidney- and lung-derived epithelial cells via stimulation of electrogenic Na+ transport mediated by the epithelial Na+ channel (ENaC). In this study we investigated the effects of aprotinin on ENaC single channel properties using transepithelial fluctuation analysis in the amphibian kidney epithelium, A6. Aprotinin caused a time- and concentration-dependent inhibition (84 ± 10.5%) in the amiloride-sensitive sodium transport (INa) with a time constant of 18 min and half maximal inhibition constant of 1 μM. Analysis of amiloride analogue blocker–induced fluctuations in INa showed linear rate–concentration plots with identical blocker on and off rates in control and aprotinin-inhibited conditions. Verification of open-block kinetics allowed for the use of a pulse protocol method (Helman, S.I., X. Liu, K. Baldwin, B.L. Blazer-Yost, and W.J. Els. 1998. Am. J. Physiol. 274:C947–C957) to study the same cells under different conditions as well as the reversibility of the aprotinin effect on single channel properties. Aprotinin caused reversible changes in all three single channel properties but only the change in the number of open channels was consistent with the inhibition of INa. A 50% decrease in INa was accompanied by 50% increases in the single channel current and open probability but an 80% decrease in the number of open channels. Washout of aprotinin led to a time-dependent restoration of INa as well as the single channel properties to the control, pre-aprotinin, values. We conclude that protease regulation of INa is mediated by changes in the number of open channels in the apical membrane. The increase in the single channel current caused by protease inhibition can be explained by a hyperpolarization of the apical membrane potential as active Na+ channels are retrieved. The paradoxical increase in channel open probability caused by protease inhibition will require further

  10. Construction of dengue virus protease expression plasmid and in vitro protease assay for screening antiviral inhibitors.

    PubMed

    Lai, Huiguo; Teramoto, Tadahisa; Padmanabhan, Radhakrishnan

    2014-01-01

    Dengue virus serotypes 1-4 (DENV1-4) are mosquito-borne human pathogens of global significance causing ~390 million cases annually worldwide. The virus infections cause in general a self-limiting disease, known as dengue fever, but occasionally also more severe forms, especially during secondary infections, dengue hemorrhagic fever and dengue shock syndrome causing ~25,000 deaths annually. The DENV genome contains a single-strand positive sense RNA, approximately 11 kb in length. The 5'-end has a type I cap structure. The 3'-end has no poly(A) tail. The viral RNA has a single long open reading frame that is translated by the host translational machinery to yield a polyprotein precursor. Processing of the polyprotein precursor occurs co-translationally by cellular proteases and posttranslationally by the viral serine protease in the endoplasmic reticulum (ER) to yield three structural proteins (capsid (C), precursor membrane (prM), and envelope (E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The active viral protease consists of both NS2B, an integral membrane protein in the ER, and the N-terminal part of NS3 (180 amino acid residues) that contains the trypsin-like serine protease domain having a catalytic triad of H51, D75, and S135. The C-terminal part of NS3, ~170-618 amino acid residues, encodes an NTPase/RNA helicase and 5'-RNA triphosphatase activities; the latter enzyme is required for the first step in 5'-capping. The cleavage sites of the polyprotein by the viral protease consist of two basic amino acid residues such as KR, RR, or QR, followed by short chain amino acid residues, G, S, or T. Since the cleavage of the polyprotein by the viral protease is absolutely required for assembly of the viral replicase, blockage of NS2B/NS3pro activity provides an effective means for designing dengue virus (DENV) small-molecule therapeutics. Here we describe the screening of small-molecule inhibitors against DENV2 protease. PMID

  11. Mapping chemical structure-activity information of HAART-drug cocktails over complex networks of AIDS epidemiology and socioeconomic data of U.S. counties.

    PubMed

    Herrera-Ibatá, Diana María; Pazos, Alejandro; Orbegozo-Medina, Ricardo Alfredo; Romero-Durán, Francisco Javier; González-Díaz, Humberto

    2015-06-01

    Using computational algorithms to design tailored drug cocktails for highly active antiretroviral therapy (HAART) on specific populations is a goal of major importance for both pharmaceutical industry and public health policy institutions. New combinations of compounds need to be predicted in order to design HAART cocktails. On the one hand, there are the biomolecular factors related to the drugs in the cocktail (experimental measure, chemical structure, drug target, assay organisms, etc.); on the other hand, there are the socioeconomic factors of the specific population (income inequalities, employment levels, fiscal pressure, education, migration, population structure, etc.) to study the relationship between the socioeconomic status and the disease. In this context, machine learning algorithms, able to seek models for problems with multi-source data, have to be used. In this work, the first artificial neural network (ANN) model is proposed for the prediction of HAART cocktails, to halt AIDS on epidemic networks of U.S. counties using information indices that codify both biomolecular and several socioeconomic factors. The data was obtained from at least three major sources. The first dataset included assays of anti-HIV chemical compounds released to ChEMBL. The second dataset is the AIDSVu database of Emory University. AIDSVu compiled AIDS prevalence for >2300 U.S. counties. The third data set included socioeconomic data from the U.S. Census Bureau. Three scales or levels were employed to group the counties according to the location or population structure codes: state, rural urban continuum code (RUCC) and urban influence code (UIC). An analysis of >130,000 pairs (network links) was performed, corresponding to AIDS prevalence in 2310 counties in U.S. vs. drug cocktails made up of combinations of ChEMBL results for 21,582 unique drugs, 9 viral or human protein targets, 4856 protocols, and 10 possible experimental measures. The best model found with the original

  12. The effect of HIV coinfection, HAART and TB treatment on cytokine/chemokine responses to Mycobacterium tuberculosis (Mtb) antigens in active TB patients and latently Mtb infected individuals.

    PubMed

    Kassa, Desta; de Jager, Wilco; Gebremichael, Gebremedhin; Alemayehu, Yodit; Ran, Leonie; Fransen, Justin; Wolday, Dawit; Messele, Tsehaynesh; Tegbaru, Belete; Ottenhoff, Tom H M; van Baarle, Debbie

    2016-01-01

    Identification of Mtb specific induced cytokine/chemokine host biomarkers could assist in developing novel diagnostic, prognostic and therapeutic tools for TB. Levels of IFN-γ, IL-2, IL-17, IL-10, IP-10 and MIP-1α were measured in supernatants of whole blood stimulated with Mtb specific fusion protein ESAT-6/CFP-10 using xMAP technology. The study groups were HIV positive TB patients (HIV(+)TB(+)), HIV negative TB patients (HIV(-)TB(+)), HIV positive tuberculin skin test positive (TST+) (HIV(+)TST(+)), HIV negative TST+ (HIV(-)TST(+)), and HIV(-)TST(-) individuals. Compared to HIV(-)TST(-), latent TB infection led to increased levels of IP-10, IFN-γ and IL-17, while levels of IL-2 and IP-10 were increased with active TB. Levels of IFN-γ, IL-17, MIP-1α, and IL-10 were increased in HIV(-)TST(+) individuals compared to HIV(-)TB(+) patients. HIV coinfection decreased the level of IFN-γ, IL-17, IP-10 and IL-2. After six months (M6) of anti-TB treatment (ATT) in HIV(-)TB(+) patients, IFN-γ, IL-10, and MIP-1α levels normalized. After M6 and M18 of ATT plus HAART in HIV(+)TB(+) patients, levels of MIP-1α and IL-10 normalized, while this was not the case for IFN-γ, IL-2, IL-17, and IP-10 levels. In HIV(+)TST(+) patients on HAART, levels of IFN-γ, IL-17, IL-10 and MIP-1α normalized, while no change in the levels of IL-2 and IP-10 were observed. In conclusion, the simultaneous measurement of IFN-γ, IL-17 and IP-10 may assist in diagnosing LTBI; IL-2 and IP-10 may assist in diagnosing active TB; while IFN-γ, IL-17, MIP-1α, and IL-10 levels could help to discriminate LTBI and active TB. In addition, IL-10 and MIP-1α levels could help to monitor responses to TB treatment and HAART.

  13. Epidemiological and clinical features, response to HAART, and survival in HIV-infected patients diagnosed at the age of 50 or more

    PubMed Central

    Nogueras, MaMercedes; Navarro, Gemma; Antón, Esperança; Sala, Montserrat; Cervantes, Manel; Amengual, MaJosé; Segura, Ferran

    2006-01-01

    Background Over the last years, the mean age of subjects with HIV infection and AIDS is increasing. Moreover, some epidemiological and clinical differences between younger and older HIV-infected individuals have been observed. However, since introduction of HAART therapy, there are controversial results regarding their response to HAART. The aim of the present study is to evaluate epidemiological and clinical features, response to HAART, and survival in elderly HIV-infected patients with regard to younger HIV-infected patients. Methods A prospective cohort study (1998–2003) was performed on patients from Sabadell Hospital, in Northeast of Spain. The cohort includes newly attended HIV-infected patients since January 1, 1998. For the purpose of this analysis, data was censured at December 31, 2003. Taking into account age at time of diagnosis, it was considered 36 HIV-positive people aged 50 years or more (Group 1, G1) and 419 HIV-positive people aged 13–40 years (Group 2, G2). Epidemiological, clinical, biological and therapy data are recorded. Statistical analysis was performed using Chi-squared test and Fisher exact test, Mann-Whitney U test, Kaplan-Meier, Log Rank test, and Two-Way ANOVA from random factors. Results G1 showed higher proportion of men than G2. The most common risk factors in G1 were heterosexual transmission (P = 0.01) and having sex with men or women (P < 0.001). G1 and G2 show parallel profiles through the time regarding immunological response (P = 0.989) and virological response (P = 0.074). However, older people showed lower CD4 cell counts at first clinic visit (P < 0.001) and, eventually, they did not achieve the same counts as G2. G1 presented faster progression to AIDS (P < 0.001) and shorter survival (P < 0.001). Conclusion Older patients have different epidemiological features. Their immunological and virological responses are good. However, older patients do not achieve the same CD4 cell counts likely due to they have lower counts

  14. Mapping chemical structure-activity information of HAART-drug cocktails over complex networks of AIDS epidemiology and socioeconomic data of U.S. counties.

    PubMed

    Herrera-Ibatá, Diana María; Pazos, Alejandro; Orbegozo-Medina, Ricardo Alfredo; Romero-Durán, Francisco Javier; González-Díaz, Humberto

    2015-06-01

    Using computational algorithms to design tailored drug cocktails for highly active antiretroviral therapy (HAART) on specific populations is a goal of major importance for both pharmaceutical industry and public health policy institutions. New combinations of compounds need to be predicted in order to design HAART cocktails. On the one hand, there are the biomolecular factors related to the drugs in the cocktail (experimental measure, chemical structure, drug target, assay organisms, etc.); on the other hand, there are the socioeconomic factors of the specific population (income inequalities, employment levels, fiscal pressure, education, migration, population structure, etc.) to study the relationship between the socioeconomic status and the disease. In this context, machine learning algorithms, able to seek models for problems with multi-source data, have to be used. In this work, the first artificial neural network (ANN) model is proposed for the prediction of HAART cocktails, to halt AIDS on epidemic networks of U.S. counties using information indices that codify both biomolecular and several socioeconomic factors. The data was obtained from at least three major sources. The first dataset included assays of anti-HIV chemical compounds released to ChEMBL. The second dataset is the AIDSVu database of Emory University. AIDSVu compiled AIDS prevalence for >2300 U.S. counties. The third data set included socioeconomic data from the U.S. Census Bureau. Three scales or levels were employed to group the counties according to the location or population structure codes: state, rural urban continuum code (RUCC) and urban influence code (UIC). An analysis of >130,000 pairs (network links) was performed, corresponding to AIDS prevalence in 2310 counties in U.S. vs. drug cocktails made up of combinations of ChEMBL results for 21,582 unique drugs, 9 viral or human protein targets, 4856 protocols, and 10 possible experimental measures. The best model found with the original

  15. Viral proteases as targets for drug design.

    PubMed

    Skoreński, Marcin; Sieńczyk, Marcin

    2013-01-01

    In order to productively infect a host, viruses must enter the cell and force host cell replication mechanisms to produce new infectious virus particles. The success of this process unfortunately results in disease progression and, in the case of infection with many viral species, may cause mortality. The discoveries of Louis Pasteur and Edward Jenner led to one of the greatest advances in modern medicine - the development of vaccines that generate long-lasting memory immune responses to combat viral infection. Widespread use of vaccines has reduced mortality and morbidity associated with viral infection and, in some cases, has completely eradicated virus from the human population. Unfortunately, several viral species maintain a significant ability to mutate and "escape" vaccine-induced immune responses. Thus, novel anti-viral agents are required for treatment and prevention of viral disease. Targeting proteases that are crucial in the viral life cycle has proven to be an effective method to control viral infection, and this avenue of investigation continues to generate anti-viral treatments. Herein, we provide the reader with a brief history as well as a comprehensive review of the most recent advances in the design and synthesis of viral protease inhibitors. PMID:23016690

  16. Broad-Spectrum Allosteric Inhibition of Herpesvirus Proteases

    PubMed Central

    2015-01-01

    Herpesviruses rely on a homodimeric protease for viral capsid maturation. A small molecule, DD2, previously shown to disrupt dimerization of Kaposi’s sarcoma-associated herpesvirus protease (KSHV Pr) by trapping an inactive monomeric conformation and two analogues generated through carboxylate bioisosteric replacement (compounds 2 and 3) were shown to inhibit the associated proteases of all three human herpesvirus (HHV) subfamilies (α, β, and γ). Inhibition data reveal that compound 2 has potency comparable to or better than that of DD2 against the tested proteases. Nuclear magnetic resonance spectroscopy and a new application of the kinetic analysis developed by Zhang and Poorman [Zhang, Z. Y., Poorman, R. A., et al. (1991) J. Biol. Chem. 266, 15591–15594] show DD2, compound 2, and compound 3 inhibit HHV proteases by dimer disruption. All three compounds bind the dimer interface of other HHV proteases in a manner analogous to binding of DD2 to KSHV protease. The determination and analysis of cocrystal structures of both analogues with the KSHV Pr monomer verify and elaborate on the mode of binding for this chemical scaffold, explaining a newly observed critical structure–activity relationship. These results reveal a prototypical chemical scaffold for broad-spectrum allosteric inhibition of human herpesvirus proteases and an approach for the identification of small molecules that allosterically regulate protein activity by targeting protein–protein interactions. PMID:24977643

  17. Effect of proteases on the. beta. -thromboglobulin radioimmunoassay

    SciTech Connect

    Donlon, J.A.; Helgeson, E.A.; Donlon, M.A.

    1985-02-11

    Rat peritoneal mast cells and mast cell granules were evaluated by radioimmunoassay for the presence of ..beta..-thromboglobulin and platelet factor 4. The initial assays indicated that a ..beta..-thromboglobulin cross reacting material was released from mast cells by compound 48/80 in a similar dose-dependent manner as histamine release. The material was also found to be associated with purified granules. However, the use of protease inhibitors in the buffers completely abolished the positive assays. Further evaluation of the effects of various proteases on the ..beta..-thromboglobulin assay indicated that elastase would also generate a false positive assay which could then be neutralized by the use of ..cap alpha../sub 1/-antitrypsin as a protease inhibitor. There was no protease effect on the platelet factor 4 radioimmunoassay which always showed no detectable amounts with mast cells, granules or proteases. These results clearly indicate the artifactual positive assays which can arise when using certain radioimmunoassay tests in the presence of cell proteases. The use of protease inhibitors is a necessary control when applying a radioimmunoassay to a system with potentially active proteases. 24 references, 2 figures, 4 tables.

  18. Expression and characterization of Coprothermobacter proteolyticus alkaline serine protease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    TECHNICAL ABSTRACT A putative protease gene (aprE) from the thermophilic bacterium Coprothermobacter proteolyticus was cloned and expressed in Bacillus subtilis. The enzyme was determined to be a serine protease based on inhibition by PMSF. Biochemical characterization demonstrated the enzyme had...

  19. Correlation between viral load, plasma levels of CD4 - CD8 T lymphocytes and AIDS-related oral diseases: a multicentre study on 30 HIV+ children in the HAART era.

    PubMed

    Nesti, M; Carli, E; Giaquinto, C; Rampon, O; Nastasio, S; Giuca, M R

    2012-01-01

    This experimental retrospective multicenter study carried out on 30 seropositive children treated with Highly Active Antiretroviral Therapy (HAART), between the ages of 18 months and 14 years, in the clinical categories Centers for Disease Control (CDC) classification 1993 A (mildly symptomatic), B (moderately symptomatic) and C (severely symptomatic) aims to: 1) clinically and immunologically demonstrate the therapeutic benefits of HAART; 2) monitor the frequency of AIDS-related oral diseases in seropositive children with HAART therapy; 3) monitor the plasma levels of total CD4, CD4 percent, CD8 percent, CD4-CD8 lymphocytes and viral load from 1997 to 30 April, 2011. The statistic methods used are the analysis of covariance and the Bonferroni Test. More than 100 AIDS-related oral diseases were found in the study samples, the most frequent being: oral candidiasis, oropharyngeal candidiasis, HSV-1 herpetic esophagyitis, herpetic gingivolstomatitis (RHOG), recurrent aphthous stomatitis (RAS), parotid swelling, oral hairy leukoplakia (OHL), Herpes simplex 1 (HSV-1), linear gingival erythema (LGE), necrotizing gingivitis (NUG), facial lipodistrophy, facial-cervical lymphadenopathy (FCL), xerostomia, dysgeusia, hyposmia, oral mucosa hyperpigmentation (OMP). The Bonferroni test showed a significant difference between the mean plasma values (mpVTL) of total CD4, CD4 percentage, CD4-CD8 T lymphocytes and Viral Load (VL) of the various oral diseases found in the study samples. The therapeutic benefits of HAART are: immune reconstitution; reduction of the HIV/AIDS-related stomatology diseases; prevention and cure of the AIDS correlated neoplasias; reduction in maternal-fetal transmission of the HIV virus. The negative effects of HAART in relation to odontostomatolgy are: increase in oral lesions from HPV; xerostomia; dysgeusia/ageusia, hyposmia, perioral paresthesia; hyperpigmentation of oral mucosa; facial lipodystrophy, recurrent aphthous stomatitis (RAS). No case of

  20. Alkaline protease production by a strain of marine yeasts

    NASA Astrophysics Data System (ADS)

    Ping, Wang; Zhenming, Chi; Chunling, Ma

    2006-07-01

    Yeast strain 10 with high yield of protease was isolated from sediments of saltern near Qingdao, China. The protease had the highest activity at pH 9.0 and 45°C. The optimal medium for the maximum alkaline protease production of strain 10 was 2.5g soluble starch and 2.0g NaNO3 in 100mL seawater with initial pH 6.0. The optimal cultivation conditions for the maximum protease production were temperature 24.5°C, aeration rate 8.0L min-1 and agitation speed 150r min-1 Under the optimal conditions, 623.1 U mg-1 protein of alkaline protease was reached in the culture within 30h of fermentation.

  1. Purification and characterization of an alkaline protease from Acetes chinensis

    NASA Astrophysics Data System (ADS)

    Xu, Jiachao; Liu, Xin; Li, Zhaojie; Xu, Jie; Xue, Changhu; Gao, Xin

    2005-07-01

    An alkaline protease from Acetes chinensis was purified and characterized in this study. The steps of purification include ammonium sulfate precipitation, ion-exchange chromatography with Q-sepharose Fast Flow, gel filtration chromatography with S300 and the second ion-exchange chromatography with Q-sepharose Fast Flow. The protease was isolated and purified, which was present and active on protein substrates (azocasein and casein). The specific protease activity was 17.15 folds and the recovery was 4.67. The molecular weight of the protease was estimated at 23.2 kD by SDS-PAGE. With azocasein as the susbstrate, the optimal temperature was 55°C and the optimal pH value was 5.5. Ion Ca2+ could enhance the proteolytic activity of the protease, while Cu2+, EDTA and PMSF could inhibit its activity.

  2. The maize cystatin CC9 interacts with apoplastic cysteine proteases.

    PubMed

    van der Linde, Karina; Mueller, André N; Hemetsberger, Christoph; Kashani, Farnusch; van der Hoorn, Renier A L; Doehlemann, Gunther

    2012-11-01

    In a recent study we identified corn cystain9 (CC9) as a novel compatibility factor for the interaction of the biotrophic smut fungus Ustilago maydis with its host plant maize. CC9 is transcriptionally induced during the compatible interaction with U. maydis and localizes in the maize apoplast where it inhibits apoplastic papain-like cysteine proteases. The proteases are activated during incompatible interaction and salicylic acid (SA) treatment and, in turn, are sufficient to induce SA signaling including PR-gene expression. Therefore the inhibition of apoplastic papain-like cysteine proteases by CC9 is essential to suppress host immunity during U. maydis infection. Here were present new experimental data on the cysteine protease-cystatin interaction and provide an in silco analysis of plant cystatins and the identified apoplastic cysteine proteases.

  3. Poliovirus protease 3C(pro) kills cells by apoptosis.

    PubMed

    Barco, A; Feduchi, E; Carrasco, L

    2000-01-20

    The tetracycline-based Tet-Off expression system has been used to analyze the effects of poliovirus protease 3C(pro) on human cells. Stable HeLa cell clones that express this poliovirus protease under the control of an inducible, tightly regulated promoter were obtained. Tetracycline removal induces synthesis of 3C protease, followed by drastic morphological alterations and cellular death. Degradation of cellular DNA in nucleosomes and generation of apoptotic bodies are observed from the second day after 3C(pro) induction. The cleavage of poly(ADP-ribose) polymerase, an enzyme involved in DNA repair, occurs after induction of 3C(pro), indicating caspase activation by this poliovirus protease. The 3C(pro)-induced apoptosis is blocked by the caspase inhibitor z-VAD-fmk. Our findings suggest that the protease 3C is responsible for triggering apoptosis in poliovirus-infected cells by a mechanism that involves caspase activation.

  4. The CCL3L1-CCR5 genotype influences the development of AIDS, but not HIV susceptibility or the response to HAART

    SciTech Connect

    Bhattacharya, Tanmoy; Stanton, Jennifer; Kim, Eun - Young; Kunstman, Kevin; Phair, John; Jacobson, Lisa P; Wolinsky, Steven M

    2008-01-01

    A selective advantage against infectious diseases such as HIV/AIDS is associated with differences in the genes relevant to immunity and virus replication. The CC chemokine receptor 5 (CCR5), the principal coreceptor for HIV, and its chemokine ligands, including CCL3L1, influences the CD4+ target cells susceptibility to infection. The CCL3L1 gene is in a region of segmental duplication on the q-arm of human chromosome 17. Increased numbers of CCL3L1 gene copies that affect the gene expression phenotype might have substantial protective effects. Here we show that the population-specific CCL3L1 gene copy number and the CCR5 {Delta}32 protein-inactivating deletion that categorizes the CCL3L1-CCR5 genotype do not influence HIV/AIDS susceptibility or the robustness of immune recovery after the initiation of highly active antiretroviral therapy (HAART).

  5. Positive selection of digestive Cys proteases in herbivorous Coleoptera.

    PubMed

    Vorster, Juan; Rasoolizadeh, Asieh; Goulet, Marie-Claire; Cloutier, Conrad; Sainsbury, Frank; Michaud, Dominique

    2015-10-01

    Positive selection is thought to contribute to the functional diversification of insect-inducible protease inhibitors in plants in response to selective pressures exerted by the digestive proteases of their herbivorous enemies. Here we assessed whether a reciprocal evolutionary process takes place on the insect side, and whether ingestion of a positively selected plant inhibitor may translate into a measurable rebalancing of midgut proteases in vivo. Midgut Cys proteases of herbivorous Coleoptera, including the major pest Colorado potato beetle (Leptinotarsa decemlineata), were first compared using a codon-based evolutionary model to look for the occurrence of hypervariable, positively selected amino acid sites among the tested sequences. Hypervariable sites were found, distributed within -or close to- amino acid regions interacting with Cys-type inhibitors of the plant cystatin protein family. A close examination of L. decemlineata sequences indicated a link between their assignment to protease functional families and amino acid identity at positively selected sites. A function-diversifying role for positive selection was further suggested empirically by in vitro protease assays and a shotgun proteomic analysis of L. decemlineata Cys proteases showing a differential rebalancing of protease functional family complements in larvae fed single variants of a model cystatin mutated at positively selected amino acid sites. These data confirm overall the occurrence of hypervariable, positively selected amino acid sites in herbivorous Coleoptera digestive Cys proteases. They also support the idea of an adaptive role for positive selection, useful to generate functionally diverse proteases in insect herbivores ingesting functionally diverse, rapidly evolving dietary cystatins. PMID:26264818

  6. Leptin replacement therapy for the treatment of non-HAART associated lipodystrophy syndromes: a meta-analysis into the effects of leptin on metabolic and hepatic endpoints.

    PubMed

    Rodríguez, Alexander J; Neeman, Teresa; Giles, Aaron G; Mastronardi, Claudio A; Paz Filho, Gilberto

    2014-11-01

    The clinical manifestations of lipodystrophy syndromes (LS) are hypoleptinemia, hyperglycemia, insulin resistance, dyslipidemia and hepatic steatosis. Leptin replacement therapy (LRT) is effective at improving these pathologies. Currently, there are no data compiling the evidence from the literature, and demonstrating the effect of LRT in LS patients. A systematic review of the MEDLINE and Cochrane Library databases was conducted to identify studies assessing the effect of LRT on metabolic and hepatic endpoints in patients with LS not associated with highly active antiretroviral therapy (HAART) use. Standardized mean differences (SMD) and 95% confidence intervals of pooled results were calculated for overall changes in glucose homeostasis, lipid profile, and hepatic physiology, using an inverse-variance random-effects model. After screening, 12 studies were included for review. Meta-analysis of results from 226 patients showed that LRT decreased fasting glucose [0.75 SMD units (range 0.36-1.13), p=0.0001], HbA1c [0.49 (0.17-0.81), p=0.003], triglycerides [1.00 (0.69-1.31), p<0.00001], total cholesterol [0.62 (0.21-1.02), p=0.003], liver volume [1.06 (0.51-1.61), p=0.0002] and AST [0.41 (0.10-0.73) p=0.01]. In patients with non-HAART LS, LRT improves the outcome of several metabolic and hepatic parameters. Studies were limited by small populations and therefore large prospective trials are needed to validate these findings.

  7. Vγ9Vδ2 T-Cell Polyfunctionality Is Differently Modulated in HAART-Treated HIV Patients according to CD4 T-Cell Count

    PubMed Central

    Casetti, Rita; De Simone, Gabriele; Sacchi, Alessandra; Rinaldi, Alessandra; Viola, Domenico; Agrati, Chiara; Bordoni, Veronica; Cimini, Eleonora; Tumino, Nicola; Besi, Francesca; Martini, Federico

    2015-01-01

    Alteration of γδ T-cell distribution and function in peripheral blood is among the earliest defects during HIV-infection. We asked whether the polyfunctional response could also be affected, and how this impairment could be associated to CD4 T-cell count. To this aim, we performed a cross-sectional study on HIV-infected individuals. In order to evaluate the polyfunctional-Vγ9Vδ2 T-cell response after phosphoantigen-stimulation, we assessed the cytokine/chemokine production and cytotoxicity by flow-cytometry in HAART-treated-HIV+ persons and healthy-donors. During HIV-infection Vγ9Vδ2-polyfunctional response quality is affected, since several Vγ9Vδ2 T-cell subsets resulted significantly lower in HIV+ patients in respect to healthy donors. Interestingly, we found a weak positive correlation between Vγ9Vδ2 T-cell-response and CD4 T-cell counts. By dividing the HIV+ patients according to CD4 T-cell count, we found that Low-CD4 patients expressed a lower number of two Vγ9Vδ2 T-cell subsets expressing MIP-1β in different combinations with other molecules (CD107a/IFNγ) in respect to High-CD4 individuals. Our results show that the Vγ9Vδ2 T-cell-response quality in Low-CD4 patients is specifically affected, suggesting a direct link between innate Vγ9Vδ2 T-cells and CD4 T-cell count. These findings suggest that Vγ9Vδ2 T-cell quality may be indirectly influenced by HAART therapy and could be included in a new therapeutical strategy which would perform an important role in fighting HIV infection. PMID:26161861

  8. Vγ9Vδ2 T-Cell Polyfunctionality Is Differently Modulated in HAART-Treated HIV Patients according to CD4 T-Cell Count.

    PubMed

    Casetti, Rita; De Simone, Gabriele; Sacchi, Alessandra; Rinaldi, Alessandra; Viola, Domenico; Agrati, Chiara; Bordoni, Veronica; Cimini, Eleonora; Tumino, Nicola; Besi, Francesca; Martini, Federico

    2015-01-01

    Alteration of γδ T-cell distribution and function in peripheral blood is among the earliest defects during HIV-infection. We asked whether the polyfunctional response could also be affected, and how this impairment could be associated to CD4 T-cell count. To this aim, we performed a cross-sectional study on HIV-infected individuals. In order to evaluate the polyfunctional-Vγ9Vδ2 T-cell response after phosphoantigen-stimulation, we assessed the cytokine/chemokine production and cytotoxicity by flow-cytometry in HAART-treated-HIV+ persons and healthy-donors. During HIV-infection Vγ9Vδ2-polyfunctional response quality is affected, since several Vγ9Vδ2 T-cell subsets resulted significantly lower in HIV+ patients in respect to healthy donors. Interestingly, we found a weak positive correlation between Vγ9Vδ2 T-cell-response and CD4 T-cell counts. By dividing the HIV+ patients according to CD4 T-cell count, we found that Low-CD4 patients expressed a lower number of two Vγ9Vδ2 T-cell subsets expressing MIP-1β in different combinations with other molecules (CD107a/IFNγ) in respect to High-CD4 individuals. Our results show that the Vγ9Vδ2 T-cell-response quality in Low-CD4 patients is specifically affected, suggesting a direct link between innate Vγ9Vδ2 T-cells and CD4 T-cell count. These findings suggest that Vγ9Vδ2 T-cell quality may be indirectly influenced by HAART therapy and could be included in a new therapeutical strategy which would perform an important role in fighting HIV infection.

  9. Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications.

    PubMed Central

    Bazan, J F; Fletterick, R J

    1988-01-01

    Proteases that are encoded by animal picornaviruses and plant como- and potyviruses form a related group of cysteine-active-center enzymes that are essential for virus maturation. We show that these proteins are homologous to the family of trypsin-like serine proteases. In our model, the active-site nucleophile of the trypsin catalytic triad, Ser-195, is changed to a Cys residue in these viral proteases. The other two residues of the triad, His-57 and Asp-102, are otherwise absolutely conserved in all the viral protease sequences. Secondary structure analysis of aligned sequences suggests the location of the component strands of the twin beta-barrel trypsin fold in the viral proteases. Unexpectedly, the 2a and 3c subclasses of viral cysteine proteases are, respectively, homologous to the small and large structural subclasses of trypsin-like serine proteases. This classification allows the molecular mapping of residues from viral sequences onto related tertiary structures; we precisely identify amino acids that are strong determinants of specificity for both small and large viral cysteine proteases. Images PMID:3186696

  10. Active Site Characterization of Proteases Sequences from Different Species of Aspergillus.

    PubMed

    Morya, V K; Yadav, Virendra K; Yadav, Sangeeta; Yadav, Dinesh

    2016-09-01

    A total of 129 proteases sequences comprising 43 serine proteases, 36 aspartic proteases, 24 cysteine protease, 21 metalloproteases, and 05 neutral proteases from different Aspergillus species were analyzed for the catalytically active site residues using MEROPS database and various bioinformatics tools. Different proteases have predominance of variable active site residues. In case of 24 cysteine proteases of Aspergilli, the predominant active site residues observed were Gln193, Cys199, His364, Asn384 while for 43 serine proteases, the active site residues namely Asp164, His193, Asn284, Ser349 and Asp325, His357, Asn454, Ser519 were frequently observed. The analysis of 21 metalloproteases of Aspergilli revealed Glu298 and Glu388, Tyr476 as predominant active site residues. In general, Aspergilli species-specific active site residues were observed for different types of protease sequences analyzed. The phylogenetic analysis of these 129 proteases sequences revealed 14 different clans representing different types of proteases with diverse active site residues.

  11. Unexpected Activity of a Novel Kunitz-type Inhibitor: INHIBITION OF CYSTEINE PROTEASES BUT NOT SERINE PROTEASES.

    PubMed

    Smith, David; Tikhonova, Irina G; Jewhurst, Heather L; Drysdale, Orla C; Dvořák, Jan; Robinson, Mark W; Cwiklinski, Krystyna; Dalton, John P

    2016-09-01

    Kunitz-type (KT) protease inhibitors are low molecular weight proteins classically defined as serine protease inhibitors. We identified a novel secreted KT inhibitor associated with the gut and parenchymal tissues of the infective juvenile stage of Fasciola hepatica, a helminth parasite of medical and veterinary importance. Unexpectedly, recombinant KT inhibitor (rFhKT1) exhibited no inhibitory activity toward serine proteases but was a potent inhibitor of the major secreted cathepsin L cysteine proteases of F. hepatica, FhCL1 and FhCL2, and of human cathepsins L and K (Ki = 0.4-27 nm). FhKT1 prevented the auto-catalytic activation of FhCL1 and FhCL2 and formed stable complexes with the mature enzymes. Pulldown experiments from adult parasite culture medium showed that rFhKT1 interacts specifically with native secreted FhCL1, FhCL2, and FhCL5. Substitution of the unusual P1 Leu(15) within the exposed reactive loop of FhKT1 for the more commonly found Arg (FhKT1Leu(15)/Arg(15)) had modest adverse effects on the cysteine protease inhibition but conferred potent activity against the serine protease trypsin (Ki = 1.5 nm). Computational docking and sequence analysis provided hypotheses for the exclusive binding of FhKT1 to cysteine proteases, the importance of the Leu(15) in anchoring the inhibitor into the S2 active site pocket, and the inhibitor's selectivity toward FhCL1, FhCL2, and human cathepsins L and K. FhKT1 represents a novel evolutionary adaptation of KT protease inhibitors by F. hepatica, with its prime purpose likely in the regulation of the major parasite-secreted proteases and/or cathepsin L-like proteases of its host.

  12. PEGylated substrates of NSP4 protease: A tool to study protease specificity

    NASA Astrophysics Data System (ADS)

    Wysocka, Magdalena; Gruba, Natalia; Grzywa, Renata; Giełdoń, Artur; Bąchor, Remigiusz; Brzozowski, Krzysztof; Sieńczyk, Marcin; Dieter, Jenne; Szewczuk, Zbigniew; Rolka, Krzysztof; Lesner, Adam

    2016-03-01

    Herein we present the synthesis of a novel type of peptidomimetics composed of repeating diaminopropionic acid residues modified with structurally diverse heterobifunctional polyethylene glycol chains (abbreviated as DAPEG). Based on the developed compounds, a library of fluorogenic substrates was synthesized. Further library deconvolution towards human neutrophil serine protease 4 (NSP4) yielded highly sensitive and selective internally quenched peptidomimetic substrates. In silico analysis of the obtained peptidomimetics revealed the presence of an interaction network with distant subsites located on the enzyme surface.

  13. Highly potent fibrinolytic serine protease from Streptomyces.

    PubMed

    Uesugi, Yoshiko; Usuki, Hirokazu; Iwabuchi, Masaki; Hatanaka, Tadashi

    2011-01-01

    We introduce a highly potent fibrinolytic serine protease from Streptomyces omiyaensis (SOT), which belongs to the trypsin family. The fibrinolytic activity of SOT was examined using in vitro assays and was compared with those of known fibrinolytic enzymes such as plasmin, tissue-type plasminogen activator (t-PA), urokinase, and nattokinase. Compared to other enzymes, SOT showed remarkably higher hydrolytic activity toward mimic peptides of fibrin and plasminogen. The fibrinolytic activity of SOT is about 18-fold higher than that of plasmin, and is comparable to that of t-PA by fibrin plate assays. Furthermore, SOT had some plasminogen activator-like activity. Results show that SOT and nattokinase have very different fibrinolytic and fibrinogenolytic modes, engendering significant synergetic effects of SOT and nattokinase on fibrinolysis. These results suggest that SOT presents important possibilities for application in the therapy of thrombosis.

  14. Design of translactam HCMV protease inhibitors as potent antivirals.

    PubMed

    Borthwick, Alan D

    2005-07-01

    Human cytomegalovirus (HCMV) is an important pathogen for which there is a significant unmet medical need. New HCMV antivirals, active against novel molecular targets, are undoubtedly needed as the currently available drugs ganciclovir, cidofovir, and foscarnet, which are all viral DNA inhibitors, suffer from limited effectiveness, mainly due to the development of drug resistance, poor bioavailability, and toxicity. One of the newer molecular targets that has been exploited in the search for better drug candidates is HCMV protease. Our deltaAla HCMV protease (wild type variant with the internal cleavage site deleted) was cloned and expressed in E. coli. This viral enzyme was used to develop HCMV protease assays to evaluate potential inhibitors. The chirally pure (SRS)-alpha-methyl pyrrolidine-5,5-trans-lactam template was synthesized, which together with the natural substrate requirements of HCMV protease and detailed SAR, was used to design potent and selective mechanism based inhibitors of HCMV protease. The mechanism of action of these inhibitors of HCMV protease was investigated by ESI/MS, and the X-ray crystal structure of the HCMV protease was used to refine our selective viral enzyme inhibitors to obtain plasma stable antivirals. A novel ELISA antiviral assay was developed which, together with a cytotoxicity assay, enabled us to discover anti-HCMV drug candidates equivalent in potency to ganciclovir that had good pharmacokinetics in the dog and good brain and ocular penetration in the guinea pig.

  15. Cold-adapted proteases as an emerging class of therapeutics.

    PubMed

    Fornbacke, Marcus; Clarsund, Mats

    2013-06-01

    Proteases have been used in medicine for several decades and are an established and well tolerated class of therapeutic agent. These proteases were sourced from mammals or bacteria that exist or have adapted to moderate temperatures (mesophilic organisms); however, proteases derived from organisms from cold environments-cold-adapted or psychrophilic proteases-generally have high specific activity, low substrate affinity, and high catalytic rates at low and moderate temperatures. Made possible by greater flexibility, psychrophilic enzymes interact with and transform the substrate at lower energy costs. Cold-adapted proteases have been used in a wide range of applications, including industrial functions, textiles, cleaning/hygiene products, molecular biology, environmental bioremediations, consumer food products, cosmetics, and pharmaceutical production. In addition to these applications, they have also shown promise as therapeutic modalities for cosmeceutical applications (by reducing glabellar [frown] lines) and a number of disease conditions, including bacterial infections (by disrupting biofilms to prevent bacterial infection), topical wound management (when used as a debridement agent to remove necrotic tissue and fibrin clots), oral/dental health management (by removing plaque and preventing periodontal disease), and in viral infections (by reducing the infectivity of viruses, such as human rhinovirus 16 and herpes simplex virus). Psychrophilic proteases with greater activity and stability (than the original organism-derived variant) have been developed; this coupled with available manufacturing recombinant production techniques suggests that cold-adapted proteases have a promising future as a distinct therapeutic class with diverse clinical applications.

  16. Laundry detergent compatibility of the alkaline protease from Bacillus cereus.

    PubMed

    Banik, Rathindra Mohan; Prakash, Monika

    2004-01-01

    The endogenous protease activity in various commercially available laundry detergents of international companies was studied. The maximum protease activity was found at 50 degrees C in pH range 10.5-11.0 in all the tested laundry detergents. The endogenous protease activity in the tested detergents retained up to 70% on incubation at 40 degrees C for 1 h, whereas less than 30% activity was only found on incubation at 50 degrees C for 1 h. The alkaline protease from an alkalophilic strain of Bacillus cereus was studied for its compatibility in commercial detergents. The cell free fermented broth from shake flask culture of the organism showed maximum activity at pH 10.5 and 50 degrees C. The protease from B. cereus showed much higher residual activity (more than 80%) on incubation with laundry detergents at 50 degrees C for 1 h or longer. The protease enzyme from B. cereus was found to be superior over the endogenous proteases present in the tested commercial laundry detergents in comparison to the enzyme stability during the washing at higher temperature, e.g., 40-50 degrees C.

  17. Pioneer oral streptococci produce immunoglobulin A1 protease.

    PubMed Central

    Cole, M F; Evans, M; Fitzsimmons, S; Johnson, J; Pearce, C; Sheridan, M J; Wientzen, R; Bowden, G

    1994-01-01

    As part of a longitudinal study of the relationship between bacterial colonization and the secretory immune response, 367 isolates of pioneer viridans streptococci collected from 40 breast- and bottle-fed neonates within the first month postpartum were tested for the production of immunoglobulin A1 (IgA1) protease and glycosidases. Fifty percent of the streptococci isolated produced IgA1 protease, including all isolates of Streptococcus oralis and S. sanguis, 60.7% of S. mitis biovar 1 isolates, and some isolates that could not be identified. Three cleavage patterns of alpha 1 heavy chains were observed. Six isolates of S. mitis biovar 1 that did not produce IgA1 protease attacked the alpha 1 chain. Incubation of IgA1 protease-negative S. mitis biovar 1 isolates with IgA1, either prior to or together with S. sanguis, rendered the IgA1 paraprotein resistant to cleavage by the IgA1 protease of S. sanguis. The ability of some pioneer streptococci in the human oral cavity to produce IgA1 protease and of others to modify the susceptibility of IgA1 to cleavage by IgA1 protease perhaps enhances their ability to survive in this habitat. Images PMID:8188337

  18. Salt stress represses production of extracellular proteases in Bacillus pumilus.

    PubMed

    Liu, R F; Huang, C L; Feng, H

    2015-05-11

    Bacillus pumilus is able to secrete subtilisin-like prote-ases, one of which has been purified and characterized biochemically, demonstrating great potential for use in industrial applications. In the current study, the biosynthesis and transcription of extracellular pro-teases in B. pumilus (BA06) under salt stress were investigated using various methods, including a proteolytic assay, zymogram analysis, and real-time PCR. Our results showed that total extracellular proteolytic activity, both in fermentation broth and on milk-containing agar plates, was considerably repressed by salt in a dosage-dependent manner. As Bacillus species usually secret multiple extracellular proteases, a vari-ety of individual extracellular protease encoding genes were selected for real-time PCR analysis. It was shown that proteases encoded by the aprE and aprX genes were the major proteases in the fermentation broth in terms of their transcripts in B. pumilus. Further, transcription of aprE, aprX, and epr genes was indeed repressed by salt stress. In con-trast, transcription of other genes (e.g., vpr and wprA) was not repressed or significantly affected by the salt. Conclusively, salt stress represses total extracellular proteolytic activity in B. pumilus, which can largely be ascribed to suppression of the major protease-encoding genes (aprE, aprX) at the transcriptional level. In contrast, transcription of other pro-tease-encoding genes (e.g., vpr, wprA) was not repressed by salt stress.

  19. Cold-adapted proteases as an emerging class of therapeutics.

    PubMed

    Fornbacke, Marcus; Clarsund, Mats

    2013-06-01

    Proteases have been used in medicine for several decades and are an established and well tolerated class of therapeutic agent. These proteases were sourced from mammals or bacteria that exist or have adapted to moderate temperatures (mesophilic organisms); however, proteases derived from organisms from cold environments-cold-adapted or psychrophilic proteases-generally have high specific activity, low substrate affinity, and high catalytic rates at low and moderate temperatures. Made possible by greater flexibility, psychrophilic enzymes interact with and transform the substrate at lower energy costs. Cold-adapted proteases have been used in a wide range of applications, including industrial functions, textiles, cleaning/hygiene products, molecular biology, environmental bioremediations, consumer food products, cosmetics, and pharmaceutical production. In addition to these applications, they have also shown promise as therapeutic modalities for cosmeceutical applications (by reducing glabellar [frown] lines) and a number of disease conditions, including bacterial infections (by disrupting biofilms to prevent bacterial infection), topical wound management (when used as a debridement agent to remove necrotic tissue and fibrin clots), oral/dental health management (by removing plaque and preventing periodontal disease), and in viral infections (by reducing the infectivity of viruses, such as human rhinovirus 16 and herpes simplex virus). Psychrophilic proteases with greater activity and stability (than the original organism-derived variant) have been developed; this coupled with available manufacturing recombinant production techniques suggests that cold-adapted proteases have a promising future as a distinct therapeutic class with diverse clinical applications. PMID:25135820

  20. Exploring a new serine protease from Cucumis sativus L.

    PubMed

    Nafeesa, Zohara; Shivalingu, B R; Vivek, H K; Priya, B S; Swamy, S Nanjunda

    2015-03-01

    Coagulation is an important physiological process in hemostasis which is activated by sequential action of proteases. This study aims to understand the involvement of aqueous fruit extract of Cucumis sativus L. (AqFEC) European burp less variety in blood coagulation cascade. AqFEC hydrolyzed casein in a dose-dependent manner. The presence of protease activity was further confirmed by casein zymography which revealed the possible presence of two high molecular weight protease(s). The proteolytic activity was inhibited only by phenyl methyl sulphonyl fluoride suggesting the presence of serine protease(s). In a dose-dependent manner, AqFEC also hydrolysed Aα and Bβ subunits of fibrinogen, whereas it failed to degrade the γ subunit of fibrinogen even at a concentration as high as 100 μg and incubation time up to 4 h. AqFEC reduced the clotting time of citrated plasma by 87.65%. The protease and fibrinogenolytic activity of AqFEC suggests its possible role in stopping the bleeding and ensuing wound healing process.

  1. Cysteine Protease Inhibitors as Chemotherapy: Lessons from a Parasite Target

    NASA Astrophysics Data System (ADS)

    Selzer, Paul M.; Pingel, Sabine; Hsieh, Ivy; Ugele, Bernhard; Chan, Victor J.; Engel, Juan C.; Bogyo, Matthew; Russell, David G.; Sakanari, Judy A.; McKerrow, James H.

    1999-09-01

    Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite's lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

  2. Staphylococcal proteases aid in evasion of the human complement system.

    PubMed

    Jusko, Monika; Potempa, Jan; Kantyka, Tomasz; Bielecka, Ewa; Miller, Halie K; Kalinska, Magdalena; Dubin, Grzegorz; Garred, Peter; Shaw, Lindsey N; Blom, Anna M

    2014-01-01

    Staphylococcus aureus is an opportunistic pathogen that presents severe health care concerns due to the prevalence of multiple antibiotic-resistant strains. New treatment strategies are urgently needed, which requires an understanding of disease causation mechanisms. Complement is one of the first lines of defense against bacterial pathogens, and S. aureus expresses several specific complement inhibitors. The effect of extracellular proteases from this bacterium on complement, however, has been the subject of limited investigation, except for a recent report regarding cleavage of the C3 component by aureolysin (Aur). We demonstrate here that four major extracellular proteases of S. aureus are potent complement inhibitors. Incubation of human serum with the cysteine proteases staphopain A and staphopain B, the serine protease V8 and the metalloproteinase Aur resulted in a drastic decrease in the hemolytic activity of serum, whereas two staphylococcal serine proteases D and E, had no effect. These four proteases were found to inhibit all pathways of complement due to the efficient degradation of several crucial components. Furthermore, S. aureus mutants lacking proteolytic enzymes were found to be more efficiently killed in human blood. Taken together, the major proteases of S. aureus appear to be important for pathogen-mediated evasion of the human complement system.

  3. Extracellular proteases modify cell wall turnover in Bacillus subtilis.

    PubMed Central

    Jolliffe, L K; Doyle, R J; Streips, U N

    1980-01-01

    The rate of turnover of peptidoglycan in exponentially growing cultures of Bacillus subtilis was observed to be sensitive to extracellular protease. In protease-deficient mutants the rates of cell wall turnover were greater than that of wild-type strain 168, whereas hyperprotease-producing strains exhibited decreased rates of peptidoglycan turnover. The rate of peptidogylcan turnover in a protease-deficient strain was decreased when the mutant was grown in the presence of a hyperprotease-producing strain. The addition of phenylmethylsulfonyl fluoride, a serine protease inhibitor, to cultures of hyperprotease-producing strains increased their rates of cell wall turnover. Isolated cell walls of all protease mutants contained autolysin levels equal to or greater than that of wild-type strain 168. The presence of filaments, or cells with incomplete septa, was observed in hyperprotease-producing strains or when a protease-deficient strain was grown in the presence of subtilisin. The results suggest that the turnover of cell walls in B. subtilis may be regulated by extracellular proteases. Images PMID:6102558

  4. Membrane Proteases and Aminoglycoside Antibiotic Resistance ▿ †

    PubMed Central

    Hinz, Aaron; Lee, Samuel; Jacoby, Kyle; Manoil, Colin

    2011-01-01

    We present genetic studies that help define the functional network underlying intrinsic aminoglycoside resistance in Pseudomonas aeruginosa. Our analysis shows that proteolysis, particularly that controlled by the membrane protease FtsH, is a major determinant of resistance. First, we examined the consequences of inactivating genes controlled by AmgRS, a two-component regulator required for intrinsic tobramycin resistance. Three of the gene products account for resistance: a modulator of FtsH protease (YccA), a membrane protease (HtpX), and a membrane protein of unknown function (PA5528). Second, we screened mutations inactivating 66 predicted proteases and related functions. Insertions inactivating two FtsH protease accessory factors (HflK and HflC) and a cytoplasmic protease (HslUV) increased tobramycin sensitivity. Finally, we generated an ftsH deletion mutation. The mutation dramatically increased aminoglycoside sensitivity. Many of the functions whose inactivation increased sensitivity appeared to act independently, since multiple mutations led to additive or synergistic effects. Up to 500-fold increases in tobramycin sensitivity were observed. Most of the mutations also were highly pleiotropic, increasing sensitivity to a membrane protein hybrid, several classes of antibiotics, alkaline pH, NaCl, and other compounds. We propose that the network of proteases provides robust protection from aminoglycosides and other substances through the elimination of membrane-disruptive mistranslation products. PMID:21764915

  5. Fluorometric CCHFV OTU protease assay with potent inhibitors.

    PubMed

    Kocabas, Fatih; Aslan, Galip S

    2015-10-01

    Crimean-Congo hemorrhagic fever virus (CCHFV) is a deadly virus that has been listed in the Category C as a potential bioterror agent. There are no specific therapies against CCHFV, which urges identification of potential therapeutic targets and development of CCHFV therapies. CCHFV OTU protease takes an important role in viral invasion through antagonizing NF-κB signaling. Inhibition of CCHFV OTU protease by small molecules warrants an exciting potential as antiviral therapeutics. Here we report the expression and purification of a C-His-tagged recombinant CCHFV OTU protease in E. coli BL21 (DE3) host strain. Activity of the refolded purified recombinant viral OTU protease has been validated with a UB-AMC fluorescent assay. In addition, we show a dose-dependent inhibition of the viral OTU protease by two small molecules. This study provides a reliable approach for recombinant expression and purification of CCHFV OTU protease, and demonstrates validation of OTU protease activity and its inhibition based on a UB-AMC florescent assay.

  6. Dynamics of T-cell subsets and their relationship with oral and systemic opportunistic infections in HIV/AIDS patients during the first year of HAART in Guangxi, China.

    PubMed

    Zhang, Linlin; Huang, Yuxiao; Liu, Zhenmin; Liu, Wei; Qin, Qi; Tao, Renchuan

    2015-07-01

    To analyze the dynamic changes in Th1, Th2, Tc1, and Tc2 of HIV/AIDS patients during the first year of highly active antiretroviral therapy (HAART) and to explore their relationship with oral and systemic opportunistic infections, a cohort study was carried out among HIV/AIDS patients in Guangxi, China. Ninety HIV/AIDS patients and 30 healthy controls (HC) were included. The enrolled HIV/AIDS patients were examined at baseline and after 3, 6, and 12 months of HAART. On each visit, oral and systemic opportunistic infections were recorded, oral Candida load and plasma viral load (VL) were counted, differential T-cell counts and flow cytometric analysis of T-cell subsets were performed. During the first year of HAART, the total number of opportunistic infections decreased steadily with the change in oral candidiasis (OC) most representatively. A significant Th1→Th2 switch (Th1/Th2 ratio 0.23 ± 0.12, HC 1.45 ± 0.38) and slight Tc1→Tc2 shift (Tc1/Tc2 ratio 0.93 ± 0.29, HC 1.13 ± 0.33) were found at baseline, and both received slow mitigation after HAART. LgCFU and clinical OC were correlated positively with both LgVL and clinical stage (P < 0.05) at baseline. LgCFU was also correlated positively with clinical stage at all four time points (P < 0.05). In multiple factor analysis, Th1 was confirmed to be correlated negatively with LgVL (Std.B = -0.295, P = 0.025) and LgCFU (Std.B = -0.227, P < 0.001) at baseline. After HAART, LgCFU and clinical stage were only correlated negatively with CD4 when all factors were included. These results suggest that oral candidiasis and oral Candida load could be useful clinical markers in the evaluation of HIV/AIDS patients. Th1 may play an important role against oral and systemic opportunistic infections. Tc1 and Tc2 both showed positive roles in the control of viremia without HAART. J. Med. Virol. 87:1158-1167, 2015. © 2015 Wiley Periodicals, Inc.

  7. A Very Low Geno2pheno False Positive Rate Is Associated with Poor Viro-Immunological Response in Drug-Naïve Patients Starting a First-Line HAART

    PubMed Central

    Armenia, Daniele; Soulie, Cathia; Di Carlo, Domenico; Fabeni, Lavinia; Gori, Caterina; Forbici, Federica; Svicher, Valentina; Bertoli, Ada; Sarmati, Loredana; Giuliani, Massimo; Latini, Alessandra; Boumis, Evangelo; Zaccarelli, Mauro; Bellagamba, Rita; Andreoni, Massimo; Marcelin, Anne-Geneviève; Calvez, Vincent; Antinori, Andrea; Ceccherini-Silberstein, Francesca; Perno, Carlo-Federico; Santoro, Maria Mercedes

    2014-01-01

    Background We previously found that a very low geno2pheno false positive rate (FPR ≤2%) defines a viral population associated with low CD4 cell count and the highest amount of X4-quasispecies. In this study, we aimed at evaluating whether FPR ≤2% might impact on the viro-immunological response in HIV-1 infected patients starting a first-line HAART. Methods The analysis was performed on 305 HIV-1 B subtype infected drug-naïve patients who started their first-line HAART. Baseline FPR (%) values were stratified according to the following ranges: ≤2; 2–5; 5–10; 10–20; 20–60; >60. The impact of genotypically-inferred tropism on the time to achieve immunological reconstitution (a CD4 cell count gain from HAART initiation ≥150 cells/mm3) and on the time to achieve virological success (the first HIV-RNA measurement <50 copies/mL from HAART initiation) was evaluated by survival analyses. Results Overall, at therapy start, 27% of patients had FPR ≤10 (6%, FPR ≤2; 7%, FPR 2–5; 14%, FPR 5–10). By 12 months of therapy the rate of immunological reconstitution was overall 75.5%, and it was significantly lower for FPR ≤2 (54.1%) in comparison to other FPR ranks (78.8%, FPR 2–5; 77.5%, FPR 5–10; 71.7%, FPR 10–20; 81.8%, FPR 20–60; 75.1%, FPR >60; p = 0.008). The overall proportion of patients achieving virological success was 95.5% by 12 months of therapy. Multivariable Cox analyses showed that patients having pre-HAART FPR ≤2% had a significant lower relative adjusted hazard [95% C.I.] both to achieve immunological reconstitution (0.37 [0.20–0.71], p = 0.003) and to achieve virological success (0.50 [0.26–0.94], p = 0.031) than those with pre-HAART FPR >60%. Conclusions Beyond the genotypically-inferred tropism determination, FPR ≤2% predicts both a poor immunological reconstitution and a lower virological response in drug-naïve patients who started their first-line therapy. This parameter could be useful to identify patients

  8. Factors Associated with Lack of Viral Suppression at Delivery among HAART-Naïve HIV-Positive Women in the International Maternal Pediatric Adolescent AIDS Clinical Trials Group (IMPAACT) P1025 Study

    PubMed Central

    Katz, Ingrid T.; Leister, Erin; Kacanek, Deborah; Hughes, Michael D.; Bardeguez, Arlene; Livingston, Elizabeth; Stek, Alice; Shapiro, David E.; Tuomala, Ruth

    2014-01-01

    Background High delivery maternal plasma HIV-1 RNA level (viral load, VL) is a risk factor for mother to child transmission and poor maternal health. Objective To identify factors associated with detectable VL at delivery despite initiation of highly active antiretroviral therapy (HAART) during pregnancy. Design Multicenter observational study. Setting 67 US AIDS clinical research sites. Patients HIV-1-positive pregnant women who initiated HAART during pregnancy. Measurements Descriptive summaries and associations between socio-demographic, HIV disease, treatment and pregnancy-related risk factors and detectable VL (>400copies/mL) at delivery. Results Between October 2002 and December 2011, 671 women met inclusion criteria and 13% had detectable VL at delivery. Factors associated with detectable VL included multiparity (16.4% vs 8% nulliparous, p=0.002), black non-Hispanic ethnicity (17.6% vs 6.6% Hispanic and 6.6% white/non-Hispanic, p<0.001), 11th grade or less education (17.6% vs.12.1% high school graduate and 6.7% some college or higher, p=0.013), and initiation of HAART in third trimester (23.9% vs 12.3% second and 8.6% first, p=0.002), timing of HIV diagnosis prior to current pregnancy (16.1% vs 11% during current pregnancy, p=0.051), and timing of first prenatal visit in 3rd trimester (33.3% vs 14.3% second and 10.5% first, p=0.002). Women who experienced treatment interruptions or reported poor medication adherence during pregnancy were more likely to have detectable VL at delivery than women with no interruptions or who reported better adherence. Limitations Women entered the study at varying times during pregnancy and for this and other reasons there was incomplete data on many covariates. Conclusions In this large U.S.-based cohort of HIV-1 positive women, 13% of women who initiated HAART during pregnancy had detectable VL at delivery. The timing of HAART initiation and prenatal care along with medication adherence during pregnancy appear to be

  9. Protease inhibition as new therapeutic strategy for GI diseases

    PubMed Central

    Vergnolle, Nathalie

    2016-01-01

    The GI tract is the most exposed organ to proteases, both in physiological and pathophysiological conditions. For digestive purposes, the lumen of the upper GI tract contains large amounts of pancreatic proteases, but studies have also demonstrated increased proteolytic activity into mucosal tissues (both in the upper and lower GI tract), associated with pathological conditions. This review aims at outlining the evidences for dysregulated proteolytic homeostasis in GI diseases and the pathogenic mechanisms of increased proteolytic activity. The therapeutic potential of protease inhibition in GI diseases is discussed, with a particular focus on IBDs, functional GI disorders and colorectal cancer. PMID:27196587

  10. In vitro digestion with proteases producing MHC class II ligands.

    PubMed

    Tohmé, Mira; Maschalidi, Sophia; Manoury, Bénédicte

    2013-01-01

    Proteases generate peptides that bind to MHC class II molecules to interact with a wide diversity of CD4(+) T cells. They are expressed in dedicated organelles: endosomes and lysosomes of professional antigen presenting cells (pAPCs) such as B cells, macrophages, and dendritic cells. The identification of endosomal proteases which produce antigenic peptides is important, for example, for better vaccination and to prevent autoimmune diseases. Here, we describe a panel of technics (in vitro digestion assays of protein with recombinant proteases or purified endosomes/lysosomes, T cell stimulation) to monitor the production of MHC class II ligands. PMID:23329510

  11. 21 CFR 184.1027 - Mixed carbohydrase and protease enzyme product.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Mixed carbohydrase and protease enzyme product... Substances Affirmed as GRAS § 184.1027 Mixed carbohydrase and protease enzyme product. (a) Mixed carbohydrase and protease enzyme product is an enzyme preparation that includes carbohydrase and protease...

  12. Detection of Legume Protease Inhibitors by the Gel-X-ray Film Contact Print Technique

    ERIC Educational Resources Information Center

    Mulimani, Veerappa H.; Sudheendra, Kulkarni; Giri, Ashok P.

    2002-01-01

    Redgram (Cajanus cajan L.) extracts have been analyzed for the protease inhibitors using a new, sensitive, simple, and rapid method for detection of electrophoretically separated protease inhibitors. The detection involves equilibrating the gel successively in the protease assay buffer and protease solution, rinsing the gel in assay buffer, and…

  13. Characterization of the protease activity of detergents: laboratory practicals for studying the protease profile and activity of various commercial detergents.

    PubMed

    Valls, Cristina; Pujadas, Gerard; Garcia-Vallve, Santi; Mulero, Miquel

    2011-07-01

    Detergent enzymes account for about 30% of the total worldwide production of enzymes and are one of the largest and most successful applications of modern industrial biotechnology. Proteases can improve the wash performance of household, industrial, and institutional laundry detergents used to remove protein-based stains such as blood, grass, body fluids, and food soils. This article describes two easy and cheap laboratory exercises to study the presence, profile, and basic enzymology of detergent proteases. These laboratory practicals are based on the determination of the detergent protease activity of various commercial detergents using the N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine p-nitroanilide method and the bovine serum albumin degradation capacity. Students are also required to elucidate the enzymatic subtype of detergent proteases by studying the inhibitory potential of several types of protease inhibitors revealed by the same experimental methodology. Additionally, the results of the exercises can be used to provide additional insights on elementary enzymology by studying the influence of several important parameters on protease activity such as temperature (in this article) and the influence of pH and effects of surfactants and oxidizers (proposed). Students also develop laboratory skills, problem-solving capacities, and the ability to write a laboratory report. The exercises are mainly designed for an advanced undergraduate project in the biochemistry and biotechnology sciences. Globally, these laboratory practicals show students the biotechnological applications of proteases in the detergent industry and also reinforce important enzymology concepts.

  14. Potent Antiviral HIV-1 Protease Inhibitor GRL-02031 Adapts to the Structures of Drug Resistant Mutants with Its P1;#8242;-Pyrrolidinone Ring

    SciTech Connect

    Chang, Yu-Chung E.; Yu, XiaXia; Zhang, Ying; Tie, Yunfeng; Wang, Yuan-Fang; Yashchuk, Sofiya; Ghosh, Arun K.; Harrison, Robert W.; Weber, Irene T.

    2012-11-14

    GRL-02031 (1) is an HIV-1 protease (PR) inhibitor containing a novel P1' (R)-aminomethyl-2-pyrrolidinone group. Crystal structures at resolutions of 1.25-1.55 {angstrom} were analyzed for complexes of 1 with the PR containing major drug resistant mutations, PR{sub I47V}, PR{sub L76V}, PR{sub V82A}, and PR{sub N88D}. Mutations of I47V and V82A alter residues in the inhibitor-binding site, while L76V and N88D are distal mutations having no direct contact with the inhibitor. Substitution of a smaller amino acid in PR{sub I47V} and PR{sub L76V} and the altered charge of PR{sub N88D} are associated with significant local structural changes compared to the wild-type PR{sub WT}, while substitution of alanine in PR{sub V82A} increases the size of the S1' subsite. The P1' pyrrolidinone group of 1 accommodates to these local changes by assuming two different conformations. Overall, the conformation and interactions of 1 with PR mutants resemble those of PR{sub WT} with similar inhibition constants in good agreement with the antiviral potency on multidrug resistant HIV-1.

  15. Heat resistant proteases produced in milk by psychrotrophic bacteria of dairy origin.

    PubMed

    Adams, D M; Barach, J T; Speck, M L

    1975-06-01

    Production of heat resistant proteases by psychrotrophs growing in milk, resistance of such proteases to ultrahigh temperature treatments and action of these enzymes on milk were studied. All of the psychrotrophs obtained from raw milk produced proteases that survived 149 C for 10s. Seventy to ninety percent of the raw milk samples contained psychrotrophs capable of producing heat resistant proteases. The protease chosen as a model was resistant to heat treatments at 110 to 150 C, and the inactivation parameters suggested that thermal destruction of heat resistant proteases would damage the milk severely. The casein content and pH of normal milk were suitable for protease action, and the protease was quite active at normal and elevated room temperatures. The protease rapidly spoiled sterile milk with the development of bitter flavor, clearing, or coagulation; and the susceptibility of sterile milk to protease increased during storage of the milk.

  16. Expression and secretion of heterologous proteases by Corynebacterium glutamicum.

    PubMed Central

    Billman-Jacobe, H; Wang, L; Kortt, A; Stewart, D; Radford, A

    1995-01-01

    Genes encoding the basic protease of Dichelobacter nodosus (bprV) and the subtilisin of Bacillus subtilis (aprE) were cloned and expressed in Corynebacterium glutamicum. In each case, enzymatically active protein was detected in the supernatants of liquid cultures. While the secretion of subtilisin was directed by its own signal peptide, the natural signal peptide of the bprV basic protease did not facilitate secretion. A hybrid aprE-bprV gene in which the promoter and signal peptide coding sequences of subtilisin replaced those of bprV could be expressed, and basic protease was secreted by C. glutamicum. Expression of these proteases in C. glutamicum provides an opportunity to compare protein secretion from this gram-positive host with that from other gram-positive and gram-negative bacteria. PMID:7747974

  17. Genotype dependent QSAR for HIV-1 protease inhibition.

    PubMed

    Boutton, Carlo W; De Bondt, Hendrik L; De Jonge, Marc R

    2005-03-24

    The development of drug-resistant viruses limits the therapeutic success of anti-HIV therapies. Some of these genetic HIV-variants display complex mutational patterns in their pol gene that codes for protease and reverse transcriptase, the most investigated molecular targets for antiretroviral therapy. In this paper, we present a computational structure-based approach to predict the resistance of a HIV-1 protease strain to amprenavir by calculating the interaction energy of the drug with HIV-1 protease. By considering the interaction energy per residue, we can identify what residue mutations contribute to drug-resistance. This approach is presented here as a structure-based tool for the prediction of resistance of HIV-1 protease toward amprenavir, with a view to use the drug-protein interaction-energy pattern in a lead-optimization procedure for the discovery of new anti-HIV drugs. PMID:15771454

  18. Improving Viral Protease Inhibitors to Counter Drug Resistance.

    PubMed

    Kurt Yilmaz, Nese; Swanstrom, Ronald; Schiffer, Celia A

    2016-07-01

    Drug resistance is a major problem in health care, undermining therapy outcomes and necessitating novel approaches to drug design. Extensive studies on resistance to viral protease inhibitors, particularly those of HIV-1 and hepatitis C virus (HCV) protease, revealed a plethora of information on the structural and molecular mechanisms underlying resistance. These insights led to several strategies to improve viral protease inhibitors to counter resistance, such as exploiting the essential biological function and leveraging evolutionary constraints. Incorporation of these strategies into structure-based drug design can minimize vulnerability to resistance, not only for viral proteases but for other quickly evolving drug targets as well, toward designing inhibitors one step ahead of evolution to counter resistance with more intelligent and rational design. PMID:27090931

  19. Toxoplasma gondii aspartic protease 1 is not essential in tachyzoites.

    PubMed

    Polonais, Valerie; Shea, Michael; Soldati-Favre, Dominique

    2011-08-01

    Aspartic proteases are important virulence factors for pathogens and are recognized as attractive drug targets. Seven aspartic proteases (ASPs) have been identified in Toxoplasma gondii genome. Bioinformatics and phylogenetic analyses regroup them into five monophyletic groups. Among them, TgASP1, a coccidian specific aspartic protease related to the food vacuole plasmepsins, is associated with the secretory pathway in non-dividing cells and relocalizes in close proximity to the nascent inner membrane complex (IMC) of daughter cells during replication. Despite a potential role for TgASP1 in IMC formation, the generation of a conventional knockout of the TgASP1 gene revealed that this protease is not required for T. gondii tachyzoite survival or for proper IMC biogenesis.

  20. Characterization of the immunoglobulin A protease of Ureaplasma urealyticum.

    PubMed Central

    Spooner, R K; Russell, W C; Thirkell, D

    1992-01-01

    Ureaplasma urealyticum strains of all serotypes express a specific human immunoglobulin A1 protease that cleaves immunoglobulin A1 to produce intact Fab and Fc fragments. The use of a variety of inhibitors suggests that the enzyme is a serine protease. N-terminal sequencing of the Fc digestion product showed that the enzyme cleaves between the proline and threonine residues 235 and 236 in the hinge region of the heavy chain of immunoglobulin A1. Images PMID:1587621

  1. Proteomic Substrate Identification for Membrane Proteases in the Brain

    PubMed Central

    Müller, Stephan A.; Scilabra, Simone D.; Lichtenthaler, Stefan F.

    2016-01-01

    Cell-cell communication in the brain is controlled by multiple mechanisms, including proteolysis. Membrane-bound proteases generate signaling molecules from membrane-bound precursor proteins and control the length and function of cell surface membrane proteins. These proteases belong to different families, including members of the “a disintegrin and metalloprotease” (ADAM), the beta-site amyloid precursor protein cleaving enzymes (BACE), membrane-type matrix metalloproteases (MT-MMP) and rhomboids. Some of these proteases, in particular ADAM10 and BACE1 have been shown to be essential not only for the correct development of the mammalian brain, but also for myelination and maintaining neuronal connections in the adult nervous system. Additionally, these proteases are considered as drug targets for brain diseases, including Alzheimer’s disease (AD), schizophrenia and cancer. Despite their biomedical relevance, the molecular functions of these proteases in the brain have not been explored in much detail, as little was known about their substrates. This has changed with the recent development of novel proteomic methods which allow to identify substrates of membrane-bound proteases from cultured cells, primary neurons and other primary brain cells and even in vivo from minute amounts of mouse cerebrospinal fluid (CSF). This review summarizes the recent advances and highlights the strengths of the individual proteomic methods. Finally, using the example of the Alzheimer-related proteases BACE1, ADAM10 and γ-secretase, as well as ADAM17 and signal peptide peptidase like 3 (SPPL3), we illustrate how substrate identification with novel methods is instrumental in elucidating broad physiological functions of these proteases in the brain and other organs. PMID:27790089

  2. Amplified detection of protease activity using porous silicon nanostructures

    NASA Astrophysics Data System (ADS)

    Orosco, Manuel

    This dissertation will focus on harnessing the optical properties of porous silicon to sense protease activity. Electrochemical etching of polished silicon wafers produces porous silicon with unique optical properties such as Fabry-Perot fringes or a dielectric mirror reflecting specific wavelengths. Porous silicon optical transducers are coupled to a biochemical reaction (protease activity) and optically measured in a label-free manner. The first chapter is an introductory chapter discussing the current methods of detecting protease activity. Also discussed is the use of porous silicon for label-free sensing. The second chapter discusses the use of thin protein layers that are spin coated on the surface of a porous silicon film and excluded from the porous matrix based on size. When active proteases are introduced to the protein layer, small peptide fragments are generated, causing a change in refractive index from low to high. This can be used as a tool to monitor protease activity and amplify the signal to the naked eye. To extend on the second chapter, a double layered porous silicon film with the first layer have large pores and the second layer etched below having small pores was used for sensing protease activity. Proteases are adsorbed into the first layer and introduction of whole protein substrate produces small peptide fragments that can enter the second layer (changing the effective optical thickness). The fourth chapter describes a method of using luminescent transducers coupled to protein films. An "on-off" sensor using protein coated luminescent porous silicon was used to detect a decrease in the intensity of luminescence due to degradation of the protein film. An "off-on" sensor involved a fluorescent dye housed in the porous film and capped with a protein coating. The release of the dye is caused by the action of a protease causing an increase in fluorescent intensity from the dye.

  3. 2-D zymographic analysis of Broccoli (Brassica oleracea L. var. Italica) florets proteases: follow up of cysteine protease isotypes in the course of post-harvest senescence.

    PubMed

    Rossano, Rocco; Larocca, Marilena; Riccio, Paolo

    2011-09-01

    Zymographic analysis of Broccoli florets (Brassica oleracea L. var. Italica) revealed the presence of acidic metallo-proteases, serine proteases and cysteine proteases. Under conditions which were denaturing for the other proteases, the study was restricted to cysteine proteases. 2-D zymography, a technique that combines IEF and zymography was used to show the presence of 11 different cysteine protease spots with molecular mass of 44 and 47-48kDa and pIs ranging between 4.1 and 4.7. pI differences could be ascribed to different degrees of phosphorylation that partly disappeared in the presence of alkaline phosphatase. Post-harvest senescence of Broccoli florets was characterized by decrease in protein and chlorophyll contents and increase of protease activity. In particular, as determined by 2-D zymography, the presence of cysteine protease clearly increased during senescence, a finding that may represent a useful tool for the control of the aging process.

  4. Dual origin of gut proteases in Formosan subterranean termites (Coptotermes formosanus Shiraki) (Isoptera: Rhinotermitidae).

    PubMed

    Sethi, Amit; Xue, Qing-Gang; La Peyre, Jerome F; Delatte, Jennifer; Husseneder, Claudia

    2011-07-01

    Cellulose digestion in lower termites, mediated by carbohydrases originating from both termite and endosymbionts, is well characterized. In contrast, limited information exists on gut proteases of lower termites, their origins and roles in termite nutrition. The objective of this study was to characterize gut proteases of the Formosan subterranean termite (Coptotermes formosanus Shiraki) (Isoptera: Rhinotermitidae). The protease activity of extracts from gut tissues (fore-, mid- and hindgut) and protozoa isolated from hindguts of termite workers was quantified using hide powder azure as a substrate and further characterized by zymography with gelatin SDS-PAGE. Midgut extracts showed the highest protease activity followed by the protozoa extracts. High level of protease activity was also detected in protozoa culture supernatants after 24 h incubation. Incubation of gut and protozoa extracts with class-specific protease inhibitors revealed that most of the proteases were serine proteases. All proteolytic bands identified after gelatin SDS-PAGE were also inhibited by serine protease inhibitors. Finally, incubation with chromogenic substrates indicated that extracts from fore- and hindgut tissues possessed proteases with almost exclusively trypsin-like activity while both midgut and protozoa extracts possessed proteases with trypsin-like and subtilisin/chymotrypsin-like activities. However, protozoa proteases were distinct from midgut proteases (with different molecular mass). Our results suggest that the Formosan subterranean termite not only produces endogenous proteases in its gut tissues, but also possesses proteases originating from its protozoan symbionts.

  5. Proteases of germinating winged-bean (Psophocarpus tetragonolobus) seeds: purification and characterization of an acidic protease.

    PubMed

    Usha, R; Singh, M

    1996-01-15

    Two major classes of protease are shown to occur in germinating winged-bean (Psophocarpus tetragonolobus) seeds, by assaying extracts at pH 8.0 and pH 5.1 with [14C]gelatin as substrate. At pH 8.0, the activity profile of the enzyme shows a steady rise throughout the period of germination, whereas the activity at the acidic pH is very low up to day 5 and then increases sharply reaching a peak on day 11, followed by an equally sharp decline. The winged-bean acidic protease (WbAP) has been purified to apparent homogeneity, as attested by a single protein band on both PAGE and SDS/PAGE. WbAP is a monomeric enzyme with a molecular mass of 35 kDa and a pH optimum of 6.0. It is a thiol protease that does not belong to the papain family and it has tightly bound Ca2+ as shown by 45Ca(2+)-exchange studies. Besides gelatin and casein, it hydrolyses a 29 kDa winged-bean protein, indicating a prospective physiological role for it in storage-protein mobilization. Immunoblot analysis shows that it occurs only in the seeds and sprouting tubers of this plant and also that it is synthesized in developing seeds just before desiccation. It appears that the newly synthesized enzyme is inactive, and activation takes place around day 6 of germination. However, neither the mechanism of activation nor the signal that triggers it is clearly understood.

  6. The crystal structure of GXGD membrane protease FlaK

    SciTech Connect

    Hu, Jian; Xue, Yi; Lee, Sangwon; Ha, Ya

    2011-09-20

    The GXGD proteases are polytopic membrane proteins with catalytic activities against membrane-spanning substrates that require a pair of aspartyl residues. Representative members of the family include preflagellin peptidase, type 4 prepilin peptidase, presenilin and signal peptide peptidase. Many GXGD proteases are important in medicine. For example, type 4 prepilin peptidase may contribute to bacterial pathogenesis, and mutations in presenilin are associated with Alzheimer's disease. As yet, there is no atomic-resolution structure in this protease family. Here we report the crystal structure of FlaK, a preflagellin peptidase from Methanococcus maripaludis, solved at 3.6 {angstrom} resolution. The structure contains six transmembrane helices. The GXGD motif and a short transmembrane helix, helix 4, are positioned at the centre, surrounded by other transmembrane helices. The crystal structure indicates that the protease must undergo conformational changes to bring the GXGD motif and a second essential aspartyl residue from transmembrane helix 1 into close proximity for catalysis. A comparison of the crystal structure with models of presenilin derived from biochemical analysis reveals three common transmembrane segments that are similarly arranged around the active site. This observation reinforces the idea that the prokaryotic and human proteases are evolutionarily related. The crystal structure presented here provides a framework for understanding the mechanism of the GXGD proteases, and may facilitate the rational design of inhibitors that target specific members of the family.

  7. The Crystal Structure of GXGD Membrane Protease FlaK

    SciTech Connect

    J Hu; Y Xue; S Lee; Y Ha

    2011-12-31

    The GXGD proteases are polytopic membrane proteins with catalytic activities against membrane-spanning substrates that require a pair of aspartyl residues. Representative members of the family include preflagellin peptidase, type 4 prepilin peptidase, presenilin and signal peptide peptidase. Many GXGD proteases are important in medicine. For example, type 4 prepilin peptidase may contribute to bacterial pathogenesis, and mutations in presenilin are associated with Alzheimer's disease. As yet, there is no atomic-resolution structure in this protease family. Here we report the crystal structure of FlaK, a preflagellin peptidase from Methanococcus maripaludis, solved at 3.6 {angstrom} resolution. The structure contains six transmembrane helices. The GXGD motif and a short transmembrane helix, helix 4, are positioned at the centre, surrounded by other transmembrane helices. The crystal structure indicates that the protease must undergo conformational changes to bring the GXGD motif and a second essential aspartyl residue from transmembrane helix 1 into close proximity for catalysis. A comparison of the crystal structure with models of presenilin derived from biochemical analysis reveals three common transmembrane segments that are similarly arranged around the active site. This observation reinforces the idea that the prokaryotic and human proteases are evolutionarily related. The crystal structure presented here provides a framework for understanding the mechanism of the GXGD proteases, and may facilitate the rational design of inhibitors that target specific members of the family.

  8. The roles of intramembrane proteases in protozoan parasites.

    PubMed

    Sibley, L David

    2013-12-01

    Intramembrane proteolysis is widely conserved throughout different forms of life, with three major types of proteases being known for their ability to cleave peptide bonds directly within the transmembrane domains of their substrates. Although intramembrane proteases have been extensively studied in humans and model organisms, they have only more recently been investigated in protozoan parasites, where they turn out to play important and sometimes unexpected roles. Signal peptide peptidases are involved in endoplasmic reticulum (ER) quality control and signal peptide degradation from exported proteins. Recent studies suggest that repurposing inhibitors developed for blocking presenilins may be useful for inhibiting the growth of Plasmodium, and possibly other protozoan parasites, by blocking signal peptide peptidases. Rhomboid proteases, originally described in the fly, are also widespread in parasites, and are especially expanded in apicomplexans. Their study in parasites has revealed novel roles that expand our understanding of how these proteases function. Within this diverse group of parasites, rhomboid proteases contribute to processing of adhesins involved in attachment, invasion, intracellular replication, phagocytosis, and immune evasion, placing them at the vertex of host-parasite interactions. This article is part of a Special Issue entitled: Intramembrane Proteases.

  9. Insights into the Cyanobacterial Deg/HtrA Proteases

    PubMed Central

    Cheregi, Otilia; Wagner, Raik; Funk, Christiane

    2016-01-01

    Proteins are the main machinery for all living processes in a cell; they provide structural elements, regulate biochemical reactions as enzymes, and are the interface to the outside as receptors and transporters. Like any other machinery proteins have to be assembled correctly and need maintenance after damage, e.g., caused by changes in environmental conditions, genetic mutations, and limitations in the availability of cofactors. Proteases and chaperones help in repair, assembly, and folding of damaged and misfolded protein complexes cost-effective, with low energy investment compared with neo-synthesis. Despite their importance for viability, the specific biological role of most proteases in vivo is largely unknown. Deg/HtrA proteases, a family of serine-type ATP-independent proteases, have been shown in higher plants to be involved in the degradation of the Photosystem II reaction center protein D1. The objective of this review is to highlight the structure and function of their cyanobacterial orthologs. Homology modeling was used to find specific features of the SynDeg/HtrA proteases of Synechocystis sp. PCC 6803. Based on the available data concerning their location and their physiological substrates we conclude that these Deg proteases not only have important housekeeping and chaperone functions within the cell, but also are needed for remodeling the cell exterior. PMID:27252714

  10. Characterizing Protease Specificity: How Many Substrates Do We Need?

    PubMed

    Schauperl, Michael; Fuchs, Julian E; Waldner, Birgit J; Huber, Roland G; Kramer, Christian; Liedl, Klaus R

    2015-01-01

    Calculation of cleavage entropies allows to quantify, map and compare protease substrate specificity by an information entropy based approach. The metric intrinsically depends on the number of experimentally determined substrates (data points). Thus a statistical analysis of its numerical stability is crucial to estimate the systematic error made by estimating specificity based on a limited number of substrates. In this contribution, we show the mathematical basis for estimating the uncertainty in cleavage entropies. Sets of cleavage entropies are calculated using experimental cleavage data and modeled extreme cases. By analyzing the underlying mathematics and applying statistical tools, a linear dependence of the metric in respect to 1/n was found. This allows us to extrapolate the values to an infinite number of samples and to estimate the errors. Analyzing the errors, a minimum number of 30 substrates was found to be necessary to characterize substrate specificity, in terms of amino acid variability, for a protease (S4-S4') with an uncertainty of 5 percent. Therefore, we encourage experimental researchers in the protease field to record specificity profiles of novel proteases aiming to identify at least 30 peptide substrates of maximum sequence diversity. We expect a full characterization of protease specificity helpful to rationalize biological functions of proteases and to assist rational drug design. PMID:26559682

  11. Signaling pathways activated by a protease allergen in basophils

    PubMed Central

    Rosenstein, Rachel K.; Bezbradica, Jelena S.; Yu, Shuang; Medzhitov, Ruslan

    2014-01-01

    Allergic diseases represent a significant burden in industrialized countries, but why and how the immune system responds to allergens remain largely unknown. Because many clinically significant allergens have proteolytic activity, and many helminths express proteases that are necessary for their life cycles, host mechanisms likely have evolved to detect the proteolytic activity of helminth proteases, which may be incidentally activated by protease allergens. A cysteine protease, papain, is a prototypic protease allergen that can directly activate basophils and mast cells, leading to the production of cytokines, including IL-4, characteristic of the type 2 immune response. The mechanism of papain’s immunogenic activity remains unknown. Here we have characterized the cellular response activated by papain in basophils. We find that papain-induced IL-4 production requires calcium flux and activation of PI3K and nuclear factor of activated T cells. Interestingly, papain-induced IL-4 production was dependent on the immunoreceptor tyrosine-based activation motif (ITAM) adaptor protein Fc receptor γ-chain, even though the canonical ITAM signaling was not activated by papain. Collectively, these data characterize the downstream signaling pathway activated by a protease allergen in basophils. PMID:25369937

  12. Molecular diversity of HIV-1 and surveillance of transmitted drug resistance variants among treatment Naïve patients, 5 years after active introduction of HAART in Kuala Lumpur, Malaysia.

    PubMed

    Ong, Lai Yee; Razak, Siti Nur Humaira; Lee, Yeat Mei; Sri La Sri Ponnampalavanar, Sasheela; Syed Omar, Sharifah Faridah; Azwa, Raja Iskandar; Tee, Kok Keng; Kamarulzaman, Adeeba

    2014-01-01

    Expansion of antiretroviral treatment programs have led to the growing concern for the development of antiretroviral drug resistance. The aims were to assess the prevalence of drug resistant HIV-1 variants and to identify circulating subtypes among HAART-naïve patients. Plasma specimens from N = 100 HIV+ HAART-naïve adult were collected between March 2008 and August 2010 and viral RNA were extracted for nested PCR and sequenced. PR-RT sequences were protein aligned and checked for transmitted drug resistance mutations. Phylogenetic reconstruction and recombination analysis were performed to determine the genotypes. Based on the WHO consensus guidelines, none of the recruited patients had any transmitted drug resistance mutations. When analyzed against the Stanford guidelines, 35% of patients had at least one reported mutation that may reduce drug susceptibility to PI (24%), NRTI (5%), and NNRTI (14%). The commonly detected mutation that may affect current first line therapy was V179D (3%), which may lead to reduced susceptibility to NNRTI. The predominant circulating HIV-1 genotypes were CRF01_AE (51%) and CRF33_01B (17%). The prevalence of unique recombinant forms (URF) was 7%; five distinct recombinant structures involving CRF01_AE and subtype B' were observed, among them a cluster of three isolates that could form a novel circulating recombinant form (CRF) candidate. Transmitted drug resistance prevalence among HAART-naïve patients was low in this cohort of patients in Kuala Lumpur despite introduction of HAART 5 years ago. Owing to the high genetic diversity, continued molecular surveillance can identify the persistent emergence of HIV-1 URF and novel CRF with significant epidemiological impact.

  13. Incidences and risk factors of first-line HAART discontinuation: a limitation to the success of the "seek, test, treat, and retain" strategy?

    PubMed

    Keita, Momory; Perbost, Isabelle; Pugliese-Wehrlen, Sylvia; Abel, Sylvie; Pugliese, Pascal; Enel, Patricia; Cuzin, Lise; Lang, Thierry; Delpierre, Cyrille

    2014-01-01

    To evaluate the incidence and risk factors of first-highly active antiretroviral therapy (HAART) modifications/interruptions and their causes in a cohort of newly-treated patients by using a competing risk model. In nine centers of the French cohort Dat'AIDS, in 1 year and 2 years of censorship, a competing risk analysis was implemented in HIV1 patients aged 18 years or older first-treated between September 2002 and March 2012. In 4669 patients, 3628 modifications (77.7%) were observed (median: 13.5 months). Cumulative incidence in 1 year: 46.8% [45.4-48.3]; in 2 years: 65.3% [63.8-66.8]. Intolerance (n = 1167; 32.3%): in 1 year, except first-treated from 2002 to 2005, modifications were not different: 2002-2003 (24.6%) 2004-2005 (26.1%), 2006-2007 (19.4%), 2008-2009 (18.8%) and 2010-2011 (15.7%). Women, AIDS patients, and those aged 50 years and older had an excess risk. Therapeutic simplification (n = 1037; 28.6%): in 1 year, except first-treated from 2002 to 2003, modifications were not different: 2002-2003 (9.0%), 2004-2005 (16.0%), 2006-2007 (11.0%), 2008-2009 (15.7%) and 2010-2011 (10.0%). Conversely to injecting-drug-users and AIDS patients, women and first-treated with non-nucleosides had an excess risk. Therapeutic failure (n = 189; 5.2%): contrary to first-treated between 2002 and 2003 or 2008 and 2009, in 1 year as in 2 years, modifications were not different. In 1 year, 1.9% for 2004-2005, 1.6% for 2006-2007 and 1.2% for 2010-2011. Maximum viral load ≥5.0 log10 copies/ml and CD4 <200 cells/mm(3) had a high probability. The study of first-HAART modifications suggests that in 1-year follow-up, intolerance incidence in the recent calendar year is still as frequent as the previous period which may constitute a limitation to the success of the seek, test, treat, and retain. PMID:24517736

  14. PEGylated substrates of NSP4 protease: A tool to study protease specificity

    PubMed Central

    Wysocka, Magdalena; Gruba, Natalia; Grzywa, Renata; Giełdoń, Artur; Bąchor, Remigiusz; Brzozowski, Krzysztof; Sieńczyk, Marcin; Dieter, Jenne; Szewczuk, Zbigniew; Rolka, Krzysztof; Lesner, Adam

    2016-01-01

    Herein we present the synthesis of a novel type of peptidomimetics composed of repeating diaminopropionic acid residues modified with structurally diverse heterobifunctional polyethylene glycol chains (abbreviated as DAPEG). Based on the developed compounds, a library of fluorogenic substrates was synthesized. Further library deconvolution towards human neutrophil serine protease 4 (NSP4) yielded highly sensitive and selective internally quenched peptidomimetic substrates. In silico analysis of the obtained peptidomimetics revealed the presence of an interaction network with distant subsites located on the enzyme surface. PMID:26955973

  15. A Novel Tricyclic Ligand-Containing Nonpeptidic HIV-1 Protease Inhibitor, GRL-0739, Effectively Inhibits the Replication of Multidrug-Resistant HIV-1 Variants and Has a Desirable Central Nervous System Penetration Property In Vitro

    PubMed Central

    Amano, Masayuki; Tojo, Yasushi; Salcedo-Gómez, Pedro Miguel; Parham, Garth L.; Nyalapatla, Prasanth R.; Das, Debananda; Ghosh, Arun K.

    2015-01-01

    We report here that GRL-0739, a novel nonpeptidic HIV-1 protease inhibitor containing a tricycle (cyclohexyl-bis-tetrahydrofuranylurethane [THF]) and a sulfonamide isostere, is highly active against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC50], 0.0019 to 0.0036 μM), with minimal cytotoxicity (50% cytotoxic concentration [CC50], 21.0 μM). GRL-0739 blocked the infectivity and replication of HIV-1NL4-3 variants selected by concentrations of up to 5 μM ritonavir or atazanavir (EC50, 0.035 to 0.058 μM). GRL-0739 was also highly active against multidrug-resistant clinical HIV-1 variants isolated from patients who no longer responded to existing antiviral regimens after long-term antiretroviral therapy, as well as against the HIV-2ROD variant. The development of resistance against GRL-0739 was substantially delayed compared to that of amprenavir (APV). The effects of the nonspecific binding of human serum proteins on the anti-HIV-1 activity of GRL-0739 were insignificant. In addition, GRL-0739 showed a desirable central nervous system (CNS) penetration property, as assessed using a novel in vitro blood-brain barrier model. Molecular modeling demonstrated that the tricyclic ring and methoxybenzene of GRL-0739 have a larger surface and make greater van der Waals contacts with protease than in the case of darunavir. The present data demonstrate that GRL-0739 has desirable features as a compound with good CNS-penetrating capability for treating patients infected with wild-type and/or multidrug-resistant HIV-1 variants and that the newly generated cyclohexyl-bis-THF moiety with methoxybenzene confers highly desirable anti-HIV-1 potency in the design of novel protease inhibitors with greater CNS penetration profiles. PMID:25691652

  16. Protease Production by Different Thermophilic Fungi

    NASA Astrophysics Data System (ADS)

    Macchione, Mariana M.; Merheb, Carolina W.; Gomes, Eleni; da Silva, Roberto

    A comparative study was carried out to evaluate protease production in solid-state fermentation (SSF) and submerged fermentation (SmF) by nine different thermophilic fungi — Thermoascus aurantiacus Miehe, Thermomyces lanuginosus, T. lanuginosus TO.03, Aspergillus flavus 1.2, Aspergillus sp. 13.33, Aspergillus sp. 13.34, Aspergillus sp. 13.35, Rhizomucor pusillus 13.36 and Rhizomucor sp. 13.37 — using substrates containing proteins to induce enzyme secretion. Soybean extract (soybean milk), soybean flour, milk powder, rice, and wheat bran were tested. The most satisfactory results were obtained when using wheat bran in SSF. The fungi that stood out in SSF were T. lanuginosus, T. lanuginosus TO.03, Aspergillus sp. 13.34, Aspergillus sp. 13.35, and Rhizomucor sp. 13.37, and those in SmF were T. aurantiacus, T. lanuginosus TO.03, and 13.37. In both fermentation systems, A. flavus 1.2 and R. pusillus 13.36 presented the lowest levels of proteolytic activity.

  17. Cystatin protease inhibitors and immune functions.

    PubMed

    Zavasnik-Bergant, Tina

    2008-05-01

    Cystatins are natural tight-binding reversible inhibitors of cysteine proteases. They are wide spread in all living organisms (mammals, nematodes, arthropods etc.) and are involved in various biological processes where they regulate normal proteolysis and also take part in disease pathology. Many cystatins show changes in expression and/or localization, as well as changes in secretion, following certain stimuli acting on immune cells. In immune cells, cystatins interfere with antigen processing and presentation, phagocytosis, expression of cytokines and nitric oxide and these ways modify the immune response. Further, it has been suggested that cystatin-type molecules secreted from parasites down-modulate the host immune response. Precise understanding of the regulatory roles on proteolytic enzymes of endogenous and exogenous cystatins, such as those from parasites, will provide us with valuable insight into how immune response could be modulated to treat a specific disease. This review covers some specific functions of individual cystatins, with a particular focus on the relevance of cystatins to the immune response.

  18. Pyrazinone protease inhibitor metabolites from Photorhabdus luminescens.

    PubMed

    Park, Hyun Bong; Crawford, Jason M

    2016-08-01

    Photorhabdus luminescens is a bioluminescent entomopathogenic bacterium that undergoes phenotypic variation and lives in mutualistic association with nematodes of the family Heterorhabditidae. The pair infects and kills insects, and during their coordinated lifecycle, the bacteria produce an assortment of specialized metabolites to regulate its mutualistic and pathogenic roles. As part of our search for new specialized metabolites from the Photorhabdus genus, we examined organic extracts from P. luminescens grown in an amino-acid-rich medium based on the free amino-acid levels found in the circulatory fluid of its common insect prey, the Galleria mellonella larva. Reversed-phase HPLC/UV/MS-guided fractionation of the culture extracts led to the identification of two new pyrazinone metabolites, lumizinones A (1) and B (2), together with two N-acetyl dipeptides (3 and 4). The lumizinones were produced only in the phenotypic variant associated with nematode development and insect pathogenesis. Their chemical structures were elucidated by analysis of 1D and 2D NMR and high-resolution ESI-QTOF-MS spectral data. The absolute configurations of the amino acids in 3 and 4 were determined by Marfey's analysis. Compounds 1-4 were evaluated for their calpain protease inhibitory activity, and lumizinone A (1) showed inhibition with an IC50 (half-maximal inhibitory concentration) value of 3.9 μm. PMID:27353165

  19. Fragment-Based Screen against HIV Protease

    PubMed Central

    Perryman, A. L.; Zhang, Q.; Soutter, H. H.; Rosenfeld, R.; McRee, D. E.; Olson, A. J.; Elder, J. E.; Stout, C. D.

    2009-01-01

    We have employed a fragment-based screen against wild-type (NL4-3) HIV protease (PR) using the Active Sight fragment library and X-ray crystallography. The experiments reveal two new binding sites for small molecules. PR was co-crystallized with fragments, or crystals were soaked in fragment solutions, using five crystal forms, and 378 data sets were collected to 2.3-1.3 Å resolution. Fragment binding induces a distinct conformation and specific crystal form of TL-3 inhibited PR during co-crystallization. One fragment, 2-methylcyclohexanol, binds in the ‘exo site’ adjacent to the Gly16Gly17Gln18 loop where the amide of Gly17 is a specific hydrogen bond donor, and hydrophobic contacts occur with the side chains of Lys14 and Leu63. Another fragment, indole-6-carboxylic acid, binds on the ‘outside/top of the flap’ via hydrophobic contacts with Trp42, Pro44, Met46, and Lys55, a hydrogen bond with Val56, and a salt-bridge with Arg57. 2-acetyl-benzothiophene also binds at this site. This study is the first fragment-based crystallographic screen against HIV PR, and the first time that fragments were screened against an inhibitor-bound drug target to search for compounds that both bind to novel sites and stabilize the inhibited conformation of the target. PMID:20659109

  20. Fragment-based screen against HIV protease.

    PubMed

    Perryman, Alexander L; Zhang, Qing; Soutter, Holly H; Rosenfeld, Robin; McRee, Duncan E; Olson, Arthur J; Elder, John E; Stout, C David

    2010-03-01

    We have employed a fragment-based screen against wild-type (NL4-3) HIV protease (PR) using the Active Sight fragment library and X-ray crystallography. The experiments reveal two new binding sites for small molecules. PR was co-crystallized with fragments, or crystals were soaked in fragment solutions, using five crystal forms, and 378 data sets were collected to 2.3-1.3 A resolution. Fragment binding induces a distinct conformation and specific crystal form of TL-3 inhibited PR during co-crystallization. One fragment, 2-methylcyclohexanol, binds in the 'exo site' adjacent to the Gly(16)Gly(17)Gln(18)loop where the amide of Gly(17)is a specific hydrogen bond donor, and hydrophobic contacts occur with the side chains of Lys(14)and Leu(63). Another fragment, indole-6-carboxylic acid, binds on the 'outside/top of the flap' via hydrophobic contacts with Trp(42), Pro(44), Met(46), and Lys(55), a hydrogen bond with Val(56), and a salt-bridge with Arg(57). 2-acetyl-benzothiophene also binds at this site. This study is the first fragment-based crystallographic screen against HIV PR, and the first time that fragments were screened against an inhibitor-bound drug target to search for compounds that both bind to novel sites and stabilize the inhibited conformation of the target.

  1. Protease induced plasticity: matrix metalloproteinase-1 promotes neurostructural changes through activation of protease activated receptor 1

    PubMed Central

    Allen, Megan; Ghosh, Suhasini; Ahern, Gerard P.; Villapol, Sonia; Maguire-Zeiss, Kathleen A.; Conant, Katherine

    2016-01-01

    Matrix metalloproteinases (MMPs) are a family of secreted endopeptidases expressed by neurons and glia. Regulated MMP activity contributes to physiological synaptic plasticity, while dysregulated activity can stimulate injury. Disentangling the role individual MMPs play in synaptic plasticity is difficult due to overlapping structure and function as well as cell-type specific expression. Here, we develop a novel system to investigate the selective overexpression of a single MMP driven by GFAP expressing cells in vivo. We show that MMP-1 induces cellular and behavioral phenotypes consistent with enhanced signaling through the G-protein coupled protease activated receptor 1 (PAR1). Application of exogenous MMP-1, in vitro, stimulates PAR1 dependent increases in intracellular Ca2+ concentration and dendritic arborization. Overexpression of MMP-1, in vivo, increases dendritic complexity and induces biochemical and behavioral endpoints consistent with increased GPCR signaling. These data are exciting because we demonstrate that an astrocyte-derived protease can influence neuronal plasticity through an extracellular matrix independent mechanism. PMID:27762280

  2. Protease increases fermentation rate and ethanol yield in dry-grind ethanol production.

    PubMed

    Johnston, David B; McAloon, Andrew J

    2014-02-01

    The effects of acid protease and urea addition during the fermentation step were evaluated. The fermentations were also tested with and without the addition of urea to determine if protease altered the nitrogen requirements of the yeast. Results show that the addition of the protease had a statistically significant effect on the fermentation rate and yield. Fermentation rates and yields were improved with the addition of the protease over the corresponding controls without protease. Protease addition either with or with added urea resulted in a higher final ethanol yield than without the protease addition. Urea addition levels >1200 ppm of supplemental nitrogen inhibited ethanol production. The economic effects of the protease addition were evaluated by using process engineering and economic models developed at the Eastern Regional Research Center. The decrease in overall processing costs from protease addition was as high as $0.01/L (4 ¢/gal) of denatured ethanol produced.

  3. Expression, purification and molecular modeling of the NIa protease of Cardamom mosaic virus.

    PubMed

    Jebasingh, T; Pandaranayaka, Eswari P J; Mahalakshmi, A; Kasin Yadunandam, A; Krishnaswamy, S; Usha, R

    2013-01-01

    The NIa protease of Potyviridae is the major viral protease that processes potyviral polyproteins. The NIa protease coding region of Cardamom mosaic virus (CdMV) is amplified from the viral cDNA, cloned and expressed in Escherichia coli. NIa protease forms inclusion bodies in E.coli. The inclusion bodies are solubilized with 8 M urea, refolded and purified by Nickel-Nitrilotriacetic acid affinity chromatography. Three-dimensional modeling of the CdMV NIa protease is achieved by threading approach using the homologous X-ray crystallographic structure of Tobacco etch mosaic virus NIa protease. The model gave an insight in to the substrate specificities of the NIa proteases and predicted the complementation of nearby residues in the catalytic triad (H42, D74 and C141) mutants in the cis protease activity of CdMV NIa protease. PMID:22888800

  4. The structure of a universally employed enzyme: V8 protease from Staphylococcus aureus

    SciTech Connect

    Prasad, Lata; Leduc, Yvonne; Hayakawa, Koto; Delbaere, Louis T.J.

    2008-06-27

    V8 protease, an extracellular protease of Staphylococcus aureus, is related to the pancreatic serine proteases. The enzyme cleaves peptide bonds exclusively on the carbonyl side of aspartate and glutamate residues. Unlike the pancreatic serine proteases, V8 protease possesses no disulfide bridges. This is a major evolutionary difference, as all pancreatic proteases have at least two disulfide bridges. The structure of V8 protease shows structural similarity with several other serine proteases, specifically the epidermolytic toxins A and B from S. aureus and trypsin, in which the conformation of the active site is almost identical. V8 protease is also unique in that the positively charged N-terminus is involved in determining the substrate-specificity of the enzyme.

  5. Expression, purification and molecular modeling of the NIa protease of Cardamom mosaic virus.

    PubMed

    Jebasingh, T; Pandaranayaka, Eswari P J; Mahalakshmi, A; Kasin Yadunandam, A; Krishnaswamy, S; Usha, R

    2013-01-01

    The NIa protease of Potyviridae is the major viral protease that processes potyviral polyproteins. The NIa protease coding region of Cardamom mosaic virus (CdMV) is amplified from the viral cDNA, cloned and expressed in Escherichia coli. NIa protease forms inclusion bodies in E.coli. The inclusion bodies are solubilized with 8 M urea, refolded and purified by Nickel-Nitrilotriacetic acid affinity chromatography. Three-dimensional modeling of the CdMV NIa protease is achieved by threading approach using the homologous X-ray crystallographic structure of Tobacco etch mosaic virus NIa protease. The model gave an insight in to the substrate specificities of the NIa proteases and predicted the complementation of nearby residues in the catalytic triad (H42, D74 and C141) mutants in the cis protease activity of CdMV NIa protease.

  6. Management of HIV-related stigma and adherence to HAART: evidence from a large representative sample of outpatients attending French hospitals (ANRS-EN12-VESPA 2003).

    PubMed

    Peretti-Watel, P; Spire, B; Pierret, J; Lert, F; Obadia, Y

    2006-04-01

    This study investigated patterns of HIV disclosure to significant others (parents, siblings, children, other relatives, friends and colleagues) and describe them in terms of socio-demographic background and other characteristics, including experiences of AIDS-related discrimination. It also assessed the relationship between disclosure patterns and adherence to HAART. We used a cross-sectional survey conducted among a national representative sample of 2,932 HIV-infected people recruited in French hospitals. HIV disclosure patterns were both selective and cumulative: disclosure was more frequent for friends and siblings, while concealment prevailed concerning children, other relatives, and colleagues; but patients who disclosed their seropositivity to one significant other were also more likely to disclose it to other significant others. Patients reporting experiences of discrimination from sexual partners were less likely to be highly adherent, and we also found a significant relationship between uncontrolled disclosure and non-adherence. Patients who have opted for concealment probably consider non-adherence and uncontrolled disclosure as competing risks, but among them a significant minority loses on both counts. Counselling provided to HIV-infected people should not separate the adherence and disclosure issues, and adherence interventions should seek to help patients to manage concurrently disclosure/concealment of their seropositivity and its consequences.

  7. Protease inhibitor from Moringa oleifera with potential for use as therapeutic drug and as seafood preservative.

    PubMed

    Bijina, B; Chellappan, Sreeja; Krishna, Jissa G; Basheer, Soorej M; Elyas, K K; Bahkali, Ali H; Chandrasekaran, M

    2011-07-01

    Protease inhibitors are well known to have several applications in medicine and biotechnology. Several plant sources are known to return potential protease inhibitors. In this study plants belonging to different families of Leguminosae, Malvaceae, Rutaceae, Graminae and Moringaceae were screened for the protease inhibitor. Among them Moringa oleifera, belonging to the family Moringaceae, recorded high level of protease inhibitor activity after ammonium sulfate fractionation. M. oleifera, which grows throughout most of the tropics and having several industrial and medicinal uses, was selected as a source of protease inhibitor since so far no reports were made on isolation of the protease inhibitor. Among the different parts of M. oleifera tested, the crude extract isolated from the mature leaves and seeds showed the highest level of inhibition against trypsin. Among the various extraction media evaluated, the crude extract prepared in phosphate buffer showed maximum recovery of the protease inhibitor. The protease inhibitor recorded high inhibitory activity toward the serine proteases thrombin, elastase, chymotrypsin and the cysteine proteases cathepsin B and papain which have more importance in pharmaceutical industry. The protease inhibitor also showed complete inhibition of activities of the commercially available proteases of Bacillus licheniformis and Aspergillus oryzae. However, inhibitory activities toward subtilisin, esperase, pronase E and proteinase K were negligible. Further, it was found that the protease inhibitor could prevent proteolysis in a commercially valuable shrimp Penaeus monodon during storage indicating the scope for its application as a seafood preservative. This is the first report on isolation of a protease inhibitor from M. oleifera. PMID:23961135

  8. Protease inhibitor from Moringa oleifera with potential for use as therapeutic drug and as seafood preservative

    PubMed Central

    Bijina, B.; Chellappan, Sreeja; Krishna, Jissa G.; Basheer, Soorej M.; Elyas, K.K.; Bahkali, Ali H.; Chandrasekaran, M.

    2011-01-01

    Protease inhibitors are well known to have several applications in medicine and biotechnology. Several plant sources are known to return potential protease inhibitors. In this study plants belonging to different families of Leguminosae, Malvaceae, Rutaceae, Graminae and Moringaceae were screened for the protease inhibitor. Among them Moringa oleifera, belonging to the family Moringaceae, recorded high level of protease inhibitor activity after ammonium sulfate fractionation. M. oleifera, which grows throughout most of the tropics and having several industrial and medicinal uses, was selected as a source of protease inhibitor since so far no reports were made on isolation of the protease inhibitor. Among the different parts of M. oleifera tested, the crude extract isolated from the mature leaves and seeds showed the highest level of inhibition against trypsin. Among the various extraction media evaluated, the crude extract prepared in phosphate buffer showed maximum recovery of the protease inhibitor. The protease inhibitor recorded high inhibitory activity toward the serine proteases thrombin, elastase, chymotrypsin and the cysteine proteases cathepsin B and papain which have more importance in pharmaceutical industry. The protease inhibitor also showed complete inhibition of activities of the commercially available proteases of Bacillus licheniformis and Aspergillus oryzae. However, inhibitory activities toward subtilisin, esperase, pronase E and proteinase K were negligible. Further, it was found that the protease inhibitor could prevent proteolysis in a commercially valuable shrimp Penaeus monodon during storage indicating the scope for its application as a seafood preservative. This is the first report on isolation of a protease inhibitor from M. oleifera. PMID:23961135

  9. Kinetics of alkaline protease production by Streptomyces griseoflavus PTCC1130

    PubMed Central

    Hosseini, Seyed Vesal; Saffari, Zahra; Farhanghi, Ali; Atyabi, Seyed Mohammad; Norouzian, Dariush

    2016-01-01

    Background and Objectives: Proteases are a group of enzymes that catalyze the degradation of proteins resulting in the production of their amino acid constituents. They are the most important group of industrial enzymes which account for about 60% of total enzymes in the market and produced mainly by microorganisms. The attempts were made to study the kinetic parameters of protease produced by Streptomyces griseoflavus PTCC1130. Materials and Methods: Streptomyces griseoflavus PTCC1130 was grown on casein agar. Different media such as BM1, BM2, BM3 and BM4 were prepared. Data obtained from growth and protease production were subjected to kinetics evaluation. Casein was used as substrate for protease activity and the released soluble peptide bearing aromatic amino acid were quantified by Folin Cioclateaue reagent. Protein content of the enzyme and the sugar utilized by the organism were estimated by Bradford and Miller’s methods respectively. Results: Basal Medium named as BM1, BM2, BM3 and BM4(50 mL in 250 mL Erlen Meyer flasks) were screened out to evaluate protease production by Streptomyces griseoflavus PTCC1130. They were inoculated with known amount of seed culture and kept on rotary shaker. To obtain the specific growth rate, wet weight of biomass was plotted against the time. The clarified supernatant was used for the analysis of protease by measuring the soluble peptide containing aromatic amino acid residues employing Folin Cioclateaue reagent. Our results showed that maximum level of enzyme production (14035 U/L) was occurred at late exponential phase using Basal Medium supplemented with zinc sulfate (0.5g/L), casein (10g/L) at pH 6.5. Conclusions: A kinetic study of protease production by Streptomyces griseoflavus PTCC1130 provided highly quantitative information regarding the behavior of a system, which is essential to study the fermentation process. Exploitation of such kinetics analysis would be useful in commercialization of microbial enzyme

  10. Characterization of a chemostable serine alkaline protease from Periplaneta americana

    PubMed Central

    2013-01-01

    Background Proteases are important enzymes involved in numerous essential physiological processes and hold a strong potential for industrial applications. The proteolytic activity of insects’ gut is endowed by many isoforms with diverse properties and specificities. Thus, insect proteases can act as a tool in industrial processes. Results In the present study, purification and properties of a serine alkaline protease from Periplaneta americana and its potential application as an additive in various bio-formulations are reported. The enzyme was purified near to homogeneity by using acetone precipitation and Sephadex G-100 gel filtration chromatography. Enzyme activity was increased up to 4.2 fold after gel filtration chromatography. The purified enzyme appeared as single protein-band with a molecular mass of ~ 27.8 kDa in SDS-PAGE. The optimum pH and temperature for the proteolytic activity for purified protein were found around pH 8.0 and 60°C respectively. Complete inhibition of the purified enzyme by phenylmethylsulfonyl fluoride confirmed that the protease was of serine-type. The purified enzyme revealed high stability and compatibility towards detergents, oxidizing, reducing, and bleaching agents. In addition, enzyme also showed stability towards organic solvents and commercial detergents. Conclusion Several important properties of a serine protease from P. Americana were revealed. Moreover, insects can serve as excellent and alternative source of industrially important proteases with unique properties, which can be utilized as additives in detergents, stain removers and other bio-formulations. Properties of the P. americana protease accounted in the present investigation can be exploited further in various industrial processes. As an industrial prospective, identification of enzymes with varying essential properties from different insect species might be good approach and bioresource. PMID:24229392

  11. Proteases in egg, miracidium and adult of Fasciola gigantica. Characterization of serine and cysteine proteases from adult.

    PubMed

    Mohamed, Saleh A; Fahmy, Afaf S; Mohamed, Tarek M; Hamdy, Soha M

    2005-10-01

    Proteolytic activity of 0-12 day old eggs, miracidium and adult worm of Fasciola gigantica was assessed and proteases were partially purified by DEAE-Sepharose and CM-cellulose columns. Four forms of protease were separated, PIa, PIb, PIc and PII. Purifications were completed for PIc and PII using Sephacryl S-200 chromatography. A number of natural and synthetic proteins were tested as substrates for F. gigantica PIc and PII. The two proteases had moderate activity levels toward azoalbumin and casein compared to azocasein, while gelatin, hemoglobin, albumin and fibrin had very low affinity toward the two enzymes. Amidolytic substrates are more specific to protease activity. PIc had higher affinity toward BAPNA-HCl (N-benzoyl-arginine-p-nitroanilide-HCl) and BTPNA-HCl (N-benzoyl-tyrosine-p-nitroanilide-HCl) at pH 8.0 indicating that the enzyme was a serine protease. However, PII had higher affinity toward BAPNA at pH 6.5 in the presence of sulfhydryl groups (beta-mercaptoethanol) indicating that the enzyme was a cysteine protease. The effect of specific protease inhibitors on these enzymes was studied. The results confirmed that proteases PIc and PII could be serine and cysteine proteases, respectively. The molecular weights of F. gigantica PIc and PII were 60,000 and 25,000, respectively. F. gigantica PIc and PII had pH optima at 7.5 and 5.5 and K(M) of 2 and 5 mg azocasein/mL, respectively. For amidolytic substrates, PIc had K(M) of 0.3 mM BAPNA/mL and 0.5 mM BTPNA/mL at pH 8.0 and PII had K(M) of 0.6 mM BAPNA/mL at pH 6.5 with reducing agent. F. gigantica PIc and PII had the same optimum temperature at 50 degrees C and were stable up to 40 degrees C. All examined metal cations tested had inhibitory effects toward the two enzymes. From substrate specificity and protease inhibitor studies, PIc and PII could be designated as serine PIc and cysteine PII, respectively. PMID:16102991

  12. Protease nexin-1 regulates retinal vascular development.

    PubMed

    Selbonne, Sonia; Francois, Deborah; Raoul, William; Boulaftali, Yacine; Sennlaub, Florian; Jandrot-Perrus, Martine; Bouton, Marie-Christine; Arocas, Véronique

    2015-10-01

    We recently identified protease nexin-1 (PN-1) or serpinE2, as a possibly underestimated player in maintaining angiogenic balance. Here, we used the well-characterized postnatal vascular development of newborn mouse retina to further investigate the role and the mechanism of action of PN-1 in physiological angiogenesis. The development of retinal vasculature was analysed by endothelial cell staining with isolectin B4. PN-1-deficient (PN-1(-/-)) retina displayed increased vascularization in the postnatal period, with elevated capillary thickness and density, compared to their wild-type littermate (WT). Moreover, PN-1(-/-) retina presented more veins/arteries than WT retina. The kinetics of retinal vasculature development, retinal VEGF expression and overall retinal structure were similar in WT and PN-1(-/-) mice, but we observed a hyperproliferation of vascular cells in PN-1(-/-) retina. Expression of PN-1 was analysed by immunoblotting and X-Gal staining of retinas from mice expressing beta-galactosidase under a PN-1 promoter. PN-1 was highly expressed in the first week following birth and then progressively decreased to a low level in adult retina where it localized on the retinal arteries. PCR arrays performed on mouse retinal RNA identified two angiogenesis-related factors, midkine and Smad5, that were overexpressed in PN-1(-/-) newborn mice and this was confirmed by RT-PCR. Both the higher vascularization and the overexpression of midkine and Smad5 mRNA were also observed in gastrocnemius muscle of PN-1(-/-) mice, suggesting that PN-1 interferes with these pathways. Together, our results demonstrate that PN-1 strongly limits physiological angiogenesis and suggest that modulation of PN-1 expression could represent a new way to regulate angiogenesis.

  13. Plasma micronutrient concentrations are altered by antiretroviral therapy and lipid-based nutrient supplements in lactating HIV-infected Malawian women

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methods: Plasma micronutrient concentrations were measured in a subsample (n = 690) of Breastfeeding, Antiretrovirals, and Nutrition (BAN) study participants who were randomly assigned at delivery to receive HAART, LNS, HAART+LNS, or no HAART/no LNS (control). HAART consisted of protease inhibitor–b...

  14. Contribution of Gag and Protease to HIV-1 Phenotypic Drug Resistance in Pediatric Patients Failing Protease Inhibitor-Based Therapy

    PubMed Central

    Giandhari, Jennifer; Basson, Adriaan E.; Sutherland, Katherine; Parry, Chris M.; Cane, Patricia A.; Coovadia, Ashraf; Kuhn, Louise; Hunt, Gillian

    2016-01-01

    Protease inhibitors (PIs) are used as a first-line regimen in HIV-1-infected children. Here we investigated the phenotypic consequences of amino acid changes in Gag and protease on lopinavir (LPV) and ritonavir (RTV) susceptibility among pediatric patients failing PI therapy. The Gag-protease from isolates from 20 HIV-1 subtype C-infected pediatric patients failing an LPV and/or RTV-based regimen was phenotyped using a nonreplicative in vitro assay. Changes in sensitivity to LPV and RTV relative to that of the matched baseline (pretherapy) sample were calculated. Gag and protease amino acid substitutions associated with PI failure were created in a reference clone by site-directed mutagenesis and assessed. Predicted phenotypes were determined using the Stanford drug resistance algorithm. Phenotypic resistance or reduced susceptibility to RTV and/or LPV was observed in isolates from 10 (50%) patients, all of whom had been treated with RTV. In most cases, this was associated with protease resistance mutations, but substitutions at Gag cleavage and noncleavage sites were also detected. Gag amino acid substitutions were also found in isolates from three patients with reduced drug susceptibilities who had wild-type protease. Site-directed mutagenesis confirmed that some amino acid changes in Gag contributed to PI resistance but only in the presence of major protease resistance-associated substitutions. The isolates from all patients who received LPV exclusively were phenotypically susceptible. Baseline isolates from the 20 patients showed a large (47-fold) range in the 50% effective concentration of LPV, which accounted for most of the discordance seen between the experimentally determined and the predicted phenotypes. Overall, the inclusion of the gag gene and the use of matched baseline samples provided a more comprehensive assessment of the effect of PI-induced amino acid changes on PI resistance. The lack of phenotypic resistance to LPV supports the continued use of

  15. Regulation of Extracellular Protease Production in Bacillus cereus T: Characterization of Mutants Producing Altered Amounts of Protease

    PubMed Central

    Aronson, A. I.; Angelo, N.; Holt, S. C.

    1971-01-01

    Twenty-nine mutants of Bacillus cereus T were selected on casein agar for their inability to produce large amounts of extracellular protease. They all formed spores, and 27 were also auxotrophs for purines or pyrimidines. Upon reversion to prototrophy, a large fraction regained the capacity to produce protease. Conversely, reversion to normal protease production resulted in loss of the purine or pyrimidine requirement in a large fraction of the revertants. One spontaneous low-protease-producing pyrimidine auxotroph studied in detail grew as well as the wild type and produced spores which were identical to those produced by the wild type on the basis of heat resistance, dipicolinic acid content, density, and appearance in the electron microscope. The rate of protein turnover in the mutant was the same as the wild type. The mutant did grow poorly, however, when casein was the principal carbon source. A mutant excreting 5 to 10 times as much protease as the wild type was isolated as a secondary mutation from the hypoproducer discussed above. Loss of the pyrimidine requirement in this case did not alter the regulation of protease production. Although the secondary mutant grew somewhat faster in most media than the wild type, the final cell yield was lower. The spores of this mutant appeared to have excess coat on the basis of both electron microscopic and chemical studies. There appear to be closely related but distinct catabolic controls for both extracellular protease and spore formation. These controls can be dissociated as for the hypoproducers but can also appear integrated as for the hyperprotease producer. Images PMID:4104235

  16. Proteolytic Activation of the Protease-activated Receptor (PAR)-2 by the Glycosylphosphatidylinositol-anchored Serine Protease Testisin*

    PubMed Central

    Driesbaugh, Kathryn H.; Buzza, Marguerite S.; Martin, Erik W.; Conway, Gregory D.; Kao, Joseph P. Y.; Antalis, Toni M.

    2015-01-01

    Protease-activated receptors (PARs) are a family of seven-transmembrane, G-protein-coupled receptors that are activated by multiple serine proteases through specific N-terminal proteolytic cleavage and the unmasking of a tethered ligand. The majority of PAR-activating proteases described to date are soluble proteases that are active during injury, coagulation, and inflammation. Less investigation, however, has focused on the potential for membrane-anchored serine proteases to regulate PAR activation. Testisin is a unique trypsin-like serine protease that is tethered to the extracellular membrane of cells through a glycophosphatidylinositol (GPI) anchor. Here, we show that the N-terminal domain of PAR-2 is a substrate for testisin and that proteolytic cleavage of PAR-2 by recombinant testisin activates downstream signaling pathways, including intracellular Ca2+ mobilization and ERK1/2 phosphorylation. When testisin and PAR-2 are co-expressed in HeLa cells, GPI-anchored testisin specifically releases the PAR-2 tethered ligand. Conversely, knockdown of endogenous testisin in NCI/ADR-Res ovarian tumor cells reduces PAR-2 N-terminal proteolytic cleavage. The cleavage of PAR-2 by testisin induces activation of the intracellular serum-response element and NFκB signaling pathways and the induction of IL-8 and IL-6 cytokine gene expression. Furthermore, the activation of PAR-2 by testisin results in the loss and internalization of PAR-2 from the cell surface. This study reveals a new biological substrate for testisin and is the first demonstration of the activation of a PAR by a serine protease GPI-linked to the cell surface. PMID:25519908

  17. Diversity of both the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the South China sea.

    PubMed

    Zhou, Ming-Yang; Chen, Xiu-Lan; Zhao, Hui-Lin; Dang, Hong-Yue; Luan, Xi-Wu; Zhang, Xi-Ying; He, Hai-Lun; Zhou, Bai-Cheng; Zhang, Yu-Zhong

    2009-10-01

    Protease-producing bacteria are known to play an important role in degrading sedimentary particular organic nitrogen, and yet, their diversity and extracellular proteases remain largely unknown. In this paper, the diversity of the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the South China Sea was investigated. The richness of the cultivable protease-producing bacteria reached 10(6) cells/g in all sediment samples. Analysis of the 16S rRNA gene sequences revealed that the predominant cultivated protease-producing bacteria are Gammaproteobacteria affiliated with the genera Pseudoalteromonas, Alteromonas, Marinobacter, Idiomarina, Halomonas, Vibrio, Shewanella, Pseudomonas, and Rheinheimera, with Alteromonas (34.6%) and Pseudoalteromonas (28.2%) as the predominant groups. Inhibitor analysis showed that nearly all the extracellular proteases from the bacteria are serine proteases or metalloproteases. Moreover, these proteases have different hydrolytic ability to different proteins, reflecting they may belong to different kinds of serine proteases or metalloproteases. To our knowledge, this study represents the first report of the diversity of bacterial proteases in deep-sea sediments.

  18. Protease Inhibitors in View of Peptide Substrate Databases

    PubMed Central

    2016-01-01

    Protease substrate profiling has nowadays almost become a routine task for experimentalists, and the knowledge on protease peptide substrates is easily accessible via the MEROPS database. We present a shape-based virtual screening workflow using vROCS that applies the information about the specificity of the proteases to find new small-molecule inhibitors. Peptide substrate sequences for three to four substrate positions of each substrate from the MEROPS database were used to build the training set. Two-dimensional substrate sequences were converted to three-dimensional conformations through mutation of a template peptide substrate. The vROCS query was built from single amino acid queries for each substrate position considering the relative frequencies of the amino acids. The peptide-substrate-based shape-based virtual screening approach gives good performance for the four proteases thrombin, factor Xa, factor VIIa, and caspase-3 with the DUD-E data set. The results show that the method works for protease targets with different specificity profiles as well as for targets with different active-site mechanisms. As no structure of the target and no information on small-molecule inhibitors are required to use our approach, the method has significant advantages in comparison with conventional structure- and ligand-based methods. PMID:27247997

  19. Characterization, biomedical and agricultural applications of protease inhibitors: A review.

    PubMed

    Shamsi, Tooba Naz; Parveen, Romana; Fatima, Sadaf

    2016-10-01

    This review describes Protease Inhibitors (PIs) which target or inhibit proteases, protein digesting enzymes. These proteases play a crucial task in many biological events including digestion, blood coagulation, apoptosis etc. Regardless of their crucial roles, they need to be checked regularly by PIs as their excess may possibly damage host organism. On basis of amino acid composition of PIs where Protease-PI enzymatic reactions occur i.e. serine, cysteine, and aspartic acid, they are classified. Nowadays, various PIs are being worked upon to fight various parasitic or viral diseases including malaria, schistosomiasis, colds, flu', dengue etc. They prevent an ongoing process begun by carcinogen exposure by keeping a check on metastasis. They also possess potential to reduce carcinogen-induced, increased levels of gene amplification to almost normal levels. Some PIs can principally be used for treatment of hypertension and congestive heart failure by blocking conversion of angiotensin I to angiotensin II for example Angiotensin-converting enzyme inhibitors (ACEIs). Also PIs target amyloid β-peptide (Aβ) level in brain which is prime responsible for development of Alzheimer's Disease (AD). Also, PIs inhibit enzymatic activity of HIV-1 Protease Receptor (PR) by preventing cleavage events in Gag and Gag-Pol that result in production of non-virulent virus particles.

  20. Mitochondrial cereblon functions as a Lon-type protease.

    PubMed

    Kataoka, Kosuke; Nakamura, China; Asahi, Toru; Sawamura, Naoya

    2016-07-15

    Lon protease plays a major role in the protein quality control system in mammalian cell mitochondria. It is present in the mitochondrial matrix, and degrades oxidized and misfolded proteins, thereby protecting the cell from various extracellular stresses, including oxidative stress. The intellectual disability-associated and thalidomide-binding protein cereblon (CRBN) contains a large, highly conserved Lon domain. However, whether CRBN has Lon protease-like function remains unknown. Here, we determined if CRBN has a protective function against oxidative stress, similar to Lon protease. We report that CRBN partially distributes in mitochondria, suggesting it has a mitochondrial function. To specify the mitochondrial role of CRBN, we mitochondrially expressed CRBN in human neuroblastoma SH-SY5Y cells. The resulting stable SH-SY5Y cell line showed no apparent effect on the mitochondrial functions of fusion, fission, and membrane potential. However, mitochondrially expressed CRBN exhibited protease activity, and was induced by oxidative stress. In addition, stably expressed cells exhibited suppressed neuronal cell death induced by hydrogen peroxide. These results suggest that CRBN functions specifically as a Lon-type protease in mitochondria.

  1. Emerging roles for diverse intramembrane proteases in plant biology.

    PubMed

    Adam, Zach

    2013-12-01

    Progress in the field of regulated intramembrane proteolysis (RIP) in recent years has made its impact on plant biology as well. Although this field within plant research is still in its infancy, some interesting observations have started to emerge. Gene encoding orthologs of rhomboid proteases, site-2 proteases (S2P), presenilin/γ-secretases, and signal peptide peptidases are found in plant genomes and some of these gene products were identified in different plant cell membranes. The lack of chloroplast-located rhomboid proteases was associated with reduced fertility and aberrations in flower morphology. Mutations in homologues of S2P resulted in chlorophyll deficiency and impaired chloroplast development. An S2P was also implicated in the response to ER stress through cleavage of ER-membrane bZIP transcription factors, allowing their migration to the nucleus and activation of the transcription of BiP chaperones. Other membrane-bound transcription factors of the NAC and PHD families were also demonstrated to undergo RIP and relocalization to the nucleus. These and other new data are expected to shed more light on the roles of intramembrane proteases in plant biology in the future. This article is part of a Special Issue entitled: Intramembrane Proteases.

  2. Alkaline protease from Thermoactinomyces sp. RS1 mitigates industrial pollution.

    PubMed

    Verma, Amit; Ansari, Mohammad W; Anwar, Mohmmad S; Agrawal, Ruchi; Agrawal, Sanjeev

    2014-05-01

    Proteases have found a wide application in the several industrial processes, such as laundry detergents, protein recovery or solubilization, prion degradation, meat tenderizations, and in bating of hides and skins in leather industries. But the main hurdle in industrial application of proteases is their economical production on a large scale. The present investigation aimed to exploit the locally available inexpensive agricultural and household wastes for alkaline protease production using Thermoactinomyces sp. RS1 via solid-state fermentation (SSF) technique. The alkaline enzyme is potentially useful as an additive in commercial detergents to mitigate pollution load due to extensive use of caustic soda-based detergents. Thermoactinomyces sp. RS1 showed good protease production under SSF conditions of 55 °C, pH 9, and 50 % moisture content with potato peels as solid substrate. The presented findings revealed that crude alkaline protease produced by Thermoactinomyces sp. RS1 via SSF is of potential application in silver recovery from used X-ray films.

  3. Extracellular proteases are released by ciliates in defined seawater microcosms.

    PubMed

    Thao, Ngo Vy; Nozawa, Akino; Obayashi, Yumiko; Kitamura, Shin-Ichi; Yokokawa, Taichi; Suzuki, Satoru

    2015-08-01

    The biodegradation of proteins in seawater requires various proteases which are commonly thought to be mainly derived from heterotrophic bacteria. We, however, found that protists showed a high protease activity and continuously produced trypsin-type enzymes. The free-living marine heterotrophic ciliate Paranophrys marina together with an associated bacterium was isolated and used for microcosm incubation with different concentrations of killed bacteria as food for 10 days. The results showed that the co-existence of the ciliate with its associated bacterium produced a significant protease activity in both cell-associated and cell-free fractions while that in the associated bacterium only microcosm was negligible. The protease profiles are different between cell-associated and cell-free fractions, and a trypsin-type enzyme hydrolyzing Boc-Val-Leu-Lys-MCA was detected throughout the period in the presence of ciliates. This suggests that ciliates release proteases into the surrounding environment which could play a role in protein digestion outside cells. It has been previously suggested that bacteria are the major transformers in seawater. We here present additional data which indicates that protists, or at least ciliates with their specific enzymes, are a potential player in organic matter degradation in water columns.

  4. Plant collagenase: unique collagenolytic activity of cysteine proteases from ginger.

    PubMed

    Kim, Misook; Hamilton, Susan E; Guddat, Luke W; Overall, Christopher M

    2007-12-01

    Two cysteine proteases, GP2 and GP3, have been isolated from ginger rhizomes (Zingiber officinale). GP2 is virtually identical to a previously identified ginger protease GPII [K.H. Choi, and R.A. Laursen, Amino-acid sequence and glycan structures of cysteine proteases with proline specificity from ginger rhizome Zingiber officinale, Eur. J. Biochem. 267 (2000) 1516-1526.], and cleaves native type I collagen at multiple discrete sites, which are in the interior of the triple helical region of this molecule. In reaction with proline-containing peptides GP2 shows preference for Pro in the P2 position, and at least 10-fold higher efficiency of hydrolysis than papain. Comparison of models of GP2 and GP3 with the crystal structure of papain shows that the three enzymes have different S2 pocket structures. The S2 pocket in GP2 and GP3 is half the size of that of papain. GP2 is the only reported plant cysteine protease with a demonstrated ability to hydrolyse native collagen. The results support a role for ginger proteases as an alternative to papain, in commercial applications such as meat tenderization, where collagen is the target substrate. PMID:17920199

  5. Characterization, biomedical and agricultural applications of protease inhibitors: A review.

    PubMed

    Shamsi, Tooba Naz; Parveen, Romana; Fatima, Sadaf

    2016-10-01

    This review describes Protease Inhibitors (PIs) which target or inhibit proteases, protein digesting enzymes. These proteases play a crucial task in many biological events including digestion, blood coagulation, apoptosis etc. Regardless of their crucial roles, they need to be checked regularly by PIs as their excess may possibly damage host organism. On basis of amino acid composition of PIs where Protease-PI enzymatic reactions occur i.e. serine, cysteine, and aspartic acid, they are classified. Nowadays, various PIs are being worked upon to fight various parasitic or viral diseases including malaria, schistosomiasis, colds, flu', dengue etc. They prevent an ongoing process begun by carcinogen exposure by keeping a check on metastasis. They also possess potential to reduce carcinogen-induced, increased levels of gene amplification to almost normal levels. Some PIs can principally be used for treatment of hypertension and congestive heart failure by blocking conversion of angiotensin I to angiotensin II for example Angiotensin-converting enzyme inhibitors (ACEIs). Also PIs target amyloid β-peptide (Aβ) level in brain which is prime responsible for development of Alzheimer's Disease (AD). Also, PIs inhibit enzymatic activity of HIV-1 Protease Receptor (PR) by preventing cleavage events in Gag and Gag-Pol that result in production of non-virulent virus particles. PMID:26955746

  6. Predictors of virologic response to ritonavir-boosted protease inhibitors.

    PubMed

    Marcelin, Anne-Genevieve; Flandre, Philippe; Peytavin, Gilles; Calvez, Vincent

    2005-01-01

    The primary mechanism of resistance to protease inhibitors involves the stepwise accumulation of mutations that alter and block the substrate binding site of HIV protease. The large degree of cross-resistance among the different protease inhibitors is a source of considerable concern for the management of patients after treatment failure. Although the output of HIV-resistance tests has been based on therapeutically arbitrary criteria, there is now an ongoing move towards correlating test interpretation with virologic outcomes on treatment. This approach is undeniably superior, in principle, for tests intended to guide drug choices. However, the predictive accuracy of a given stratagem that links genotype or phenotype to drug response is strongly influenced by the study design, data capture and the analytical methodology used to derive it. There is no definitively superior methodology for generating a genotype-response association for use in interpreting a resistance test, and the various approaches used to date all have their strengths and weaknesses. Combining the information of therapeutic drug monitoring and resistance tests is likely to be of greatest clinical utility in antiretroviral-experienced patients harboring HIV strains with reduced susceptibility. The combination of pharmacologic and virologic parameters as a predictor of the virologic response has been merged into the parameter known as "inhibitory quotient". This article discusses the potential interest of the use of inhibitory quotients as an approach for enhancing the potency and durability of boosted protease inhibitors against protease inhibitor-resistant viruses. PMID:16425962

  7. Extracellular proteases are released by ciliates in defined seawater microcosms.

    PubMed

    Thao, Ngo Vy; Nozawa, Akino; Obayashi, Yumiko; Kitamura, Shin-Ichi; Yokokawa, Taichi; Suzuki, Satoru

    2015-08-01

    The biodegradation of proteins in seawater requires various proteases which are commonly thought to be mainly derived from heterotrophic bacteria. We, however, found that protists showed a high protease activity and continuously produced trypsin-type enzymes. The free-living marine heterotrophic ciliate Paranophrys marina together with an associated bacterium was isolated and used for microcosm incubation with different concentrations of killed bacteria as food for 10 days. The results showed that the co-existence of the ciliate with its associated bacterium produced a significant protease activity in both cell-associated and cell-free fractions while that in the associated bacterium only microcosm was negligible. The protease profiles are different between cell-associated and cell-free fractions, and a trypsin-type enzyme hydrolyzing Boc-Val-Leu-Lys-MCA was detected throughout the period in the presence of ciliates. This suggests that ciliates release proteases into the surrounding environment which could play a role in protein digestion outside cells. It has been previously suggested that bacteria are the major transformers in seawater. We here present additional data which indicates that protists, or at least ciliates with their specific enzymes, are a potential player in organic matter degradation in water columns. PMID:26115436

  8. Three Pseudomonas aeruginosa strains with different protease profiles.

    PubMed

    Andrejko, Mariola; Zdybicka-Barabas, Agnieszka; Janczarek, Monika; Cytryńska, Małgorzata

    2013-01-01

    The proteolytic activity of three Pseudomonas aeruginosa strains, ATCC 27853 - a reference strain, and two clinical isolates was tested. The activity was examined after culturing the bacteria in two different growth media: the minimal M9 medium and rich Luria-Bertani broth (LB). Based on zymograms and protease activity specific assays, it was concluded that the reference strain produced three proteolytic enzymes in the LB medium: protease IV, elastase B and elastase A, while alkaline protease was only produced in the M9 medium. The clinical isolates of P. aeruginosa produced elastase B and alkaline protease when grown in the LB medium and the minimal M9 medium, respectively. PCR analysis confirmed the presence of both the lasB gene encoding elastase B and aprA coding for alkaline protease in the genomes of the three P. aeruginosa strains analyzed. The expression of these genes coding for two important P. aeruginosa virulence factors was dependent on the growth conditions in all the strains studied. The contribution of the extracellular proteinases to the virulence of P. aeruginosa strains used in this study was investigated using an insect model, the greater wax moth Galleria mellonella.

  9. A tunable, modular approach to fluorescent protease-activated reporters.

    PubMed

    Wu, Peng; Nicholls, Samantha B; Hardy, Jeanne A

    2013-04-01

    Proteases are one of the most important and historically utilized classes of drug targets. To effectively interrogate this class of proteins, which encodes nearly 2% of the human proteome, it is necessary to develop effective and cost-efficient methods that report on their activity both in vitro and in vivo. We have developed a robust reporter of caspase proteolytic activity, called caspase-activatable green fluorescent protein (CA-GFP). The caspases play central roles in homeostatic regulation, as they execute programmed cell death, and in drug design, as caspases are involved in diseases ranging from cancer to neurodegeneration. CA-GFP is a genetically encoded dark-to-bright fluorescent reporter of caspase activity in in vitro, cell-based, and animal systems. Based on the CA-GFP platform, we developed reporters that can discriminate the activities of caspase-6 and -7, two highly related proteases. A second series of reporters, activated by human rhinovirus 3C protease, demonstrated that we could alter the specificity of the reporter by reengineering the protease recognition sequence. Finally, we took advantage of the spectrum of known fluorescent proteins to generate green, yellow, cyan, and red reporters, paving the way for multiplex protease monitoring. PMID:23561537

  10. Hydrophobic Core Flexibility Modulates Enzyme Activity in HIV-1 Protease

    SciTech Connect

    Mittal, Seema; Cai, Yufeng; Nalam, Madhavi N.L.; Bolon, Daniel N.A.; Schiffer, Celia A.

    2012-09-11

    Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Oxidized and reduced crystal structures of these variants show the overall structure of the protease is retained. However, cross-linking the cysteines led to drastic loss in enzyme activity, which was regained upon reducing the disulfide cross-links. Molecular dynamics simulations showed that altered dynamics propagated throughout the enzyme from the engineered disulfide. Thus, altered flexibility within the hydrophobic core can modulate HIV-1 protease activity, supporting the hypothesis that drug resistant mutations distal from the active site can alter the balance between substrate turnover and inhibitor binding by modulating enzyme activity.

  11. Mitochondrial cereblon functions as a Lon-type protease

    PubMed Central

    Kataoka, Kosuke; Nakamura, China; Asahi, Toru; Sawamura, Naoya

    2016-01-01

    Lon protease plays a major role in the protein quality control system in mammalian cell mitochondria. It is present in the mitochondrial matrix, and degrades oxidized and misfolded proteins, thereby protecting the cell from various extracellular stresses, including oxidative stress. The intellectual disability-associated and thalidomide-binding protein cereblon (CRBN) contains a large, highly conserved Lon domain. However, whether CRBN has Lon protease-like function remains unknown. Here, we determined if CRBN has a protective function against oxidative stress, similar to Lon protease. We report that CRBN partially distributes in mitochondria, suggesting it has a mitochondrial function. To specify the mitochondrial role of CRBN, we mitochondrially expressed CRBN in human neuroblastoma SH-SY5Y cells. The resulting stable SH-SY5Y cell line showed no apparent effect on the mitochondrial functions of fusion, fission, and membrane potential. However, mitochondrially expressed CRBN exhibited protease activity, and was induced by oxidative stress. In addition, stably expressed cells exhibited suppressed neuronal cell death induced by hydrogen peroxide. These results suggest that CRBN functions specifically as a Lon-type protease in mitochondria. PMID:27417535

  12. Selection of multiple human immunodeficiency virus type 1 variants that encode viral proteases with decreased sensitivity to an inhibitor of the viral protease.

    PubMed Central

    Kaplan, A H; Michael, S F; Wehbie, R S; Knigge, M F; Paul, D A; Everitt, L; Kempf, D J; Norbeck, D W; Erickson, J W; Swanstrom, R

    1994-01-01

    Inhibitors of the human immunodeficiency virus type 1 (HIV-1) protease represent a promising addition to the available agents used to inhibit virus replication in a therapeutic setting. HIV-1 is capable of generating phenotypic variants in the face of a variety of selective pressures. The potential to generate variants with reduced sensitivity to a protease inhibitor was examined by selecting for virus growth in cell culture in the presence of the protease inhibitor A-77003. Virus variants grew out in the presence of the inhibitor, and these variants encoded proteases with reduced sensitivity to the inhibitor. Variants were identified that encoded changes in each of the three subsites of the protease that interact with the inhibitor. HIV-1 displays significant potential for altering its interaction with this protease inhibitor, suggesting the need for multiple protease inhibitors with varying specificities. Images PMID:8202533

  13. Diversity of cultivable protease-producing bacteria in sediments of Jiaozhou Bay, China

    PubMed Central

    Zhang, Xi-Ying; Han, Xiao-Xu; Chen, Xiu-Lan; Dang, Hong-Yue; Xie, Bin-Bin; Qin, Qi-Long; Shi, Mei; Zhou, Bai-Cheng; Zhang, Yu-Zhong

    2015-01-01

    Although protease-producing bacteria are key players in the degradation of organic nitrogen and essential for the nitrogen recycling in marine sediments, diversity of both these bacteria and their extracellular proteases is still largely unknown. This study investigated the diversity of the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the eutrophied Jiaozhou Bay, China through phylogenetic analysis and protease inhibitor tests. The abundance of the cultivable protease-producing bacteria was up to 104 cells/g in all six sediment samples. The cultivated protease-producing bacteria mostly belonged to the phyla Proteobacteria and Firmicutes with the predominant genera being Photobacterium (39.4%), Bacillus (25.8%), and Vibrio (19.7%). Protease inhibitor tests revealed that extracellular proteases secreted by the bacteria were mainly serine proteases and/or metalloproteases with relatively low proportions of cysteine proteases. This study represents the first comprehensive analysis on the diversity of protease-producing bacteria and their extracellular proteases in sediments of a eutrophic bay. PMID:26441943

  14. Tobacco Etch Virus protease: A shortcut across biotechnologies.

    PubMed

    Cesaratto, Francesca; Burrone, Oscar R; Petris, Gianluca

    2016-08-10

    About thirty years ago, studies on the RNA genome of Tobacco Etch Virus revealed the presence of an efficient and specific protease, called Tobacco Etch Virus protease (TEVp), that was part of the Nuclear Inclusion a (NIa) enzyme. TEVp is an efficient and specific protease of 27kDa that has become a valuable biotechnological tool. Nowadays TEVp is a unique endopeptidase largely exploited in biotechnology from industrial applications to in vitro and in vivo cellular studies. A number of TEVp mutants with different rate of cleavage, stability and specificity have been reported. Similarly, a panel of different target cleavage sites, derived from the canonical ENLYFQ-G/S site, has been established. In this review we describe these aspects of TEVp and some of its multiple applications. A particular focus is on the use and molecular biology of TEVp in living cells and organisms. PMID:27312702

  15. Tobacco Etch Virus protease: A shortcut across biotechnologies.

    PubMed

    Cesaratto, Francesca; Burrone, Oscar R; Petris, Gianluca

    2016-08-10

    About thirty years ago, studies on the RNA genome of Tobacco Etch Virus revealed the presence of an efficient and specific protease, called Tobacco Etch Virus protease (TEVp), that was part of the Nuclear Inclusion a (NIa) enzyme. TEVp is an efficient and specific protease of 27kDa that has become a valuable biotechnological tool. Nowadays TEVp is a unique endopeptidase largely exploited in biotechnology from industrial applications to in vitro and in vivo cellular studies. A number of TEVp mutants with different rate of cleavage, stability and specificity have been reported. Similarly, a panel of different target cleavage sites, derived from the canonical ENLYFQ-G/S site, has been established. In this review we describe these aspects of TEVp and some of its multiple applications. A particular focus is on the use and molecular biology of TEVp in living cells and organisms.

  16. Allostery in trypsin-like proteases suggests new therapeutic strategies.

    PubMed

    Gohara, David W; Di Cera, Enrico

    2011-11-01

    Trypsin-like proteases (TLPs) are a large family of enzymes responsible for digestion, blood coagulation, fibrinolysis, development, fertilization, apoptosis and immunity. A current paradigm posits that the irreversible transition from an inactive zymogen to the active protease form enables productive interaction with substrate and catalysis. Analysis of the entire structural database reveals two distinct conformations of the active site: one fully accessible to substrate (E) and the other occluded by the collapse of a specific segment (E*). The allosteric E*-E equilibrium provides a reversible mechanism for activity and regulation in addition to the irreversible zymogen to protease conversion and points to new therapeutic strategies aimed at inhibiting or activating the enzyme. In this review, we discuss relevant examples, with emphasis on the rational engineering of anticoagulant thrombin mutants.

  17. Alkaline protease from Neurospora crassa. Purification and partial characterization

    SciTech Connect

    Lindberg, R.A.; Eirich, L.D.; Price, J.S.; Wolfinbarger, L. Jr.; Drucker, H.

    1981-01-25

    A simple purification procedure was developed for the extracellular alkaline protease from Neurospora crassa. Key steps in the purification were: (1) the choice of gelatin as the protein inducer, which induces optimally at a much lower concentration than other commonly employed protein inducers; (2) heat treatment, during which the inducer is digested by the protease; and (3) a concentration step that eliminates the usual precipitation procedures and removes much of the digested protein inducer. The preparation was homogeneous and had a molecular weight of approx. 30,500. The protease has 100% activity from pH 6.0 to 10.0, is heat labile above 45/sup 0/C, and susceptible to autodigestion. Hydrolysis of the ..beta.. chain from insulin indicates a preferential cleavage on the carboxyl group side of neutral and aromatic amino acids.

  18. An Augmented Multiple-Protease-Based Human Phosphopeptide Atlas.

    PubMed

    Giansanti, Piero; Aye, Thin Thin; van den Toorn, Henk; Peng, Mao; van Breukelen, Bas; Heck, Albert J R

    2015-06-23

    Although mass-spectrometry-based screens enable thousands of protein phosphorylation sites to be monitored simultaneously, they often do not cover important regulatory sites. Here, we hypothesized that this is due to the fact that nearly all large-scale phosphoproteome studies are initiated by trypsin digestion. We tested this hypothesis using multiple proteases for protein digestion prior to Ti(4+)-IMAC-based enrichment. This approach increases the size of the detectable phosphoproteome substantially and confirms the considerable tryptic bias in public repositories. We define and make available a less biased human phosphopeptide atlas of 37,771 unique phosphopeptides, correlating to 18,430 unique phosphosites, of which fewer than 1/3 were identified in more than one protease data set. We demonstrate that each protein phosphorylation site can be linked to a preferred protease, enhancing its detection by mass spectrometry (MS). For specific sites, this approach increases their detectability by more than 1,000-fold. PMID:26074081

  19. Substrate specificity of the ubiquitin and Ubl proteases

    PubMed Central

    Ronau, Judith A; Beckmann, John F; Hochstrasser, Mark

    2016-01-01

    Conjugation and deconjugation of ubiquitin and ubiquitin-like proteins (Ubls) to cellular proteins are highly regulated processes integral to cellular homeostasis. Most often, the C-termini of these small polypeptides are attached to lysine side chains of target proteins by an amide (isopeptide) linkage. Deubiquitinating enzymes (DUBs) and Ubl-specific proteases (ULPs) comprise a diverse group of proteases that recognize and remove ubiquitin and Ubls from their substrates. How DUBs and ULPs distinguish among different modifiers, or different polymeric forms of these modifiers, remains poorly understood. The specificity of ubiquitin/Ubl-deconjugating enzymes for particular substrates depends on multiple factors, ranging from the topography of specific substrate features, as in different polyubiquitin chain types, to structural elements unique to each enzyme. Here we summarize recent structural and biochemical studies that provide insights into mechanisms of substrate specificity among various DUBs and ULPs. We also discuss the unexpected specificities of non-eukaryotic proteases in these families. PMID:27012468

  20. Peptide synthesis in neat organic solvents with novel thermostable proteases.

    PubMed

    Toplak, Ana; Nuijens, Timo; Quaedflieg, Peter J L M; Wu, Bian; Janssen, Dick B

    2015-06-01

    Biocatalytic peptide synthesis will benefit from enzymes that are active at low water levels in organic solvent compositions that allow good substrate and product solubility. To explore the use of proteases from thermophiles for peptide synthesis under such conditions, putative protease genes of the subtilase class were cloned from Thermus aquaticus and Deinococcus geothermalis and expressed in Escherichia coli. The purified enzymes were highly thermostable and catalyzed efficient peptide bond synthesis at 80°C and 60°C in neat acetonitrile with excellent conversion (>90%). The enzymes tolerated high levels of N,N-dimethylformamide (DMF) as a cosolvent (40-50% v/v), which improved substrate solubility and gave good conversion in 5+3 peptide condensation reactions. The results suggest that proteases from thermophiles can be used for peptide synthesis under harsh reaction conditions.

  1. Protease-triggered siRNA delivery vehicles.

    PubMed

    Rozema, David B; Blokhin, Andrei V; Wakefield, Darren H; Benson, Jonathan D; Carlson, Jeffrey C; Klein, Jason J; Almeida, Lauren J; Nicholas, Anthony L; Hamilton, Holly L; Chu, Qili; Hegge, Julia O; Wong, So C; Trubetskoy, Vladimir S; Hagen, Collin M; Kitas, Eric; Wolff, Jon A; Lewis, David L

    2015-07-10

    The safe and efficacious delivery of membrane impermeable therapeutics requires cytoplasmic access without the toxicity of nonspecific cytoplasmic membrane lysis. We have developed a mechanism for control of cytoplasmic release which utilizes endogenous proteases as a trigger and results in functional delivery of small interfering RNA (siRNA). The delivery approach is based on reversible inhibition of membrane disruptive polymers with protease-sensitive substrates. Proteolytic hydrolysis upon endocytosis restores the membrane destabilizing activity of the polymers thereby allowing cytoplasmic access of the co-delivered siRNA. Protease-sensitive polymer masking reagents derived from polyethylene glycol (PEG), which inhibit membrane interactions, and N-acetylgalactosamine, which targets asialoglycoprotein receptors on hepatocytes, were synthesized and used to formulate masked polymer-siRNA delivery vehicles. The size, charge and stability of the vehicles enable functional delivery of siRNA after subcutaneous administration and, with modification of the targeting ligand, have the potential for extrahepatic targeting.

  2. Membrane-anchored serine proteases in health and disease

    PubMed Central

    Bugge, Thomas; Wu, Qingyu

    2013-01-01

    Serine proteases of the trypsin-like family have long been recognized to be critical effectors of biological processes as diverse as digestion, blood coagulation, fibrinolysis, and immunity. In recent years, a subgroup of these enzymes has been identified that are anchored directly to plasma membranes, either by a carboxy-terminal transmembrane domain (Type I), an amino-terminal transmembrane domain with a cytoplasmic extension (Type II or TTSP), or through a glycosyl-phosphatidylinositol (GPI) linkage. Recent biochemical, cellular, and in vivo analyses have now established that membrane-anchored serine proteases are key pericellular contributors to processes vital for development and the maintenance of homeostasis. This chapter will review our current knowledge of the biological and physiological functions of these proteases, their molecular substrates, and their contributions to disease. PMID:21238933

  3. Acid phosphatase and protease activities in immobilized rat skeletal muscles

    NASA Technical Reports Server (NTRS)

    Witzmann, F. A.; Troup, J. P.; Fitts, R. H.

    1982-01-01

    The effect of hind-limb immobilization on selected Iysosomal enzyme activities was studied in rat hing-limb muscles composed primarily of type 1. 2A, or 2B fibers. Following immobilization, acid protease and acid phosphatase both exhibited signifcant increases in their activity per unit weight in all three fiber types. Acid phosphatase activity increased at day 14 of immobilization in the three muscles and returned to control levels by day 21. Acid protease activity also changed biphasically, displaying a higher and earlier rise than acid phosphatase. The pattern of change in acid protease, but not acid phosphatase, closely parallels observed muscle wasting. The present data therefore demonstrate enhanced proteolytic capacity of all three fiber types early during muscular atrophy. In addition, the data suggest a dependence of basal hydrolytic and proteolytic activities and their adaptive response to immobilization on muscle fiber composition.

  4. Alpha-1 Antitrypsin Deficiency: Beyond the Protease/Antiprotease Paradigm.

    PubMed

    Cosio, Manuel G; Bazzan, Erica; Rigobello, Chiara; Tinè, Mariaenrica; Turato, Graziella; Baraldo, Simonetta; Saetta, Marina

    2016-08-01

    From the discovery that alpha-1 antitrypsin (AAT) was an effective inhibitor of neutrophil elastase originated the classic paradigm of protease/antiprotease imbalance, linking lung destruction to the unopposed effect of proteases in patients with the deficiency. Notwithstanding its importance as an antiprotease, it has become evident that alpha-1 antitrypsin has important antiinflammatory and immune-regulatory activities, which may be critically involved in lung destruction. We review here recent evidence showing that, indeed, an important adaptive immune reaction is present in lungs with AAT deficiency, similar to the one seen in severe chronic obstructive pulmonary disease with normal AAT. On the basis of recent evidence from epidemiological, clinical, and pathogenetic studies, it is likely time to move on from the original protease/antiprotease hypothesis for the production of emphysema toward a more complex paradigm, involving the antiinflammatory and immune modulating functions of AAT. PMID:27564665

  5. (Processing and targeting of the thiol protease aleurain)

    SciTech Connect

    Rogers, J.C.

    1990-01-01

    Our goal for work during the past two years under this Grant was to characterize the barley thiol protease, aleurain, to determine if it is secreted or retained intracellularly in aleurone cells, and to begin to elucidate structural features that might control targeting of the protein to its final destination. We have shown that aleurain is synthesized as a proenzyme with two N-linked oligosaccharide chains, one high mannose-type and one complex-type. Aleurain undergoes processing to mature form by removal of an Nterminal prosegment, and is retained intracellularly; it cannot be detected among proteins secreted from aleurone cells. Treatment of aleurone cells with tunicamycin to prevent glycosylation of aleurain does not prevent processing of the unglycosylated form. The N-terminal portion of aleurain's prosegment is homologous to the comparable region in two yeast vacuolar proteases, where that region is known to contain the signal necessary for targeting the proteases to the vacuole. 18 refs., 7 figs.

  6. Genetic Changes in HIV-1 Gag-Protease Associated with Protease Inhibitor-Based Therapy Failure in Pediatric Patients

    PubMed Central

    Giandhari, Jennifer; Basson, Adriaan E.; Coovadia, Ashraf; Kuhn, Louise; Abrams, Elaine J.; Strehlau, Renate; Morris, Lynn

    2015-01-01

    Abstract Studies have shown a low frequency of HIV-1 protease drug resistance mutations in patients failing protease inhibitor (PI)-based therapy. Recent studies have identified mutations in Gag as an alternate pathway for PI drug resistance in subtype B viruses. We therefore genotyped the Gag and protease genes from 20 HIV-1 subtype C-infected pediatric patients failing a PI-based regimen. Major protease resistance mutations (M46I, I54V, and V82A) were identified in eight (40%) patients, as well as Gag cleavage site (CS) mutations (at codons 373, 374, 378, 428, 431, 449, 451, and 453) in nine (45%) patients. Four of these Gag CS mutations occurred in the absence of major protease mutations at PI failure. In addition, amino acid changes were noted at Gag non-CS with some predicted to be under HLA/KIR immune-mediated pressure and/or drug selection pressure. Changes in Gag during PI failure therefore warrant further investigation of the Gag gene and its role in PI failure in HIV-1 subtype C infection. PMID:25919760

  7. Gag-Protease Sequence Evolution Following Protease Inhibitor Monotherapy Treatment Failure in HIV-1 Viruses Circulating in East Africa.

    PubMed

    Sutherland, Katherine A; Goodall, Ruth L; McCormick, Adele; Kapaata, Anne; Lyagoba, Fred; Kaleebu, Pontiano; Thiltgen, Geant; Gilks, Charles F; Spyer, Moira; Kityo, Cissy; Pillay, Deenan; Dunn, David; Gupta, Ravindra K

    2015-10-01

    Around 2.5 million HIV-infected individuals failing first-line therapy qualify for boosted protease inhibitor (bPI)-based second-line therapy globally. Major resistance mutations are rarely present at treatment failure in patients receiving bPI and the determinants of failure in these patients remain unknown. There is evidence that Gag can impact PI susceptibility. Here, we have sequenced Gag-Protease before and following failure in 23 patients in the SARA trial infected with subtypes A, C, and D viruses. Before bPI, significant variation in Protease and Gag was observed at positions previously associated with PI exposure and resistance including Gag mutations L449P, S451N, and L453P and Protease K20I and L63P. Following PI failure, previously described mutations in Protease and Gag were observed, including those at the cleavage sites such as R361K and P453L. However, the emergence of clear genetic determinants of therapy failure across patients was not observed. Larger Gag sequence datasets will be required to comprehensively identify mutational correlates of bPI failure across subtypes. PMID:26258548

  8. Alkyl hydroxybenzoic acid derivatives that inhibit HIV-1 protease dimerization.

    PubMed

    Flausino, O A; Dufau, L; Regasini, L O; Petrônio, M S; Silva, D H S; Rose, T; Bolzani, V S; Reboud-Ravaux, M

    2012-01-01

    The therapeutic potential of gallic acid and its derivatives as anti-cancer, antimicrobial and antiviral agents is well known. We have examined the mechanism by which natural gallic acid and newly synthesized gallic acid alkyl esters and related protocatechuic acid alkyl esters inhibit HIV-1 protease to compare the influence of the aromatic ring substitutions on inhibition. We used Zhang-Poorman's kinetic analysis and fluorescent probe binding to demonstrate that several gallic and protecatechuic acid alkyl esters inhibited HIV-1 protease by preventing the dimerization of this obligate homodimeric aspartic protease rather than targeting the active site. The tri-hydroxy substituted benzoic moiety in gallates was more favorable than the di-substituted one in protocatechuates. In both series, the type of inhibition, its mechanism and the inhibitory efficiency dramatically depended on the length of the alkyl chain: no inhibition with alkyl chains less than 8 carbon atoms long. Molecular dynamics simulations corroborated the kinetic data and propose that gallic esters are intercalated between the two N- and C-monomer ends. They complete the β-sheet and disrupt the dimeric enzyme. The best gallic ester (14 carbon atoms, K(id) of 320 nM) also inhibited the multi-mutated protease MDR-HM. These results will aid the rational design of future generations of non-peptide inhibitors of HIV-1 protease dimerization that inhibit multi-mutated proteases. Finally, our work suggests the wide use of gallic and protocatechuic alkyl esters to dissociate intermolecular β-sheets involved in protein-protein interactions.

  9. Analysis of the immunoglobulin A protease gene of Streptococcus sanguis.

    PubMed Central

    Gilbert, J V; Plaut, A G; Wright, A

    1991-01-01

    The amino acid sequence T-P-P-T-P-S-P-S is tandemly duplicated in the heavy chain of human immunoglobulin A1 (IgA1), the major antibody in secretions. The bacterial pathogen Streptococcus sanguis, a precursor to dental caries and a cause of bacterial endocarditis, yields IgA protease that cleaves only the Pro-Thr peptide bond in the left duplication, while the type 2 IgA proteases of the genital pathogen Neisseria gonorrhoeae and the respiratory pathogen Haemophilus influenzae cleave only the P-T bond in the right half. We have sequenced the entire S. sanguis iga gene cloned into Escherichia coli. A segment consisting of 20 amino acids tandemly repeated 10 times, of unknown function, occurs near the amino-terminal end of the enzyme encoded in E. coli. Identification of a predicted zinc-binding region in the S. sanguis enzyme and the demonstration that mutations in this region result in production of a catalytically inactive protein support the idea that the enzyme is a metalloprotease. The N. gonorrhoeae and H. influenzae enzymes were earlier shown to be serine-type proteases, while the Bacteroides melaninogenicus IgA protease was shown to be a cysteine-type enzyme. The streptococcal IgA protease amino acid sequence has no significant homology with either of the two previously determined IgA protease sequences, that of type 2 N. gonorrhoeae and type 1 H. influenzae. The differences in both structure and mechanism among these functionally analogous enzymes underscore their role in the infectious process and offer some prospect of therapeutic intervention. Images PMID:1987065

  10. [Isolation of Actinomycetes synthesizing proteases with thrombolytic activity].

    PubMed

    Lysenko, S V; Salivonik, S M

    1988-01-01

    Proteases with the thrombolytic activity were studied in 212 strains of actinomycetes isolated from different soils of the Soviet Union. The cultures belonged to the genera Micromonospora, Nocardia and Streptomyces. Proteases were synthesized by 41% of the studied actinomycetes and some of their strains completely dissolved in vitro artificially obtained blood thrombi within 120-240 min. In the Streptomyces genus, more active strains were found in the groups Flavus, Fradia and Globisporus. The groups Olivaceus, Violaceus and Viridis had less active strains. PMID:3062331

  11. The chlamydial protease CPAF: important or not, important for what?

    PubMed

    Häcker, Georg

    2014-05-01

    The protease CPAF is only found in Chlamydiales and in at least most bacteria that share with Chlamydia the biphasic life-style in a cytosolic inclusion. CPAF is intriguing: it appears to be secreted from the inclusion across the inclusion membrane into the cytosol. A bacterial protease ravaging in the cytosol of a human cell may cause a plethora of effects. Curiously, very few are known. The current discussion is bogged down by a focus on experimental artifact, while proposed functions of CPAF remain speculative. I here make the attempt to summarize what we know about CPAF.

  12. Rhomboid proteases in mitochondria and plastids: keeping organelles in shape.

    PubMed

    Jeyaraju, Danny V; Sood, Aditi; Laforce-Lavoie, Audrey; Pellegrini, Luca

    2013-02-01

    Rhomboids constitute the most widespread and conserved family of intramembrane cleaving proteases. They are key regulators of critical cellular processes in bacteria and animals, and are poised to play an equally important role also in plants. Among eukaryotes, a distinct subfamily of rhomboids, prototyped by the mammalian mitochondrial protein Parl, ensures the maintenance of the structural and functional integrity of mitochondria and plastids. Here, we discuss the studies that in the past decade have unveiled the role, regulation, and structure of this unique group of rhomboid proteases. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.

  13. Gastrointestinal absorption and biological activities of serine and cysteine proteases of animal and plant origin: review on absorption of serine and cysteine proteases.

    PubMed

    Lorkowski, Gerhard

    2012-01-01

    Research has confirmed that peptides and larger protein molecules pass through the mucosal barrier of the gastrointestinal tract. Orally administered serine and cysteine proteases of plant and animal origin also reach blood and lymph as intact, high molecular weight and physiologically active protein molecules. Their absorption may be supported by a self-enhanced paracellular transport mechanism resulting in sub-nanomolar concentration of transiently free protease molecules or, in a complex with anti-proteases, at higher concentrations. Data from pharmacokinetic investigations reveals dose linearity for maximum plasma levels of free proteases not unusual for body proteases and a high inter-individual variability. There is no interference with each other after oral administration of protease combinations, and absorption follows an unusual invasion and elimination kinetic due to slow velocity of absorption and a fast 100% protein binding to anti-proteases. Oral application of proteases leads to increased proteolytic serum activity and increased plasma concentrations of the corresponding anti-proteases. Their biological activity is determined by their proteolytic activity as free proteases on soluble peptides/proteins or cell surface receptors (e.g. protease activated receptors) and their activity in the complex formed with their specific and/or unspecific anti-proteases. The anti-protease-complexes, during immune reaction and injuries often loaded with different cytokines, are cleared from body fluids and tissue by receptor mediated endocytosis on hepatocytes and/or blood cells. Oral administration of enteric coated tablets containing proteolytic enzymes of plant and animal origin may be a safe method to stabilize, positively influence or enhance physiological and immunological processes during disease processes and in healthy consumers.

  14. Gastrointestinal absorption and biological activities of serine and cysteine proteases of animal and plant origin: review on absorption of serine and cysteine proteases

    PubMed Central

    Lorkowski, Gerhard

    2012-01-01

    Research has confirmed that peptides and larger protein molecules pass through the mucosal barrier of the gastrointestinal tract. Orally administered serine and cysteine proteases of plant and animal origin also reach blood and lymph as intact, high molecular weight and physiologically active protein molecules. Their absorption may be supported by a self-enhanced paracellular transport mechanism resulting in sub-nanomolar concentration of transiently free protease molecules or, in a complex with anti-proteases, at higher concentrations. Data from pharmacokinetic investigations reveals dose linearity for maximum plasma levels of free proteases not unusual for body proteases and a high inter-individual variability. There is no interference with each other after oral administration of protease combinations, and absorption follows an unusual invasion and elimination kinetic due to slow velocity of absorption and a fast 100% protein binding to anti-proteases. Oral application of proteases leads to increased proteolytic serum activity and increased plasma concentrations of the corresponding anti-proteases. Their biological activity is determined by their proteolytic activity as free proteases on soluble peptides/proteins or cell surface receptors (e.g. protease activated receptors) and their activity in the complex formed with their specific and/or unspecific anti-proteases. The anti-protease-complexes, during immune reaction and injuries often loaded with different cytokines, are cleared from body fluids and tissue by receptor mediated endocytosis on hepatocytes and/or blood cells. Oral administration of enteric coated tablets containing proteolytic enzymes of plant and animal origin may be a safe method to stabilize, positively influence or enhance physiological and immunological processes during disease processes and in healthy consumers. PMID:22461953

  15. Genetic Evidence Supporting the Association of Protease and Protease Inhibitor Genes with Inflammatory Bowel Disease: A Systematic Review

    PubMed Central

    Bekkering, Geertruida E.; Nüesch, Eveline; Mendes, Camila T.; Schmied, Stefanie; Wyder, Stefan; Kellen, Eliane; Villiger, Peter M.; Rutgeerts, Paul; Vermeire, Séverine; Lottaz, Daniel

    2011-01-01

    As part of the European research consortium IBDase, we addressed the role of proteases and protease inhibitors (P/PIs) in inflammatory bowel disease (IBD), characterized by chronic mucosal inflammation of the gastrointestinal tract, which affects 2.2 million people in Europe and 1.4 million people in North America. We systematically reviewed all published genetic studies on populations of European ancestry (67 studies on Crohn's disease [CD] and 37 studies on ulcerative colitis [UC]) to identify critical genomic regions associated with IBD. We developed a computer algorithm to map the 807 P/PI genes with exact genomic locations listed in the MEROPS database of peptidases onto these critical regions and to rank P/PI genes according to the accumulated evidence for their association with CD and UC. 82 P/PI genes (75 coding for proteases and 7 coding for protease inhibitors) were retained for CD based on the accumulated evidence. The cylindromatosis/turban tumor syndrome gene (CYLD) on chromosome 16 ranked highest, followed by acylaminoacyl-peptidase (APEH), dystroglycan (DAG1), macrophage-stimulating protein (MST1) and ubiquitin-specific peptidase 4 (USP4), all located on chromosome 3. For UC, 18 P/PI genes were retained (14 proteases and 4protease inhibitors), with a considerably lower amount of accumulated evidence. The ranking of P/PI genes as established in this systematic review is currently used to guide validation studies of candidate P/PI genes, and their functional characterization in interdisciplinary mechanistic studies in vitro and in vivo as part of IBDase. The approach used here overcomes some of the problems encountered when subjectively selecting genes for further evaluation and could be applied to any complex disease and gene family. PMID:21931648

  16. Teaching Foundational Topics and Scientific Skills in Biochemistry within the Conceptual Framework of HIV Protease

    ERIC Educational Resources Information Center

    Johnson, R. Jeremy

    2014-01-01

    HIV protease has served as a model protein for understanding protein structure, enzyme kinetics, structure-based drug design, and protein evolution. Inhibitors of HIV protease are also an essential part of effective HIV/AIDS treatment and have provided great societal benefits. The broad applications for HIV protease and its inhibitors make it a…

  17. A New Subtilase-Like Protease Deriving from Fusarium equiseti with High Potential for Industrial Applications.

    PubMed

    Juntunen, Kari; Mäkinen, Susanna; Isoniemi, Sari; Valtakari, Leena; Pelzer, Alexander; Jänis, Janne; Paloheimo, Marja

    2015-09-01

    A gene encoding a novel extracellular subtilisin-like protease was cloned from the ascomycete Fusarium equiseti and expressed in Trichoderma reesei. The F. equiseti protease (Fe protease) showed excellent performance in stain removal and good compatibility with several commercial laundry detergent formulations, suggesting that it has high potential for use in various industrial applications. The recombinant enzyme was purified and characterized. The temperature optimum of the Fe protease was 60 °C and it showed high activity in the pH range of 6-10, with a sharp decline in activity at pH above 10. The amino acid specificity of the Fe protease was studied using casein, cytochrome c, and ubiquitin as substrates. The Fe protease had broad substrate specificity: almost all amino acid residues were accepted at position P1, even though it showed some preference for cleavage at the C-terminal side of asparagine and histidine residues. The S4 subsite of Fe protease favors aspartic acid and threonine. The other well-characterized proteases from filamentous fungi, Proteinase K from Engyodontium album, Thermomycolin from Malbranchea sulfurea, and alkaline subtilisins from Bacillus species prefer hydrophobic amino acids in both the S1 and S4 subsites. Due to its different specificity compared to the members of the S8 family of clan SB of proteases, we consider that the Fe protease is a new protease. It does not belong to any previously defined IUBMB groups of proteases.

  18. Optimized virus disruption improves detection of HIV-1 p24 in particles and uncovers a p24 reactivity in patients with undetectable HIV-1 RNA under long-term HAART.

    PubMed

    Schüpbach, Jörg; Tomasik, Zuzana; Knuchel, Marlyse; Opravil, Milos; Günthard, Huldrych F; Nadal, David; Böni, Jürg

    2006-08-01

    HIV-1 p24 antigen (p24) measurement by signal amplification-boosted ELISA of heat-denatured plasma is being evaluated as an alternative to HIV-1 RNA quantitation in resource-poor settings. Some observations suggested that virion-associated p24 is suboptimally detected using Triton X-100-based virus dissociation buffer (kit buffer). A new reagent (SNCR buffer) containing both denaturing and non-denaturing detergents was therefore developed and evaluated. The SNCR buffer increased the measured p24 concentration about 1.5- to 3-fold in HIV-negative plasma reconstituted with purified HIV-1 particles, while not increasing the background. Among 127 samples of HIV-1-positive patients with moderate to high concentrations of HIV-1 RNA the increase was about threefold across the entire concentration range (P < 0.0001). Specificity before neutralization among prospectively tested clinical samples ruled HIV-negative was 828 of 845 (98.0%) for the SNCR buffer and 464 of 479 (96.9%) for kit buffer. Specificity after confirmatory neutralization of reactive samples or a follow-up test was 100% with either buffer. Surprisingly, the SNCR buffer revealed a p24 reactivity in 115 of 187 samples (61.5%) from adult patients exhibiting undetectable HIV-1 RNA below 5 copies/ml for a duration of 6-30 months under HAART (3.7% with kit buffer). The rate of p24 reactivity in these patients did not decrease with duration of HAART. In conclusion, the SNCR buffer improves the detection of particle-associated HIV-1 p24, thereby increasing the measured p24 concentration in samples with medium to high HIV-1 RNA. It also uncovers the presence of a p24 reactivity, whose identity remains to be determined, in a significant fraction of samples with undetectable HIV-1 RNA under long-term HAART.

  19. Protease production by immobilized mycelia of Streptomyces fradiae

    SciTech Connect

    Kokubu, T.; Karube, I.; Suzuki, S.

    1981-01-01

    Streptomyces fradiae was immobilized in polyacrylamide gel prepared from 5% total acrylamide (90% acrylamide and 10% N,N-methylenebisacrylamide). Production of protease by the immobilized mycelia was attempted in a batch system. A dilute medium containing 0.5% starch, 0.5% meat extract, and 0.5% yeast extract was employed. The reusability of the immobilized and washed mycelia was examined. The activity of protease production by washed mycelia was rapidly decreased with increasing use cycles. The activity of the immobilized mycelia increased gradually, and reached a maximum after ten use cycles. Then, the activity gradually decreased with increasing reaction cycles. This might be caused by destruction of the gels. On the other hand, the sterilization of the surface of the immobilized mycelia was effective for elongation of the lifetime. As a result, the half-life of protease production by the sterilized immobilized mycelia was about 30 days. The rate of protease production by immobilized mycelia was 12,000 U/ml/hr. This value was four times higher than that by submerged culture.

  20. Purification and characterization of a pineapple crown leaf thiol protease.

    PubMed

    Singh, L Rupachandra; Devi, Th Premila; Devi, S Kunjeshwori

    2004-02-01

    A thiol protease was isolated and purified from the crown leaf of pineapple, Ananas comosus (L.) Merr. cv. Queen, by an immunoaffinity procedure. After the purification to electrophoretic homogeneity, the enzyme was characterized with respect to some of its physico-chemical and kinetic properties. The molecular weight of the protease (22.4-22.9 kDa), Km (97 microM) and kcat (8.8 s(-1)) for its esterolytic cleavage of the synthetic protease substrate N(alpha)-CBZ-L-lysine p-nitrophenyl ester, the concentration of its thiol activator L-cysteine required for half maximal activation A0.5 (9.9 microM), optimum pH (6.5) for its proteolytic action on azocasein, T(1/2) (60 degrees C) for inactivation by heating the enzyme (35.5 microg protein/mL) in citrate buffer pH 6.0 for 15 min, and SH-group content (0.98 mol/mol enzyme) were determined. Most of these physicochemical and kinetic properties were found to be similar to those of the already well-characterized stem bromelain (EC 3.4.22.32). Thus, the immunoaffinity purified crown leaf protease appeared to be closely related to stem bromelain.

  1. Secretory immunity and the bacterial IgA proteases.

    PubMed

    Kornfeld, S J; Plaut, A G

    1981-01-01

    The characteristics and functions of microbial IgA proteases are reviewed. These enzymes represent a structurally heterogeneous group of proteins that are secreted into the extracellular environment by bacteria capable of causing human disease. The IgA proteases, which vary in their requirements for metal ions, are neutral endopeptidases whose role in the infectious process is not known but whose pronounced substrate specificity for human proteins of the IgA1 subclass has repeatedly been demonstrated. As reagents, the IgA proteases are useful in cleaving IgA molecules to yield intact Fc alpha and Fab alpha fragments that will allow the study of the structure and function of the two large regions of IgA immunoglobulin proteins. The role, if any, of these enzymes in promoting infection by pathogenic members of the genera Neisseria, Hemophilus, and Streptococcus is not known, although the secretory immune system is primarily mediated by antibodies of the IgA isotype, among which are IgA1 subclass proteins, and these proteins are susceptible to cleavage by IgA protease. The determination of the role of these enzymes in the pathogenesis of human infection must await clearer understanding of antigenicity and antibody function at secretory sites and of the relative roles of the two subclasses of human IgA in immune defense.

  2. Processing and targeting of the thiol protease aleurain: Progress report

    SciTech Connect

    Rogers, J.C.

    1988-01-01

    This study addresses the processing and targeting of the thiol protease aleurain in monocots. A probe derived from the aleurain cDNA specific for the 5'-most 400 bp (a region encoding the first 140 amino acids of the preprotein hybridized to at least 3 separate elements in the barley genome; only one represented the aleurain gene. In contrast, a probe specific for the remaining 2/23 of the cDNA (representing the protease domain) hybridized to only a single copy sequence. To know if this pattern pertained in other, closely related, monocots, we probed Southern blots of genomic DNA from maize, rye, oats, sorghum, and pearl millet with each probe. In each instance except for maize DNA, the 5' domain probe hybridizes to several fragments in addition to those identified by the protease domain probe. Presumable the darkest hybridization in each represents the fragment carrying the sequences homologous to barley aleurain. The fragments from a given restriction enzyme identified by the protease domain probe in sorghum, millet, and maize, were indistinguishable in size indicating that the gene sequences, as well as flanking DNA, are so well conserved among the group that the location of the hexanucleotide sequences have not diverged. (3 refs., 3 figs.)

  3. Post-translational control of genetic circuits using Potyvirus proteases.

    PubMed

    Fernandez-Rodriguez, Jesus; Voigt, Christopher A

    2016-07-27

    Genetic engineering projects often require control over when a protein is degraded. To this end, we use a fusion between a degron and an inactivating peptide that can be added to the N-terminus of a protein. When the corresponding protease is expressed, it cleaves the peptide and the protein is degraded. Three protease:cleavage site pairs from Potyvirus are shown to be orthogonal and active in exposing degrons, releasing inhibitory domains and cleaving polyproteins. This toolbox is applied to the design of genetic circuits as a means to control regulator activity and degradation. First, we demonstrate that a gate can be constructed by constitutively expressing an inactivated repressor and having an input promoter drive the expression of the protease. It is also shown that the proteolytic release of an inhibitory domain can improve the dynamic range of a transcriptional gate (200-fold repression). Next, we design polyproteins containing multiple repressors and show that their cleavage can be used to control multiple outputs. Finally, we demonstrate that the dynamic range of an output can be improved (8-fold to 190-fold) with the addition of a protease-cleaved degron. Thus, controllable proteolysis offers a powerful tool for modulating and expanding the function of synthetic gene circuits. PMID:27298256

  4. Generic protease detection technology for monitoring periodontal disease.

    PubMed

    Zheng, Xinwei; Cook, Joseph P; Watkinson, Michael; Yang, Shoufeng; Douglas, Ian; Rawlinson, Andrew; Krause, Steffi

    2011-01-01

    Periodontal diseases are inflammatory conditions that affect the supporting tissues of teeth and can lead to destruction of the bone support and ultimately tooth loss if untreated. Progression of periodontitis is usually site specific but not uniform, and currently there are no accurate clinical methods for distinguishing sites where there is active disease progression from sites that are quiescent. Consequently, unnecessary and costly treatment of periodontal sites that are not progressing may occur. Three proteases have been identified as suitable markers for distinguishing sites with active disease progression and quiescent sites: human neutrophil elastase, cathepsin G and MMP8. Generic sensor materials for the detection of these three proteases have been developed based on thin dextran hydrogel films cross-linked with peptides. Degradation of the hydrogel films was monitored using impedance measurements. The target proteases were detected in the clinically relevant range within a time frame of 3 min. Good specificity for different proteases was achieved by choosing appropriate peptide cross-linkers.

  5. THE ROLE OF CYSTEINE PROTEASE IN ALZHEIMER DISEASE

    PubMed Central

    Hasanbasic, Samra; Jahic, Alma; Karahmet, Emina; Sejranic, Asja; Prnjavorac, Besim

    2016-01-01

    Introduction: Cysteine protease are biological catalysts which play a pivotal role in numerous biological reactions in organism. Much of the literature is inscribed to their biochemical significance, distribution and mechanism of action. Many diseases, e.g. Alzheimer’s disease, develop due to enzyme balance disruption. Understanding of cysteine protease’s disbalance is therefor a key to unravel the new possibilities of treatment. Cysteine protease are one of the most important enzymes for protein disruption during programmed cell death. Whether protein disruption is part of cell deaths is not enough clear in any cases. Thereafter, any tissue disruption, including proteolysis, generate more or less inflammation appearance. Review: This review briefly summarizes the current knowledge about pathological mechanism’s that results in AD, with significant reference to the role of cysteine protease in it. Based on the summary, new pharmacological approach and development of novel potent drugs with selective toxicity targeting cysteine protease will be a major challenge in years to come. PMID:27482169

  6. Design, synthesis, and activity of nanocellulosic protease sensors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we contrast the molecular assembly, and biochemical utility of nanocellulosic materials prepared from cotton and wood as protease sensors. The cotton-based nanocellulosic substrates were prepared in a variety of ways to produce nanocrystals, films and aerogels, which were derivatized with eithe...

  7. graal: a Drosophila gene coding for several mosaic serine proteases.

    PubMed

    Munier, Anne Isabelle; Medzhitov, Ruslan; Janeway, Charles A; Doucet, Daniel; Capovilla, Maria; Lagueux, Marie

    2004-10-01

    Serine proteases play vital roles in several biological processes such as development and immunity. We have characterized Graal, a large multi-domain serine protease from Drosophila. Graal is spliced in at least three transcripts that are present throughout development. The domains found in Graal proteins are: chitin-binding domains (CBD), scavenger receptor cysteine-rich (SRCR) domains, low density lipoprotein receptor cysteine-rich (LDLR-CR) domains, histidine and proline-rich domains, a NGGYQPP-repeat domain and a serine protease domain. The last 2370 nucleotides of these RNAs are identical and encode a His-rich domain, two SRCR domains, two LDLR-CR domains and a protease domain. The transcription of graal is upregulated after fungal or bacterial infection. Analysis of the Iso1 (y;cn,sp,bw) strain shows that graal transcription is impaired in this fly line due to the insertion of a retrotransposon in the sixth exon. However, no phenotype could be observed consecutive to the absence of graal full length transcripts, particularly in the context of an immune challenge.

  8. Differential protease activity augments polyphagy in Helicoverpa armigera.

    PubMed

    Chikate, Y R; Tamhane, V A; Joshi, R S; Gupta, V S; Giri, A P

    2013-06-01

    Helicoverpa armigera (Lepidoptera: Noctuidae) and other polyphagous agricultural pests are extending their plant host range and emerging as serious agents in restraining crop productivity. Dynamic regulation, coupled with a diversity of digestive and detoxifying enzymes, play a crucial role in the adaptation of polyphagous insects. To investigate the functional intricacy of serine proteases in the development and polyphagy of H. armigera, we profiled the expression of eight trypsin-like and four chymotrypsin-like phylogenetically diverse mRNAs from different life stages of H. armigera reared on nutritionally distinct host plants. These analyses revealed diet- and stage-specific protease expression patterns. The trypsins expressed showed structural variations, which might result in differential substrate specificity and interaction with inhibitors. Protease profiles in the presence of inhibitors and their mass spectrometric analyses revealed insight into their differential activity. These findings emphasize the differential expression of serine proteases and their consequences for digestive physiology in promoting polyphagy in H. armigera. PMID:23432026

  9. Recent trends in protease-catalyzed peptide synthesis.

    PubMed

    Lombard, C; Saulnier, J; Wallach, J M

    2005-10-01

    Enzymatic peptide syntheses may be either thermodynamically- or kinetically-controlled. The former may be catalyzed by any proteases; the latter is limited to serine and cysteine proteases. This methodology is quite stereospecific and avoids side chain protection but is suffering of some drawbacks. Thus, only two industrial processes are by now developed: the production of aspartame and the conversion of porcine into human insulin. However, recent improvements have been carried out in different directions: 1-Search for proteases with high and/or new P'1 and P1 specificities. 2-Protease engineering to promote synthesis towards hydrolysis and to enlarge specificity. 3-Development of mimetic or "inverse" substrates to limit further hydrolysis of synthesized peptide. 4-Change of the physical state of reactants. Three axes have mainly be explored: solid-solid conversion, use of cross-linked enzyme crystals (CLEC) and enzyme immobilization. 5-Modification of experimental conditions. The principal and recent developments deal with: heterogeneous catalysis, synthesis in low water-containing organic solvents, in ionic liquids or at subzero temperatures. This review will illustrate these new orientations with examples described in the recent literature.

  10. Botulinum neurotoxin: a deadly protease with applications to human medicine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins (BoNTs) are some of the most potent biological toxins to humans. They are synthesized by the gram-positive, spore-forming bacterium Clostridium botulinum. BoNT is secreted from the bacterium as a ~150 kDa polypeptide which is cleaved by bacterial or host proteases into a ~50 kD...

  11. Protease-activated-receptor-2 affects protease-activated-receptor-1-driven breast cancer.

    PubMed

    Jaber, Mohammad; Maoz, Miriam; Kancharla, Arun; Agranovich, Daniel; Peretz, Tamar; Grisaru-Granovsky, Sorina; Uziely, Beatrice; Bar-Shavit, Rachel

    2014-07-01

    Mammalian protease-activated-receptor-1 and -2 (PAR1 and PAR2) are activated by proteases found in the flexible microenvironment of a tumor and play a central role in breast cancer. We propose in the present study that PAR1 and PAR2 act together as a functional unit during malignant and physiological invasion processes. This notion is supported by assessing pro-tumor functions in the presence of short hairpin; shRNA knocked-down hPar2 or by the use of a truncated PAR2 devoid of the entire cytoplasmic tail. Silencing of hPar2 by shRNA-attenuated thrombin induced PAR1 signaling as recapitulated by inhibiting the assembly of Etk/Bmx or Akt onto PAR1-C-tail, by thrombin-instigated colony formation and invasion. Strikingly, shRNA-hPar2 also inhibited the TFLLRN selective PAR1 pro-tumor functions. In addition, while evaluating the physiological invasion process of placenta extravillous trophoblast (EVT) organ culture, we observed inhibition of both thrombin or the selective PAR1 ligand; TFLLRNPNDK induced EVT invasion by shRNA-hPar2 but not by scrambled shRNA-hPar2. In parallel, when a truncated PAR2 was utilized in a xenograft mouse model, it inhibited PAR1-PAR2-driven tumor growth in vivo. Similarly, it also attenuated the interaction of Etk/Bmx with the PAR1-C-tail in vitro and decreased markedly selective PAR1-induced Matrigel invasion. Confocal images demonstrated co-localization of PAR1 and PAR2 in HEK293T cells over-expressing YFP-hPar2 and HA-hPar1. Co-immuno-precipitation analyses revealed PAR1-PAR2 complex formation but no PAR1-CXCR4 complex was formed. Taken together, our observations show that PAR1 and PAR2 act as a functional unit in tumor development and placenta-uterus interactions. This conclusion may have significant consequences on future breast cancer therapeutic modalities and improved late pregnancy outcome. PMID:24177339

  12. In-cell protease assay systems based on trans-localizing molecular beacon proteins using HCV protease as a model system.

    PubMed

    Kim, Jeong Hee; Lee, Min Jun; Hwang, Inhwan; Hwang, Hyun Jin

    2013-01-01

    This study describes a sensitive in-cell protease detection system that enables direct fluorescence detection of a target protease and its inhibition inside living cells. This live-cell imaging system provides a fluorescent molecular beacon protein comprised of an intracellular translocation signal sequence, a protease-specific cleavage sequence, and a fluorescent tag sequence(s). The molecular beacon protein is designed to change its intracellular localization upon cleavage by a target protease, i.e., from the cytosol to a subcellular organelle or from a subcellular organelle to the cytosol. Protease activity can be monitored at the single cell level, and accordingly the entire cell population expressing the protease can be accurately enumerated. The clear cellular change in fluorescence pattern makes this system an ideal tool for various life science and drug discovery research, including high throughput and high content screening applications.

  13. The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis.

    PubMed

    Beers, Eric P; Jones, Alan M; Dickerman, Allan W

    2004-01-01

    The Arabidopsis thaliana genome has over 550 protease sequences representing all five catalytic types: serine, cysteine, aspartic acid, metallo and threonine (MEROPS peptidase database, http://merops.sanger.ac.uk/), which probably reflect a wide variety of as yet unidentified functions performed by plant proteases. Recent indications that the 26S proteasome, a T1 family-threonine protease, is a regulator of light and hormone responsive signal transduction highlight the potential of proteases to participate in many aspects of plant growth and development. Recent discoveries that proteases are required for stomatal distribution, embryo development and disease resistance point to wider roles for four additional multigene families that include some of the most frequently studied (yet poorly understood) plant proteases: the subtilisin-like, serine proteases (family S8), the papain-like, cysteine proteases (family C1A), the pepsin-like, aspartic proteases (family A1) and the plant matrixin, metalloproteases (family M10A). In this report, 54 subtilisin-like, 30 papain-like and 59 pepsin-like proteases from Arabidopsis, are compared with S8, C1A and A1 proteases known from other plant species at the functional, phylogenetic and gene structure levels. Examples of structural conservation between S8, C1A and A1 genes from rice, barley, tomato and soybean and those from Arabidopsis are noted, indicating that some common, essential plant protease roles were established before the divergence of monocots and eudicots. Numerous examples of tandem duplications of protease genes and evidence for a variety of restricted expression patterns suggest that a high degree of specialization exists among proteases within each family. We propose that comprehensive analysis of the functions of these genes in Arabidopsis will firmly establish serine, cysteine and aspartic proteases as regulators and effectors of a wide range of plant processes.

  14. Structural Mechanisms of Inactivation in Scabies Mite Serine Protease Paralogues

    SciTech Connect

    Fischer, Katja; Langendorf, Christopher G.; Irving, James A.; Reynolds, Simone; Willis, Charlene; Beckham, Simone; Law, Ruby H.P.; Yang, Sundy; Bashtannyk-Puhalovich, Tanya A.; McGowan, Sheena; Whisstock, James C.; Pike, Robert N.; Kemp, David J.; Buckle, Ashley M.

    2009-08-07

    The scabies mite (Sarcoptes scabiei) is a parasite responsible for major morbidity in disadvantaged communities and immuno-compromised patients worldwide. In addition to the physical discomfort caused by the disease, scabies infestations facilitate infection by Streptococcal species via skin lesions, resulting in a high prevalence of rheumatic fever/heart disease in affected communities. The scabies mite produces 33 proteins that are closely related to those in the dust mite group 3 allergen and belong to the S1-like protease family (chymotrypsin-like). However, all but one of these molecules contain mutations in the conserved active-site catalytic triad that are predicted to render them catalytically inactive. These molecules are thus termed scabies mite inactivated protease paralogues (SMIPPs). The precise function of SMIPPs is unclear; however, it has been suggested that these proteins might function by binding and protecting target substrates from cleavage by host immune proteases, thus preventing the host from mounting an effective immune challenge. In order to begin to understand the structural basis for SMIPP function, we solved the crystal structures of SMIPP-S-I1 and SMIPP-S-D1 at 1.85 {angstrom} and 2.0 {angstrom} resolution, respectively. Both structures adopt the characteristic serine protease fold, albeit with large structural variations over much of the molecule. In both structures, mutations in the catalytic triad together with occlusion of the S1 subsite by a conserved Tyr200 residue is predicted to block substrate ingress. Accordingly, we show that both proteases lack catalytic function. Attempts to restore function (via site-directed mutagenesis of catalytic residues as well as Tyr200) were unsuccessful. Taken together, these data suggest that SMIPPs have lost the ability to bind substrates in a classical 'canonical' fashion, and instead have evolved alternative functions in the lifecycle of the scabies mite.

  15. Nematicidal Bacteria Associated to Pinewood Nematode Produce Extracellular Proteases

    PubMed Central

    Francisco, Romeu; Verissimo, Paula; Santos, Susana S.; Fonseca, Luís; Abrantes, Isabel M. O.; Morais, Paula V.

    2013-01-01

    Bacteria associated with the nematode Bursaphelenchus xylophilus, a pathogen of trees and the causal agent of pine wilt disease (PWD) may play a role in the disease. In order to evaluate their role (positive or negative to the tree), strains isolated from the track of nematodes from infected Pinus pinaster trees were screened, in vitro, for their nematicidal potential. The bacterial products, from strains more active in killing nematodes, were screened in order to identify and characterize the nematicidal agent. Forty-seven strains were tested and, of these, 21 strains showed capacity to produce extracellular products with nematicidal activity. All Burkholderia strains were non-toxic. In contrast, all Serratia strains except one exhibited high toxicity. Nematodes incubated with Serratia strains showed, by SEM observation, deposits of bacteria on the nematode cuticle. The most nematicidal strain, Serratia sp. A88copa13, produced proteases in the supernatant. The use of selective inhibitors revealed that a serine protease with 70 kDa was majorly responsible for the toxicity of the supernatant. This extracellular serine protease is different phylogenetically, in size and biochemically from previously described proteases. Nematicidal assays revealed differences in nematicidal activity of the proteases to different species of Bursaphelenchus, suggesting its usefulness in a primary screen of the nematodes. This study offers the basis for further investigation of PWD and brings new insights on the role bacteria play in the defense of pine trees against B. xylophilus. Understanding all the factors involved is important in order to develop strategies to control B. xylophilus dispersion. PMID:24244546

  16. Serine protease activation of near-silent epithelial Na+ channels.

    PubMed

    Caldwell, Ray A; Boucher, Richard C; Stutts, M Jackson

    2004-01-01

    The regulation of epithelial Na+ channel (ENaC) function is critical for normal salt and water balance. This regulation is achieved through cell surface insertion/retrieval of channels, by changes in channel open probability (Po), or through a combination of these processes. Epithelium-derived serine proteases, including channel activating protease (CAP) and prostasin, regulate epithelial Na+ transport, but the molecular mechanism is unknown. We tested the hypothesis that extracellular serine proteases activate a near-silent ENaC population resident in the plasma membrane. Single-channel events were recorded in outside-out patches from fibroblasts (NIH/3T3) stably expressing rat alpha-, beta-, and gamma-subunits (rENaC), before and during exposure to trypsin, a serine protease homologous to CAP and prostasin. Under baseline conditions, near-silent patches were defined as having rENaC activity (NPo) < 0.03, where N is the number of channels. Within 1-5 min of 3 microg/ml bath trypsin superfusion, NPo increased approximately 66-fold (n = 7). In patches observed to contain a single functional channel, trypsin increased Po from 0.02 +/- 0.01 to 0.57 +/- 0.03 (n = 3, mean +/- SE), resulting from the combination of an increased channel open time and decreased channel closed time. Catalytic activity was required for activation of near-silent ENaC. Channel conductance and the Na+/Li+ current ratio with trypsin were similar to control values. Modulation of ENaC Po by endogenous epithelial serine proteases is a potentially important regulator of epithelial Na+ transport, distinct from the regulation achieved by hormone-induced plasma membrane insertion of channels. PMID:12967915

  17. Genome-wide survey of prokaryotic serine proteases: Analysis of distribution and domain architectures of five serine protease families in prokaryotes

    PubMed Central

    Tripathi, Lokesh P; Sowdhamini, R

    2008-01-01

    Background Serine proteases are one of the most abundant groups of proteolytic enzymes found in all the kingdoms of life. While studies have established significant roles for many prokaryotic serine proteases in several physiological processes, such as those associated with metabolism, cell signalling, defense response and development, functional associations for a large number of prokaryotic serine proteases are relatively unknown. Current analysis is aimed at understanding the distribution and probable biological functions of the select serine proteases encoded in representative prokaryotic organisms. Results A total of 966 putative serine proteases, belonging to five families, were identified in the 91 prokaryotic genomes using various sensitive sequence search techniques. Phylogenetic analysis reveals several species-specific clusters of serine proteases suggesting their possible involvement in organism-specific functions. Atypical phylogenetic associations suggest an important role for lateral gene transfer events in facilitating the widespread distribution of the serine proteases in the prokaryotes. Domain organisations of the gene products were analysed, employing sensitive sequence search methods, to infer their probable biological functions. Trypsin, subtilisin and Lon protease families account for a significant proportion of the multi-domain representatives, while the D-Ala-D-Ala carboxypeptidase and the Clp protease families are mostly single-domain polypeptides in prokaryotes. Regulatory domains for protein interaction, signalling, pathogenesis, cell adhesion etc. were found tethered to the serine protease domains. Some domain combinations (such as S1-PDZ; LON-AAA-S16 etc.) were found to be widespread in the prokaryotic lineages suggesting a critical role in prokaryotes. Conclusion Domain architectures of many serine proteases and their homologues identified in prokaryotes are very different from those observed in eukaryotes, suggesting distinct roles

  18. Crystal structure of an intracellular protease from Pyrococcus horikoshii at 2-Å resolution

    PubMed Central

    Du, Xinlin; Choi, In-Geol; Kim, Rosalind; Wang, Weiru; Jancarik, Jaru; Yokota, Hisao; Kim, Sung-Hou

    2000-01-01

    The intracellular protease from Pyrococcus horikoshii (PH1704) and PfpI from Pyrococcus furiosus are members of a class of intracellular proteases that have no sequence homology to any other known protease family. We report the crystal structure of PH1704 at 2.0-Å resolution. The protease is tentatively identified as a cysteine protease based on the presence of cysteine (residue 100) in a nucleophile elbow motif. In the crystal, PH1704 forms a hexameric ring structure, and the active sites are formed at the interfaces between three pairs of monomers. PMID:11114201

  19. Characterization of the Protease Activity of Detergents: Laboratory Practicals for Studying the Protease Profile and Activity of Various Commercial Detergents

    ERIC Educational Resources Information Center

    Valls, Cristina; Pujadas, Gerard; Garcia-Vallve, Santi; Mulero, Miquel

    2011-01-01

    Detergent enzymes account for about 30% of the total worldwide production of enzymes and are one of the largest and most successful applications of modern industrial biotechnology. Proteases can improve the wash performance of household, industrial, and institutional laundry detergents used to remove protein-based stains such as blood, grass, body…

  20. Cloning, expression and activity analysis of a novel fibrinolytic serine protease from Arenicola cristata

    NASA Astrophysics Data System (ADS)

    Zhao, Chunling; Ju, Jiyu

    2015-06-01

    The full-length cDNA of a protease gene from a marine annelid Arenicola cristata was amplified through rapid amplification of cDNA ends technique and sequenced. The size of the cDNA was 936 bp in length, including an open reading frame encoding a polypeptide of 270 amino acid residues. The deduced amino acid sequnce consisted of pro- and mature sequences. The protease belonged to the serine protease family because it contained the highly conserved sequence GDSGGP. This protease was novel as it showed a low amino acid sequence similarity (< 40%) to other serine proteases. The gene encoding the active form of A. cristata serine protease was cloned and expressed in E. coli. Purified recombinant protease in a supernatant could dissolve an artificial fibrin plate with plasminogen-rich fibrin, whereas the plasminogen-free fibrin showed no clear zone caused by hydrolysis. This result suggested that the recombinant protease showed an indirect fibrinolytic activity of dissolving fibrin, and was probably a plasminogen activator. A rat model with venous thrombosis was established to demonstrate that the recombinant protease could also hydrolyze blood clot in vivo. Therefore, this recombinant protease may be used as a thrombolytic agent for thrombosis treatment. To our knowledge, this study is the first of reporting the fibrinolytic serine protease gene in A. cristata.

  1. Proteases of Wood Rot Fungi with Emphasis on the Genus Pleurotus

    PubMed Central

    Inácio, Fabíola Dorneles; Ferreira, Roselene Oliveira; de Araujo, Caroline Aparecida Vaz; Brugnari, Tatiane; Castoldi, Rafael; Peralta, Rosane Marina; de Souza, Cristina Giatti Marques

    2015-01-01

    Proteases are present in all living organisms and they play an important role in physiological conditions. Cell growth and death, blood clotting, and immune defense are all examples of the importance of proteases in maintaining homeostasis. There is growing interest in proteases due to their use for industrial purposes. The search for proteases with specific characteristics is designed to reduce production costs and to find suitable properties for certain industrial sectors, as well as good producing organisms. Ninety percent of commercialized proteases are obtained from microbial sources and proteases from macromycetes have recently gained prominence in the search for new enzymes with specific characteristics. The production of proteases from saprophytic basidiomycetes has led to the identification of various classes of proteases. The genus Pleurotus has been extensively studied because of its ligninolytic enzymes. The characteristics of this genus are easy cultivation techniques, high yield, low nutrient requirements, and excellent adaptation. There are few studies in the literature about proteases of Pleurotus spp. This review gathers together information about proteases, especially those derived from basidiomycetes, and aims at stimulating further research about fungal proteases because of their physiological importance and their application in various industries such as biotechnology and medicine. PMID:26180792

  2. Yeast Endoplasmic Reticulum Sequestration Screening for the Engineering of Proteases from Libraries Expressed in Yeast.

    PubMed

    Yi, Li; Taft, Joseph M; Li, Qing; Gebhard, Mark C; Georgiou, George; Iverson, Brent L

    2015-01-01

    There is significant interest in engineering proteases with desired proteolytic properties. We describe a high-throughput fluorescence-activated cell sorting (FACS) assay for detecting altered proteolytic activity of protease in yeast, at the single cell level. This assay relies on coupling yeast endoplasmic reticulum (ER) retention, yeast surface display, and FACS analysis. The method described here allows facile screening of large libraries, and of either protease or substrate variants, including the screening of protease libraries against substrate libraries. We demonstrate the application of this technique in the screening of libraries of Tobacco Etch Virus protease (TEV-P) for altered proteolytic activities. In addition, the generality of this method is also validated by other proteases such as human granzyme K and the hepatitis C virus protease, and the human Abelson tyrosine kinase. PMID:26060071

  3. Production of plant proteases in vivo and in vitro--a review.

    PubMed

    González-Rábade, Nuria; Badillo-Corona, Jesús Agustín; Aranda-Barradas, Juan Silvestre; Oliver-Salvador, María Del Carmen

    2011-01-01

    In the latest two decades, the interest received by plant proteases has increased significantly. Plant enzymes such as proteases are widely used in medicine and the food industry. Some proteases, like papain, bromelain and ficin are used in various processes such as brewing, meat softening, milk-clotting, cancer treatment, digestion and viral disorders. These enzymes can be obtained from their natural source or through in vitro cultures, in order to ensure a continuous source of plant enzymes. The focus of this review will be the production of plant proteases both in vivo and in vitro, with particular emphasis on the different types of commercially important plant proteases that have been isolated and characterized from naturally grown plants. In vitro approaches for the production of these proteases is also explored, focusing on the techniques that do not involve genetic transformation of the plants and the attempts that have been made in order to enhance the yield of the desired proteases. PMID:21889977

  4. Redefining the concept of protease-activated receptors: cathepsin S evokes itch via activation of Mrgprs

    PubMed Central

    Reddy, Vemuri B.; Sun, Shuohao; Azimi, Ehsan; Elmariah, Sarina B.; Dong, Xinzhong; Lerner, Ethan A.

    2015-01-01

    Sensory neurons expressing Mas-related G protein coupled receptors (Mrgprs) mediate histamine-independent itch. We show that the cysteine protease cathepsin S activates MrgprC11 and evokes receptor-dependent scratching in mice. In contrast to its activation of conventional protease-activated receptors, cathepsin S mediated activation of MrgprC11 did not involve the generation of a tethered ligand. We demonstrate further that different cysteine proteases selectively activate specific mouse and human Mrgpr family members. This expansion of our understanding by which proteases interact with GPCRs redefines the concept of what constitutes a protease-activated receptor. The findings also implicate proteases as ligands to members of this orphan receptor family while providing new insights into how cysteine proteases contribute to itch. PMID:26216096

  5. Purification and characterization of a serine protease from Cucumis trigonus Roxburghi.

    PubMed

    Asif-Ullah, Mufti; Kim, Key-Sun; Yu, Yeon Gyu

    2006-05-01

    Kachri fruit, Cucumis trigonus Roxburghi, contains high protease activity and has been used as meat tenderizer in the Indian subcontinent. A 67 kDa serine protease from Kachri fruit was purified by DEAE-Sepharose and CM-Sepharose chromatography, whose optimum activity was at pH 11 and 70 degrees C. Its activity was strongly inhibited by PMSF, but not by EDTA, pepstatin, or cysteine protease inhibitors. The substrate specificity of the purified protease towards synthetic peptides was comparable to cucumisin, the first characterized subtilisin class plant protease from the sarcocarp of melon fruit (Cucumis melo). These characteristics, along with the N-terminal amino acid sequence, indicated that the isolated protease from Cucumis trigonus Roxburghi is a cucumisin homologue, which belongs to the serine protease family. PMID:16603211

  6. Development of marine biotechnology as a resource for novel proteases and their role in modern biotechnology.

    PubMed

    Homaei, Ahmad; Lavajoo, Fatemeh; Sariri, Reyhaneh

    2016-07-01

    Marine environment consists of the largest sources diversified genetic pool of material with an enormous potential for a wide variety of enzymes including proteases. A protease hydrolyzes the peptide bond and most of proteases possess many industrial applications. Marine proteases differ considerably from those found in internal or external organs of invertebrates and vertebrates. In common with all enzymes, external factors such as temperature, pH and type of media are important for the activity, catalytic efficiency, stability and proper functioning of proteases. In this review valuable characteristics of proteases in marine organisms and their applications are gathered from a wide literature survey. Considering their biochemical significance and their increasing importance in biotechnology, a thorough understanding of marine proteases functioning could be of prime importance.

  7. Evolution of soldier-specific venomous protease in social aphids.

    PubMed

    Kutsukake, Mayako; Nikoh, Naruo; Shibao, Harunobu; Rispe, Claude; Simon, Jean-Christophe; Fukatsu, Takema

    2008-12-01

    In social aphids of the genus Tuberaphis a cysteine protease gene of the family cathepsin B exhibits soldier-specific expression and intestinal protease production. The product is orally excreted and injected by soldier nymphs into natural enemies, thereby exerting an insecticidal activity. In an attempt to gain insights into when and how the novel venomous protease for the altruistic caste has evolved, we investigated the soldier-specific type (S-type) and nonspecific type (N-type) cathepsin B genes from social and nonsocial aphids. All the social aphids examined, representing the genera Tuberaphis, Astegopteryx, and Cerataphis, possessed both the S-type and N-type genes. Phylogenetically distant nonsocial aphids also possessed cathepsin B genes allied to the S-type and the N-type, indicating the evolutionary origin of these genes in the common ancestor of extant aphids. In Tuberaphis species the S-type genes exhibited significant soldier-specific expression and accelerated molecular evolution whereas the N-type genes did not. In Astegopteryx and Cerataphis species, meanwhile, both the S-type and N-type genes exhibited neither remarkable soldier-specific expression nor accelerated molecular evolution. These results suggest that the S-type gene acquired the soldier-specific expression and the venom function after divergence of the genus Tuberaphis. On the structural model of the S-type protease of Tuberaphis styraci the accelerated molecular evolution was associated with the molecular surface rather than the catalytic cleft, suggesting that the venom activity was probably acquired by relatively minor modifications on the molecular surface rather than by generation of a novel active site. In Cerataphis jamuritsu the S-type gene was, although containing a stop codon, structurally almost intact and still transcribed, suggesting recent pseudogenization of the gene copy and possible relevance to relaxed functional constraint in the highly multiplied protease gene family

  8. Elimination of protease-inhibitor complexes from the arthritic joint.

    PubMed

    Ekerot, L; Ohlsson, K; Necking, L

    1985-01-01

    In the rheumatic joint, destructive proteolytic enzymes are released and counteracted by complexation to the predominant inhibitors alpha 2-macroglobulin and alpha 1-antitrypsin. The articular elimination of these complexes is thought to be of decisive importance in the protease-inhibitor interplay influencing the inhibitory capacity of the synovial fluid. Protease-alpha 2-macroglobulin complexes showed an intraarticular half-life shorter than 2 hours in arthritis and were eliminated by various routes including phagocytosis in cells of the synovial membrane and in regional lymph nodes, in addition to haematogenous resorption and uptake in the liver. The phagocytosed complexes were degraded to low-molecular-weight metabolites excretable in the urine. The articular elimination of elastase-alpha 1-antitrypsin complexes seemed to be an equivalent process, but an additional intrasynovial dissociation of the complexes was also indicated. Thus the intraarticularly released elastase seemed to be bound and eliminated in complex with alpha 2-macroglobulin. PMID:2414244

  9. Quality control of a molybdoenzyme by the Lon protease.

    PubMed

    Redelberger, David; Genest, Olivier; Arabet, Dallel; Méjean, Vincent; Ilbert, Marianne; Iobbi-Nivol, Chantal

    2013-12-11

    Molybdoenzymes contain a molybdenum cofactor in their active site to catalyze various redox reactions in all domains of life. To decipher crucial steps during their biogenesis, the TorA molybdoenzyme of Escherichia coli had played a major role to understand molybdoenzyme maturation process driven by specific chaperones. TorD, the specific chaperone of TorA, is also involved in TorA protection. Here, we show that immature TorA (apoTorA) is degraded in vivo and in vitro by the Lon protease. Lon interacts with apoTorA but not with holoTorA. Lon and TorD compete for apoTorA binding but TorD binding protects apoTorA against degradation. Lon is the first protease shown to eliminate an immature or misfolded molybdoenzyme probably by targeting its inactive catalytic site.

  10. Protease-dependent mechanisms of complement evasion by bacterial pathogens.

    PubMed

    Potempa, Michal; Potempa, Jan

    2012-09-01

    The human immune system has evolved a variety of mechanisms for the primary task of neutralizing and eliminating microbial intruders. As the first line of defense, the complement system is responsible for rapid recognition and opsonization of bacteria, presentation to phagocytes and bacterial cell killing by direct lysis. All successful human pathogens have mechanisms of circumventing the antibacterial activity of the complement system and escaping this stage of the immune response. One of the ways in which pathogens achieve this is the deployment of proteases. Based on the increasing number of recent publications in this area, it appears that proteolytic inactivation of the antibacterial activities of the complement system is a common strategy of avoiding targeting by this arm of host innate immune defense. In this review, we focus on those bacteria that deploy proteases capable of degrading complement system components into non-functional fragments, thus impairing complement-dependent antibacterial activity and facilitating pathogen survival inside the host.

  11. Occurrence of aspartyl proteases in brine after herring marinating.

    PubMed

    Szymczak, Mariusz; Lepczyński, Adam

    2016-03-01

    Herrings are marinated in a brine consisting of salt and acetic acid. During marinating, various nitrogen fractions diffuse from fish flesh to the brine, causing significant nutritional quality losses of the raw material. In this study, it has been demonstrated for the first time that proteases diffuse from the fish to the marinating brine. Using ammonium sulphate precipitation and affinity chromatography on pepstatin-A agarose bed the aspartyl proteases were purified and concentrated over 2600-fold from a marinating brine. Pepstatin-A completely inhibited the activity of the purified preparation. The preparation was active against fluorogenic substrates specific for cathepsin D and E and inactive against substrates specific for cysteine cathepsins. Depending on incubation time, the preparation showed pH-optimum at 2.0 or 4.5. The 2D SDS-PAGE separation demonstrated the presence of a few proteins with molecular weights and pI values typical of cathepsin D, E and pepsin.

  12. Intestinal proteases of free-living and parasitic astigmatid mites.

    PubMed

    Holt, Deborah C; Burgess, Stewart T G; Reynolds, Simone L; Mahmood, Wajahat; Fischer, Katja

    2013-02-01

    Among arthropod pests, mites are responsible for considerable damage to crops, humans and other animals. However, detailed physiological data on these organisms remain sparse, mainly because of their small size but possibly also because of their extreme diversity. Focusing on intestinal proteases, we draw together information from three distinct mite species that all feed on skin but have separately adapted to a free-living, a strictly ecto-parasitic and a parasitic lifestyle. A wide range of studies involving immunohistology, molecular biology, X-ray crystallography and enzyme biochemistry of mite gut proteases suggests that these creatures have diverged considerably as house dust mites, sheep scab mites and scabies mites. Each species has evolved a particular variation of a presumably ancestral repertoire of digestive enzymes that have become specifically adapted to their individual environmental requirements.

  13. Effects of Mucuna pruriens protease inhibitors on Echis carinatus venom.

    PubMed

    Hope-Onyekwere, Nnadozie Stanley; Ogueli, Godwin Ifeanyi; Cortelazzo, Alessio; Cerutti, Helena; Cito, Annarita; Aguiyi, John C; Guerranti, Roberto

    2012-12-01

    The medicinal plant Mucuna pruriens, with reputed anti-snake venom properties has been reported to contain a kunitz-type trypsin inhibitor. This study was undertaken to further evaluate the protease inhibitory potential of gpMuc, a multiform glycoprotein, and other protein fractions from M. pruriens seeds against trypsin, chymotrypsin, Echis carinatus snake venom, ecarin and thrombin. The results showed that gpMuc inhibited both trypsin and chymotrypsin activities and was thermally stable, maintaining its trypsin inhibitory activity at temperatures of up to 50°C. Its structural conformation was also maintained at pH ranges of 4-7. Immunoreactivity study confirms that it contains protease-recognizing epitope on one of its isoforms. The whole protein extract of M. pruriens seeds inhibited prothrombin activation by ecarin and whole E. carinatus venom, and also thrombin-like activity using chromogenic assay. PMID:22447581

  14. Additivity in the Analysis and Design of HIV Protease Inhibitors

    PubMed Central

    Jorissen, Robert N.; Kiran Kumar Reddy, G. S.; Ali, Akbar; Altman, Michael D.; Chellappan, Sripriya; Anjum, Saima G.; Tidor, Bruce; Schiffer, Celia A.; Rana, Tariq M.; Gilson, Michael K.

    2009-01-01

    We explore the applicability of an additive treatment of substituent effects to the analysis and design of HIV protease inhibitors. Affinity data for a set of inhibitors with a common chemical framework were analyzed to provide estimates of the free energy contribution of each chemical substituent. These estimates were then used to design new inhibitors, whose high affinities were confirmed by synthesis and experimental testing. Derivations of additive models by least-squares and ridge-regression methods were found to yield statistically similar results. The additivity approach was also compared with standard molecular descriptor-based QSAR; the latter was not found to provide superior predictions. Crystallographic studies of HIV protease-inhibitor complexes help explain the perhaps surprisingly high degree of substituent additivity in this system, and allow some of the additivity coefficients to be rationalized on a structural basis. PMID:19193159

  15. Protease-dependent mechanisms of complement evasion by bacterial pathogens

    PubMed Central

    Potempa, Michal; Potempa, Jan

    2012-01-01

    The human immune system has evolved a variety of mechanisms for the primary task of neutralizing and eliminating microbial intruders. As the first line of defense, the complement system is responsible for rapid recognition and opsonisation of bacteria, presentation to phagocytes and bacterial cell killing by direct lysis. All successful human pathogens have mechanisms of circumventing the antibacterial activity of the complement system and escaping this stage of the immune response. One of the ways in which pathogens achieve this is the deployment of proteases. Based on the increasing number of recent publications in this area, it appears that proteolytic inactivation of the antibacterial activities of the complement system is a common strategy of avoiding targeting by this arm of host innate immune defense. In this review, we focus on those bacteria that deploy proteases capable of degrading complement system components into non-functional fragments, thus impairing complement-dependent antibacterial activity and facilitating pathogen survival inside the host. PMID:22944688

  16. Host cell proteases: critical determinants of coronavirus tropism and pathogenesis

    PubMed Central

    Millet, Jean Kaoru; Whittaker, Gary R.

    2015-01-01

    Coronaviruses are a large group of enveloped, single-stranded positive-sense RNA viruses that infect a wide range of avian and mammalian species, including humans. The emergence of deadly human coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV) have bolstered research in these viral and often zoonotic pathogens. While coronavirus cell and tissue tropism, host range, and pathogenesis are initially controlled by interactions between the spike envelope glycoprotein and host cell receptor, it is becoming increasingly apparent that proteolytic activation of spike by host cell proteases also plays a critical role. Coronavirus spike proteins are the main determinant of entry as they possess both receptor binding and fusion functions. Whereas binding to the host cell receptor is an essential first step in establishing infection, the proteolytic activation step is often critical for the fusion function of spike, as it allows for controlled release of the fusion peptide into target cellular membranes. Coronaviruses have evolved multiple strategies for proteolytic activation of spike, and a large number of host proteases have been shown to proteolytically process the spike protein. These include, but are not limited to, endosomal cathepsins, cell surface transmembrane protease/serine (TMPRSS) proteases, furin, and trypsin. This review focuses on the diversity of strategies coronaviruses have evolved to proteolytically activate their fusion protein during spike protein biosynthesis and the critical entry step of their life cycle, and highlights important findings on how proteolytic activation of coronavirus spike influences tissue and cell tropism, host range and pathogenicity. PMID:25445340

  17. Interaction of proteases with legume seed inhibitors. Molecular features.

    PubMed

    de Seidl, D S

    1996-12-01

    After having found that raw black beans (Phaseolus vulgaris) were toxic, while the cooked ones constitute the basic diet of the underdeveloped peoples of the world, in the sixties, our research directed by Dr. Jaffé, concentrated mainly around the detection and identification of the heat labile toxic factors in legume seeds. A micromethod for the detection of protease inhibitors (PI) in individual seeds was developed, for the purpose of establishing that the multiple trypsin inhibitors (TI) found in the Cubagua variety were expressions of single seeds and not a mixture of a non homogenous bean lot. Six isoinhibitors were isolated and purified, all of which were "double-headed" and interacted with trypsin (T) and chymotrypsin (CHT) independently and simultaneously, as shown by electrophoresis of their binary and ternary complexes with each and both enzymes. However, their affinity for the enzymes, including elastases, was rather variable, as well as their amino acid composition which consisted of 51 units for inhibitor V, the smallest, and 83 amino acids for inhibitor I, the largest. A low molecular weight protein fraction that inhibited subtilisin (S), but recognized neither T, CHT nor pancreatic elastase was detected in 63 varieties of Phaseolus vulgaris as well as in broad beans (Vicia faba), chick peas (Cicer arietinum), jack beans (Canavalia ensiformis), kidney beans (Vigna aureus), etc., It was absent though, in soybeans (Glycine max), lentils (Lens culinaris), green peas (Pisum sativum), cowpea (Vigna sinensis) and lupine seeds (Lupinus sp). Subtilisin inhibitors (SI) were isolated from black beans, broad beans, chick peas and jack beans. Their Mr is between 8-9KD and they show a rather high stability in the presence of denaturing agents. They are specific toward microbial proteases, in addition to subtilisins, Carlsberg and BPN', they inhibit the alkaline protease from Tritirachium album (Protease K), from Aspergillus oryzae and one isolated from

  18. Proteases of Treponema denticola outer sheath and extracellular vesicles.

    PubMed Central

    Rosen, G; Naor, R; Rahamim, E; Yishai, R; Sela, M N

    1995-01-01

    Electron microscopical observations of the oral periodontopathogen Treponema denticola show the presence of extracellular vesicles bound to the bacterial surface or free in the surrounding medium. Extracellular vesicles from T. denticola ATCC 35404, 50 to 100 nm in diameter, were isolated and further characterized. Protein and proteolytic patterns of the vesicles were found to be very similar to those of isolated T. denticola outer sheaths. They were enriched with the major outer sheath polypeptides (molecular sizes, 113 to 234 kDa) and with outer sheath proteases of 91, 153, 173, and 228 kDa. These findings indicate that treponemal outer sheath vesicles contain the necessary adhesins and proteolytic arsenal for adherence to and damage of eucaryotic cells and mammalian matrix proteins. The major outer sheath- and vesicle-associated protease of T. denticola ATCC 35404 was purified and characterized. The purified enzyme had a molecular size of 91 kDa, and it dissociated into three polypeptides of 72, 38, and 35 kDa upon heating in the presence of sodium dodecyl sulfate with or without a reducing agent. The activity of the enzyme could be inhibited by diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, and phenylboronic acid. The value of the second-order rate constant of the protease inactivation by phenylmethylsulfonyl fluoride was 0.48 x 10(4) M(-1) min-1. Inhibition of the enzyme by phenylboronic acid was rapid (< 1 min) and pH dependent. These data strongly suggest that this major surface proteolytic activity belongs to a family of serine proteases. PMID:7558307

  19. Detergent-compatible proteases: microbial production, properties, and stain removal analysis.

    PubMed

    Niyonzima, Francois Niyongabo; More, Sunil

    2015-01-01

    Proteases are one of the most important commercial enzymes used in various industrial domains such as detergent and leather industries. The alkaline proteases as well as other detergent-compatible enzymes such as lipases and amylases serve now as the key components in detergent formulations. They break down various stains during fabric washing. The search for detergent-compatible proteases with better properties is a continuous exercise. The current trend is to use detergent-compatible proteases that are stable over a wide temperature range. Although the proteases showing stability at elevated pH have the capacity to be used in detergent formulations, their usage can be significant if they are also stable and compatible with detergent and detergent ingredients, and also able to remove protein stains. Despite the existence of some reviews on alkaline proteases, there is no specification for the use of alkaline proteases as detergent additives. The present review describes the detergent-compatible proteases tested as detergent additives. An overview was provided for screening, optimization, purification, and properties of detergent compatible proteases, with an emphasis on the stability and compatibility of the alkaline proteases with the detergent and detergent compounds, as well as stain removal examination methods.

  20. Optimum production and characterization of an acid protease from marine yeast Metschnikowia reukaufii W6b

    NASA Astrophysics Data System (ADS)

    Li, Jing; Peng, Ying; Wang, Xianghong; Chi, Zhenming

    2010-12-01

    The marine yeast strain W6b isolated from sediment of the South China Sea was found to produce a cell-bound acid protease. The crude acid protease produced by this marine yeast showed the highest activity at pH 3.5 and 40 °C. The optimal pH and temperature for the crude acid protease were in agreement with those for acid protease produced by the terrestrial yeasts. The optimal medium of the acid protease production was seawater containing 1.0% glucose, 1.5% casein, and 0.5% yeast extract, and the optimal cultivation conditions of the acid protease production were pH 4.0, a temperature of 25 °C and a shaking speed of 140 rmin-1. Under the optimal conditions, 72.5 UmL-1 of acid protease activity could be obtained in cell suspension within 48 h of fermentation at shake flask level. The acid protease production was induced by high-molecular-weight nitrogen sources and repressed by low-molecular-weight nitrogen sources. Skimmed-milk-clotting test showed that the crude acid protease from the cell suspension of the yeast W6b had high skimmed milk coagulability. The acid protease produced by M. reukaufii W6b may have highly potential applications in cheese, food and fermentation industries.

  1. Multiple Classes of Immune-Related Proteases Associated with the Cell Death Response in Pepper Plants

    PubMed Central

    Bae, Chungyun; Kim, Su-min; Lee, Dong Ju; Choi, Doil

    2013-01-01

    Proteases regulate a large number of biological processes in plants, such as metabolism, physiology, growth, and defense. In this study, we carried out virus-induced gene silencing assays with pepper cDNA clones to elucidate the biological roles of protease superfamilies. A total of 153 representative protease genes from pepper cDNA were selected and cloned into a Tobacco rattle virus-ligation independent cloning vector in a loss-of-function study. Silencing of 61 proteases resulted in altered phenotypes, such as the inhibition of shoot growth, abnormal leaf shape, leaf color change, and lethality. Furthermore, the silencing experiments revealed that multiple proteases play a role in cell death and immune response against avirulent and virulent pathogens. Among these 153 proteases, 34 modulated the hypersensitive cell death response caused by infection with an avirulent pathogen, and 16 proteases affected disease symptom development caused by a virulent pathogen. Specifically, we provide experimental evidence for the roles of multiple protease genes in plant development and immune defense following pathogen infection. With these results, we created a broad sketch of each protease function. This information will provide basic information for further understanding the roles of the protease superfamily in plant growth, development, and defense. PMID:23696830

  2. Effects on the Qualities of Proteolysis to Beef by Non-coating and Coating Protease Treatment

    PubMed Central

    Shin, Jung-Kue; Cho, Hyung-Yong

    2016-01-01

    This study was performed to improve the techniques used for tenderizing red meat as elderly food. Beef meat was immersed in liposome encapsulated enzyme solution and the effect of protease encapsulation on the beef properties was analyzed. The protease encapsulation properties were analyzed according to the size distribution and enzymatic activity. After enzyme reaction on the beef, the chemical properties of the meat such as pH, water holding capacity, shear rate, lipid oxidation and total volatile basic nitrogen (TVB-N) were analyzed. The pH of the beef increased during the reaction and coating protease (CP) was higher than non-coating protease (NCP). Total color differences were increased remarkably after 36 h and generally, the difference in CP was relatively lower than in NCP. WHC was significantly decreased within 24 h, and no effect from the protease coating was observed. Protease activity was significantly increased within 48 h and no differences in the enzyme coating were observed. The TVB-N value of NCP was increased within 24 h while CP was sustained for up to 36 h. The TVB-N value of protease treated meat increased after 36 h and no effect from the protease coating was detected. Consequently, liposome encapsulated protease was found to have similar properties as non-coated protease. Application of liposome seems to be an interesting option for injecting various functional materials without changing the properties of meat. PMID:27499672

  3. Effects on the Qualities of Proteolysis to Beef by Non-coating and Coating Protease Treatment.

    PubMed

    Kim, Kwang-Il; Lee, Sang-Yoon; Kim, Soo-Jin; Seo, Jae-Hee; Lee, Joong-Kyu; Shin, Jung-Kue; Cho, Hyung-Yong; Choi, Mi-Jung

    2016-01-01

    This study was performed to improve the techniques used for tenderizing red meat as elderly food. Beef meat was immersed in liposome encapsulated enzyme solution and the effect of protease encapsulation on the beef properties was analyzed. The protease encapsulation properties were analyzed according to the size distribution and enzymatic activity. After enzyme reaction on the beef, the chemical properties of the meat such as pH, water holding capacity, shear rate, lipid oxidation and total volatile basic nitrogen (TVB-N) were analyzed. The pH of the beef increased during the reaction and coating protease (CP) was higher than non-coating protease (NCP). Total color differences were increased remarkably after 36 h and generally, the difference in CP was relatively lower than in NCP. WHC was significantly decreased within 24 h, and no effect from the protease coating was observed. Protease activity was significantly increased within 48 h and no differences in the enzyme coating were observed. The TVB-N value of NCP was increased within 24 h while CP was sustained for up to 36 h. The TVB-N value of protease treated meat increased after 36 h and no effect from the protease coating was detected. Consequently, liposome encapsulated protease was found to have similar properties as non-coated protease. Application of liposome seems to be an interesting option for injecting various functional materials without changing the properties of meat. PMID:27499672

  4. [Extracellular proteases of mycelial fungi as participants of pathogenic processes].

    PubMed

    Dunaevskiĭ, Ia E; Matveeva, A R; Fatkhullina, G N; Beliakova, G A; Kolomiets, T M; Kovalenko, E D; Belozerskiĭ, M A

    2008-01-01

    The interest in proteases secreted by mycelial fungi is due to several reasons of which one of the most important is their involvement in the initiation and development of the pathogenic process. A comparison of saprophytic and phytopathogenic mycelial fungi revealed one characteristic feature, namely, the appearance of a new trypsin-like activity in phytopathogens that is absent in saprophytes. To clear up the question of whether the degree of pathogenicity of a fungus is related to the activity of secreted trypsin-like protease, several species of Fusarium of various pathogenicity were compared. In two species, F. sporotrichioides (which causes ear fusa-riosis of rye) and F. heterosporum (the causative agent of root rot in wheat), a clear correlation between the activity and pathogenicity was revealed: the more pathogenetic F. sporotrichioides exhibited a higher extracellular trypsin-like activity than the less pathogenetic species F. heterosporum. Thus, the presence of trypsin-like activity in a saprotroph-pathogen pair may be an indicator of the pathogenicity of a fungus; in some cases, the value of this activity may indicate the degree of its pathogenicity. This suggests that trypsin-like proteases specific to phytopathogens are directly involved in the pathogenetic process, probably, through interaction with the "sentry" protein or the product of the resistance gene. PMID:18672678

  5. Peptide-modified optical filters for detecting protease activity.

    PubMed

    Kilian, Kristopher A; Böcking, Till; Gaus, Katharina; Gal, Michael; Gooding, J Justin

    2007-11-01

    The organic derivatization of silicon-based nanoporous photonic crystals is presented as a method to immobilize peptides for the detection of protease enzymes in solution. A narrow-line-width rugate filter, a one-dimensional photonic crystal, is fabricated that exhibits a high-reflectivity optical resonance that is sensitive to small changes in the refractive index at the pore walls. To immobilize peptide in the pore of the photonic crystal, the hydrogen-terminated silicon surface was first modified with the alkene 10-succinimidyl undecenoate via hydrosilylation. The monolayer with the succinimide ester moiety at the distal end served the dual function of protecting the underlying silicon from oxidation as well as providing a surface suitable for subsequent derivatization with amines. The surface was further modified with 1-aminohexa(ethylene glycol) (EG(6)) to resist nonspecific adsorption of proteins common in complex biological samples. The distal hydroxyl of the EG(6) is activated using the solid-phase coupling reagent disuccinimidyl carbonate for selective immobilization of peptides as protease recognition elements. X-ray photoelectron spectroscopy analysis reveals high activation and coupling efficiency at each stage of the functionalization. Exposure of the peptide-modified crystals to the protease subtilisin in solution causes a change in the refractive index, resulting in a shift of the resonance to shorter wavelengths, indicating cleavage of organic material within the pores. The lowest detected concentration of enzyme was 37 nM (7.4 pmol in 200 microL).

  6. Milk-clotting mechanism of Dregea sinensis Hemsl. protease.

    PubMed

    Zhang, Yali; Wang, Hongyan; Tao, Liang; Huang, Ai-xiang

    2015-12-01

    Dregea sinensis Hemsl. is used as a milk coagulant to produce goat milk cakes in Yunnan, China. However, the composition of milk-clotting compounds and the related mechanism have not been reported. Crude protease was extracted from the stem, purified, and then separated with a Millipore ultrafiltration centrifuge tube. Cysteine protease (procerain B) was identified as the main milk-clotting protein through electrospray ionization mass spectrometry, and its molecular weight was 23.8 kDa. The protease can partially degrade α-casein (CN) and completely degrade β- and κ-CN, and κ-CN degradation resulted in milk clotting. The molecular weight and AA sequence of the peptide fractions were determined through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and a peptide sequencer, respectively. The enzyme cleaved κ-CN at Ala90-Gln91 and produced deputy κ-CN and caseinomacropeptide with molecular weights of 12 and 6.9 kDa, respectively. This cleavage site differed from the majority of chymosins cleaved at Phe105-Met106. PMID:26506540

  7. Evolution of the protease-activated receptor family in vertebrates

    PubMed Central

    JIN, MIN; YANG, HAI-WEI; TAO, AI-LIN; WEI, JI-FU

    2016-01-01

    Belonging to the G protein-coupled receptor (GPcr) family, the protease-activated receptors (Pars) consist of 4 members, PAR1-4. PARs mediate the activation of cells via thrombin, serine and other proteases. Such protease-triggered signaling events are thought to be critical for hemostasis, thrombosis and other normal pathological processes. In the present study, we examined the evolution of PARs by analyzing phylogenetic trees, chromosome location, selective pressure and functional divergence based on the 169 functional gene alignment sequences from 57 vertebrate gene sequences. We found that the 4 PARs originated from 4 invertebrate ancestors by phylogenetic trees analysis. The selective pressure results revealed that only PAR1 appeared by positive selection during its evolution, while the other PAR members did not. In addition, we noticed that although these PARs evolved separately, the results of functional divergence indicated that their evolutional rates were similar and their functions did not significantly diverge. The findings of our study provide valuable insight into the evolutionary history of the vertebrate PAR family. PMID:26820116

  8. Separation of proteases from yellowfin tuna spleen by ultrafiltration.

    PubMed

    Li, Zhenyu; Youravong, Wirote; H-Kittikun, Aran

    2006-12-01

    Separation of protease, trypsin and chymotrypsin from yellowfin tuna spleen extract by ultrafiltration (UF) using regenerated cellulose membranes with molecular weight cut off (MWCO) 30 and 100 kDa was studied. The 100 kDa membrane had a higher transmission of enzymes than that of the 30 kDa membrane. The enzyme transmission varied from 0.01 to 0.18 and from 0.6 to 0.8 for the 30 kDa membrane and 100 kDa membrane, respectively. The protein transmission was about 0.8 for both membranes. Increasing cross-flow rate and transmembrane pressure (TMP) increased permeate flux. The limiting fluxes at cross-flow rate 120, 240 and 360 L/h for the 30 kDa membrane were 17.3, 43.9 and 54.7 L/m2h, respectively and the limiting fluxes at the same flow rate for 100 kDa membrane were 34.1, 51.1 and 68.4 L/m2h, respectively. The separation of these proteases was achieved using the 30 kDa membrane. The purities of proteases were increased more than ten times at TMP 1.5 bar and cross-flow rate 360 L/h by diafiltration using 30 kDa membrane. PMID:16314093

  9. [Extracellular proteases of mycelial fungi as participants of pathogenic processes].

    PubMed

    Dunaevskiĭ, Ia E; Matveeva, A R; Fatkhullina, G N; Beliakova, G A; Kolomiets, T M; Kovalenko, E D; Belozerskiĭ, M A

    2008-01-01

    The interest in proteases secreted by mycelial fungi is due to several reasons of which one of the most important is their involvement in the initiation and development of the pathogenic process. A comparison of saprophytic and phytopathogenic mycelial fungi revealed one characteristic feature, namely, the appearance of a new trypsin-like activity in phytopathogens that is absent in saprophytes. To clear up the question of whether the degree of pathogenicity of a fungus is related to the activity of secreted trypsin-like protease, several species of Fusarium of various pathogenicity were compared. In two species, F. sporotrichioides (which causes ear fusa-riosis of rye) and F. heterosporum (the causative agent of root rot in wheat), a clear correlation between the activity and pathogenicity was revealed: the more pathogenetic F. sporotrichioides exhibited a higher extracellular trypsin-like activity than the less pathogenetic species F. heterosporum. Thus, the presence of trypsin-like activity in a saprotroph-pathogen pair may be an indicator of the pathogenicity of a fungus; in some cases, the value of this activity may indicate the degree of its pathogenicity. This suggests that trypsin-like proteases specific to phytopathogens are directly involved in the pathogenetic process, probably, through interaction with the "sentry" protein or the product of the resistance gene.

  10. Pathogen-Secreted Proteases Activate a Novel Plant Immune Pathway

    PubMed Central

    Cheng, Zhenyu; Li, Jian-Feng; Niu, Yajie; Zhang, Xue-Cheng; Woody, Owen Z.; Xiong, Yan; Djonović, Slavica; Millet, Yves; Bush, Jenifer; McConkey, Brendan J.; Sheen, Jen; Ausubel, Frederick M.

    2015-01-01

    Mitogen-Activated Protein Kinase (MAPK) cascades play central roles in innate immune signaling networks in plants and animals1,2. In plants, however, the molecular mechanisms of how signal perception is transduced to MAPK activation remain elusive1. We report that pathogen-secreted proteases activate a previously unknown signaling pathway in Arabidopsis thaliana involving the Gα, Gβ and Gγ subunits of heterotrimeric G-protein complexes, which function upstream of a MAPK cascade. In this pathway, Receptor for Activated C Kinase 1 (RACK1) functions as a novel scaffold that binds to the Gβ subunit as well as to all three tiers of the MAPK cascade, thereby linking upstream G protein signaling to downstream activation of a MAPK cascade. The protease-G protein-RACK1-MAPK cascade modules identified in these studies are distinct from previously described plant immune signaling pathways such as the one elicited by bacterial flagellin, in which G proteins function downstream of or in parallel to a MAPK cascade without the involvement of the RACK1 scaffolding protein. The discovery of the novel protease-mediated immune signaling pathway described here was facilitated by the use of the broad host range, opportunistic bacterial pathogen Pseudomonas aeruginosa. The ability of P. aeruginosa to infect both plants and animals makes it an excellent model to identify novel types of immunoregulatory strategies that account for its niche adaptation to diverse host tissues and immune systems. PMID:25731164

  11. Humanized-VHH Transbodies that Inhibit HCV Protease and Replication

    PubMed Central

    Jittavisutthikul, Surasak; Thanongsaksrikul, Jeeraphong; Thueng-in, Kanyarat; Chulanetra, Monrat; Srimanote, Potjanee; Seesuay, Watee; Malik, Aijaz Ahmad; Chaicumpa, Wanpen

    2015-01-01

    There is a need for safe and broadly effective anti-HCV agents that can cope with genetic multiplicity and mutations of the virus. In this study, humanized-camel VHHs to genotype 3a HCV serine protease were produced and were linked molecularly to a cell penetrating peptide, penetratin (PEN). Human hepatic (Huh7) cells transfected with the JFH-1 RNA of HCV genotype 2a and treated with the cell penetrable nanobodies (transbodies) had a marked reduction of the HCV RNA intracellularly and in their culture fluids, less HCV foci inside the cells and less amounts of HCV core antigen in culture supernatants compared with the infected cells cultured in the medium alone. The PEN-VHH-treated-transfected cells also had up-regulation of the genes coding for the host innate immune response (TRIF, TRAF3, IRF3, IL-28B and IFN-β), indicating that the cell penetrable nanobodies rescued the host innate immune response from the HCV mediated-suppression. Computerized intermolecular docking revealed that the VHHs bound to residues of the protease catalytic triad, oxyanion loop and/or the NS3 N-terminal portion important for non-covalent binding of the NS4A protease cofactor protein. The so-produced transbodies have high potential for testing further as a candidate for safe, broadly effective and virus mutation tolerable anti-HCV agents. PMID:25903832

  12. Enzyme histochemical studies of membrane proteases in rat subfornical organ.

    PubMed

    De Bault, L E; Mitro, A

    1994-12-01

    Localization of membrane proteases glutamyl aminopeptidase (EAP), microsomal alanyl aminopeptidase (mAAP), dipeptidyl peptidase IV (DPP IV) and gamma-glutamyl transpeptidase (gamma-GTP) were studied in vessels of the rat subfornical organ (SFO), ependyma which cover the surface of the SFO, and adjacent brain structures. Results of enzyme histochemical reactions showed strong activity for EAP, mAAP, and gamma-GTP, but absence of DPP IV in microvessels of SFO. The ependyma which cover the SFO was positive for gamma-GTP, but negative for other studied proteases. Our results showed that the spectrum of enzymes in the majority of the vessels of SFO is similar to that of the microvessels of the adjacent brain tissue which were positive for EAP, mAAP, and gamma-GTP, but negative for DPP IV. The relative intensity of the enzyme reactions in vessels varied from central to lateral locations in the SFO and the adjacent brain tissue. There was also a difference in the relative reaction intensity from one enzyme to the other. The presence and heterogeneous distribution of the enzymes are consistent with the hypothesis that membrane proteases of the microvascular endothelium constitute an enzyme-barrier between blood and parenchyma of the SFO and between blood and brain tissue, and may be involved in metabolism or modulation of various peptides when they contact the plasma membrane of the endothelial cells of the vessels.

  13. The functional and pathologic relevance of autophagy proteases

    PubMed Central

    Fernández, Álvaro F.; López-Otín, Carlos

    2015-01-01

    Autophagy is a well-conserved catabolic process essential for cellular homeostasis. First described in yeast as an adaptive response to starvation, this pathway is also present in higher eukaryotes, where it is triggered by stress signals such as damaged organelles or pathogen infection. Autophagy is characterized at the cellular level by the engulfment of portions of the cytoplasm in double-membrane structures called autophagosomes. Autophagosomes fuse with lysosomes, resulting in degradation of the inner autophagosomal membrane and luminal content. This process is coordinated by complex molecular systems, including the ATG8 ubiquitin–like conjugation system and the ATG4 cysteine proteases, which are implicated in the formation, elongation, and fusion of these autophagic vesicles. In this Review, we focus on the diverse functional roles of the autophagins, a protease family formed by the four mammalian orthologs of yeast Atg4. We also address the dysfunctional expression of these proteases in several pathologic conditions such as cancer and inflammation and discuss potential therapies based on their modulation. PMID:25654548

  14. Cloning, nucleotide sequence, and expression of Achromobacter protease I gene.

    PubMed

    Ohara, T; Makino, K; Shinagawa, H; Nakata, A; Norioka, S; Sakiyama, F

    1989-12-01

    Achromobacter protease I (API) is a lysine-specific serine protease which hydrolyzes specifically the lysyl peptide bond. A gene coding for API was cloned from Achromobacter lyticus M497-1. Nucleotide sequence of the cloned DNA fragment revealed that the gene coded for a single polypeptide chain of 653 amino acids. The N-terminal 205 amino acids, including signal peptide and the threonine/serine-rich C-terminal 180 amino acids are flanking the 268 amino acid-mature protein which was identified by protein sequencing. Escherichia coli carrying a plasmid containing the cloned API gene overproduced and secreted a protein of Mr 50,000 (API') into the periplasm. This protein exhibited a distinct endopeptidase activity specific for lysyl bonds as well. The N-terminal amino acid sequence of API' was the same as mature API, suggesting that the enzyme retained the C-terminal extended peptide chain. The present experiments indicate that API, an extracellular protease produced by gram-negative bacteria, is synthesized in vivo as a precursor protein bearing long extended peptide chains at both N and C termini. PMID:2684982

  15. Enhanced Thermostability of a Fungal Alkaline Protease by Different Additives

    PubMed Central

    Nirmal, Nilesh P.; Laxman, R. Seeta

    2014-01-01

    A fungal strain (Conidiobolus brefeldianus MTCC 5184) isolated from plant detritus secreted a high activity alkaline protease. Thermostability studies of the fungal alkaline protease (FAP) revealed that the protease is stable up to 50°C with 40% residual activity after one hour. Effect of various additives such as sugars, sugar alcohols, polyols, and salts, on the thermostability of FAP was evaluated. Among the additives tested, glycerol, mannitol, xylitol, sorbitol, and trehalose were found to be very effective in increasing the stability of FAP, which was found to be concentration dependent. Fivefold increase in residual activity of FAP was observed in the presence of trehalose (50%) and sorbitol (50%) at 50°C for 4 h, compared to FAP without additive. Other additives like calcium at 20 mM and 10–15% ammonium sulphate showed lower stability improvement than trehalose and sorbitol. NaCl, MgCl2, K2HPO4, and glycine were found to be poor stabilizers and showed only a marginal improvement. PEG 6000 did not show any increase in stability but was found to be slightly inhibitory. PMID:25105022

  16. Identification of papain-like cysteine proteases from the bovine piroplasm Babesia bigemina and evolutionary relationship of piroplasms C1 family of cysteine proteases.

    PubMed

    Martins, Tiago M; do Rosário, Virgílio E; Domingos, Ana

    2011-01-01

    Papain-like cysteine proteases have been shown to have essential roles in parasitic protozoa and are under study as promising drug targets. Five genes were identified by sequence similarity search to be homologous to the cysteine protease family in the ongoing Babesia bigemina genome sequencing project database and were compared with the annotated genes from the complete bovine piroplasm genomes of Babesia bovis, Theileria annulata, and Theileria parva. Multiple genome alignments and sequence analysis were used to evaluate the molecular evolution events that occurred in the C1 family of cysteine proteases in these piroplasms of veterinary importance. BbiCPL1, one of the newly identified cysteine protease genes in the B. bigemina genome was expressed in Escherichia coli and shows activity against peptide substrates. Considerable differences were observed in the cysteine protease family between Babesia and Theileria genera, and this may partially explain the diverse infection mechanisms of these tick-borne diseases. PMID:20655912

  17. Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors.

    PubMed

    Zeminian, Luciana Bonome; Padovani, Juliana Lara; Corvino, Sílvia Maria; Silva, Giovanni Faria; Pardini, Maria Inês de Moura Campos; Grotto, Rejane Maria Tommasini

    2013-02-01

    The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV) RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3) have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.

  18. Anatomical localization of protease-activated receptor-1 and protease-mediated neuroglial crosstalk on peri-synaptic astrocytic endfeet.

    PubMed

    Shavit, Efrat; Michaelson, Daniel M; Chapman, Joab

    2011-11-01

    We studied the localization, activation and function of protease-activated receptor 1 (PAR-1) at the CNS synapse utilizing rat brain synaptosomes and slices. Confocal immunofluoresence and transmission electron microscopy in brain slices with pre-embedding diaminobenzidine (DAB) immunostaining found PAR-1 predominantly localized to the peri-synaptic astrocytic endfeet. Structural confocal immunofluorescence microscopy studies of isolated synaptosomes revealed spherical structures stained with anti-PAR-1 antibody which co-stained mainly for glial-filament acidic protein compared with the neuronal markers synaptophysin and PSD-95. Immunoblot studies of synaptosomes demonstrated an appropriate major band corresponding to PAR-1 and activation of the receptor by a specific agonist peptide (SFLLRN) significantly modulated phosphorylated extracellular signal-regulated kinase. A significant membrane potential depolarization was produced by thrombin (1 U/mL) and the PAR-1 agonist (100 μM) and depolarization by high K(+) elevated extracellular thrombin-like activity in the synaptosomes preparation. The results indicate PAR-1 localized to the peri-synaptic astrocytic endfeet is most likely activated by synaptic proteases and induces cellular signaling and modulation of synaptic electrophysiology. A protease mediated neuron-glia pathway may be important in both physiological and pathological regulation of the synapse. PMID:21854391

  19. Serological Analysis of Immunogenic Properties of Recombinant Meningococcus IgA1 Protease-Based Proteins.

    PubMed

    Kotelnikova, O V; Zinchenko, A A; Vikhrov, A A; Alliluev, A P; Serova, O V; Gordeeva, E A; Zhigis, L S; Zueva, V S; Razgulyaeva, O A; Melikhova, T D; Nokel, E A; Drozhzhina, E Yu; Rumsh, L D

    2016-07-01

    Using the genome sequence of IgA1 protease of N. meningitidis of serogroup B, four recombinant proteins of different structure and molecular weight were constructed. These proteins were equal in inducing the formation of specific antibodies to IgA1 protease and had protective properties against meningococci. In the sera of immunized mice, anti-IgA1 protease antibodies were detected by whole-cell ELISA, which indicated the presence of IgA1 protease on the surface of these bacteria. We hypothesized that the protective properties of IgA1 protease-based antigens and IgA1 protease analogs could be realized not only via impairment of bacterium adhesion to the mucosa, but also via suppression of this pathogen in the organism. The presented findings seem promising for using these proteins as the basis for anti-meningococcus vaccine.

  20. Serine proteases of the human immune system in health and disease.

    PubMed

    Heutinck, Kirstin M; ten Berge, Ineke J M; Hack, C Erik; Hamann, Jörg; Rowshani, Ajda T

    2010-07-01

    Serine proteases form a large family of protein-cleaving enzymes that play an essential role in processes like blood coagulation, apoptosis and inflammation. Immune cells express a wide variety of serine proteases such as granzymes in cytotoxic lymphocytes, neutrophil elastase, cathepsin G and proteinase 3 in neutrophils and chymase and tryptase in mast cells. Regulation of proteolysis induced by these serine proteases is essential to prevent self-induced damage. Hence, there are specialized serine protease inhibitors, serpins, which are broadly distributed. Here, we discuss the function of human serine proteases in inflammation, apoptosis and tissue remodeling. Furthermore, we address their impact on development and progression of immune mediated-diseases. Understanding the mode of action of serine proteases will help to unravel molecular processes involved in immunological disorders and will facilitate the identification of new therapeutic targets.

  1. A bead-based cleavage method for large-scale identification of protease substrates

    PubMed Central

    Wang, Chunli; Ye, Mingliang; Wei, Xiaoluan; Bian, Yangyang; Cheng, Kai; Zou, Hanfa

    2016-01-01

    Proteolysis is a major form of post translational modification which occurs when a protease cleaves peptide bonds in a target protein to modify its activity. Tracking protease substrates is indispensable for understanding its cellular functions. However, it is difficult to directly identify protease substrates because the end products of proteolysis, the cleaved protein fragments, must be identified among the pool of cellular proteins. Here we present a bead-based cleavage approach using immobilized proteome as the screening library to identify protease substrates. This method enables efficient separation of proteolyzed proteins from background protein mixture. Using caspase-3 as the model protease, we have identified 1159 high confident substrates, among which, strikingly, 43.9% of substrates undergo degradation during apoptosis. The huge number of substrates and positive support of in vivo evidence indicate that the BBC method is a powerful tool for protease substrates identification. PMID:26935269

  2. A bead-based cleavage method for large-scale identification of protease substrates.

    PubMed

    Wang, Chunli; Ye, Mingliang; Wei, Xiaoluan; Bian, Yangyang; Cheng, Kai; Zou, Hanfa

    2016-01-01

    Proteolysis is a major form of post translational modification which occurs when a protease cleaves peptide bonds in a target protein to modify its activity. Tracking protease substrates is indispensable for understanding its cellular functions. However, it is difficult to directly identify protease substrates because the end products of proteolysis, the cleaved protein fragments, must be identified among the pool of cellular proteins. Here we present a bead-based cleavage approach using immobilized proteome as the screening library to identify protease substrates. This method enables efficient separation of proteolyzed proteins from background protein mixture. Using caspase-3 as the model protease, we have identified 1159 high confident substrates, among which, strikingly, 43.9% of substrates undergo degradation during apoptosis. The huge number of substrates and positive support of in vivo evidence indicate that the BBC method is a powerful tool for protease substrates identification. PMID:26935269

  3. Protease activities of Candida spp. isolated from otitis externa: preliminary result.

    PubMed

    Arsović, N A; Banko, A V; Dimitrijević, M V; Djordjević, V Z; Milovanović, J P; Arsenijević, V A

    2009-01-01

    Otomycosis is a fungal infection of the ear predominantly caused by Candida and Aspergillus spp. The possible virulence factors of Candida spp. are enzymes, such as proteases, phospholipases, phosphatases and esterase. According to our knowledge, protease production in Candida strains isolated from patients with otomycosis has not been investigated. The present study was aimed at determining in vitro protease activity in 8 strains of Candida spp. (C. parapsilosis, C. famata, C. guilliermondii and C. albicans) isolated from children with otomycosis. A majority of isolated strains 7/8 (87.5%) were protease positive. The protease activity ranged from Pz 0.61 to 0.78. Further investigation is necessary to clarify the contribution of protease production to Candida virulence associated with otomycosis.

  4. Using C. elegans to Identify the Protease Targets of Serpins In Vivo

    PubMed Central

    Bhatia, Sangeeta R.; Miedel, Mark T.; Chotoo, Cavita K.; Graf, Nathan J.; Hood, Brian L.; Conrads, Thomas P.; Silverman, Gary A.; Luke, Cliff J.

    2015-01-01

    Most serpins inhibit serine and/or cysteine proteases, and their inhibitory activities are usually defined in vitro. However, the physiological protease targets of most serpins are unknown despite many years of research. This may be due to the rapid degradation of the inactive serpin:protease complexes and/or the conditions under which the serpin inhibits the protease. The model organism Caenorhabditis elegans is an ideal system for identifying protease targets due to powerful forward and reverse genetics, as well as the ease of creating transgenic animals. Using combinatorial approaches of genetics and biochemistry in C. elegans, the true in vivo protease targets of the endogenous serpins can be elucidated. PMID:21683259

  5. The Plasticity of the β-Trefoil Fold Constitutes an Evolutionary Platform for Protease Inhibition*

    PubMed Central

    Azarkan, Mohamed; Martinez-Rodriguez, Sergio; Buts, Lieven; Baeyens-Volant, Danielle; Garcia-Pino, Abel

    2011-01-01

    Proteases carry out a number of crucial functions inside and outside the cell. To protect the cells against the potentially lethal activities of these enzymes, specific inhibitors are produced to tightly regulate the protease activity. Independent reports suggest that the Kunitz-soybean trypsin inhibitor (STI) family has the potential to inhibit proteases with different specificities. In this study, we use a combination of biophysical methods to define the structural basis of the interaction of papaya protease inhibitor (PPI) with serine proteases. We show that PPI is a multiple-headed inhibitor; a single PPI molecule can bind two trypsin units at the same time. Based on sequence and structural analysis, we hypothesize that the inherent plasticity of the β-trefoil fold is paramount in the functional evolution of this family toward multiple protease inhibition. PMID:22027836

  6. Opportunistic Infections in HIV-Infected Patients Differ Strongly in Frequencies and Spectra between Patients with Low CD4+ Cell Counts Examined Postmortem and Compensated Patients Examined Antemortem Irrespective of the HAART Era

    PubMed Central

    Powell, Marta K.; Benková, Kamila; Selinger, Pavel; Dogoši, Marek; Kinkorová Luňáčková, Iva; Koutníková, Hana; Laštíková, Jarmila; Roubíčková, Alena; Špůrková, Zuzana; Laclová, Lucie; Eis, Václav; Šach, Josef

    2016-01-01

    Objective AIDS-related mortality has changed dramatically with the onset of highly active antiretroviral therapy (HAART), which has even allowed compensated HIV-infected patients to withdraw from secondary therapy directed against opportunistic pathogens. However, in recently autopsied HIV-infected patients, we observed that associations with a broad spectrum of pathogens remain, although detailed analyses are lacking. Therefore, we focused on the possible frequency and spectrum shifts in pathogens associated with autopsied HIV-infected patients. Design We hypothesized that the pathogens frequency and spectrum changes found in HIV-infected patients examined postmortem did not recapitulate the changes found previously in HIV-infected patients examined antemortem in both the pre- and post-HAART eras. Because this is the first comprehensive study originating from Central and Eastern Europe, we also compared our data with those obtained in the West and Southwest Europe, USA and Latin America. Methods We performed autopsies on 124 HIV-infected patients who died from AIDS or other co-morbidities in the Czech Republic between 1985 and 2014. The pathological findings were retrieved from the full postmortem examinations and autopsy records. Results We collected a total of 502 host-pathogen records covering 82 pathogen species, a spectrum that did not change according to patients’ therapy or since the onset of the epidemics, which can probably be explained by the fact that even recently deceased patients were usually decompensated (in 95% of the cases, the last available CD4+ cell count was falling below 200 cells*μl-1) regardless of the treatment they received. The newly identified pathogen taxa in HIV-infected patients included Acinetobacter calcoaceticus, Aerococcus viridans and Escherichia hermannii. We observed a very limited overlap in both the spectra and frequencies of the pathogen species found postmortem in HIV-infected patients in Europe, the USA and Latin

  7. MALT1 Protease Activity Is Required for Innate and Adaptive Immune Responses

    PubMed Central

    Yu, Jong W.; Hoffman, Sandy; Beal, Allison M.; Dykon, Angela; Ringenberg, Michael A.; Hughes, Anna C.; Dare, Lauren; Anderson, Amber D.; Finger, Joshua; Kasparcova, Viera; Rickard, David; Berger, Scott B.; Ramanjulu, Joshi; Emery, John G.; Gough, Peter J.; Bertin, John; Foley, Kevin P.

    2015-01-01

    CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4+ and CD8+ T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. In vivo, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses in vivo. PMID:25965667

  8. Involvement of serine proteases in the excystation and metacystic development of Entamoeba invadens.

    PubMed

    Makioka, Asao; Kumagai, Masahiro; Kobayashi, Seiki; Takeuchi, Tsutomu

    2009-10-01

    Although the functions of cysteine proteases involved in the pathogenicity and differentiation of Entamoeba histolytica have been demonstrated, little is known about the functions of serine proteases. We examined the involvement of serine proteases in amoebic excystation and metacystic development using inhibitors specific for serine proteases. Entamoeba invadens IP-1 strain was used as the model of excystation and metacystic development of E. histolytica. Four serine protease inhibitors, phenylmethanesulfonyl fluoride (PMSF), 4-(2-aminoethyl) bezensulfonylfluoride hydrochloride, 3, 4-dichloroisocoumarin, and N-tosyl-phe-chloromethylketone, decreased the number of metacystic amoebae in a dose-dependent manner, without showing cytotoxicity to cysts. PMSF inhibited not only the increase but also the development of metacystic amoebae as determined by the change of nucleus number from four- to one-nucleate amoebae. The protease activity in cyst lysates was also inhibited by PMSF and the band of protease on gelatin sodium dodecyl sulfate polyacrylamide gel electrophoresis was weaker than controls when treated with PMSF. Three serine protease families, S28 (three types), S9 (two), and S26 (one) were retrieved from the database of E. invadens. Phylogenetic analysis revealed that amebic enzymes from the serine protease families formed different clades from those from other organisms. The expression levels of these serine proteases in cysts 5 h after the induction of excystation as assessed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) were higher than those observed prior to induction assayed by real-time RT-PCR; the increase in one type of S9 (named S9-3) expression was the highest. The expression of S9 enzymes also increased from cysts to trophozoites higher than the other family serine proteases. Thus, the results show that Entamoeba uses their serine proteases in the excystation and metacystic development, which leads to successful infection.

  9. Chaperone-protease systems in regulation and protein quality control in Bacillus subtilis.

    PubMed

    Molière, Noël; Turgay, Kürşad

    2009-11-01

    The Gram-positive model organism Bacillus subtilis is extremely well adapted to changing environmental conditions. The chaperone-protease ClpCP and other AAA+ proteases constitute an important component of the B. subtilis protein quality control system that is essential for survival during stress. In this review, we discuss recent discoveries concerning the molecular mechanism, regulation and localization of proteases and chaperones in B. subtilis.

  10. The salt-sensitive structure and zinc inhibition of Borrelia burgdorferi protease BbHtrA.

    PubMed

    Russell, Theresa M; Tang, Xiaoling; Goldstein, Jason M; Bagarozzi, Dennis; Johnson, Barbara J B

    2016-02-01

    HtrA serine proteases are highly conserved and essential ATP-independent proteases with chaperone activity. Bacteria express a variable number of HtrA homologues that contribute to the virulence and pathogenicity of bacterial pathogens. Lyme disease spirochetes possess a single HtrA protease homologue, Borrelia burgdorferi HtrA (BbHtrA). Previous studies established that, like the human orthologue HtrA1, BbHtrA is proteolytically active against numerous extracellular proteins in vitro. In this study, we utilized size exclusion chromatography and blue native polyacrylamide gel electrophoresis (BN-PAGE) to demonstrate BbHtrA oligomeric structures that were substrate independent and salt sensitive. Examination of the influence of transition metals on the activity of BbHtrA revealed that this protease is inhibited by Zn(2+) > Cu(2+) > Mn(2+). Extending this analysis to two other HtrA proteases, E. coli DegP and HtrA1, revealed that all three HtrA proteases were reversibly inhibited by ZnCl2 at all micro molar concentrations examined. Commercial inhibitors for HtrA proteases are not available and physiologic HtrA inhibitors are unknown. Our observation of conserved zinc inhibition of HtrA proteases will facilitate structural and functional studies of additional members of this important class of proteases. PMID:26480895

  11. Nine Crystal Structures Determine the Substrate Envelope of the MDR HIV-1 Protease

    SciTech Connect

    Liu, Zhigang; Wang, Yong; Brunzelle, Joseph; Kovari, Iulia A.; Kovari, Ladislau C.

    2012-03-27

    Under drug selection pressure, emerging mutations render HIV-1 protease drug resistant, leading to the therapy failure in anti-HIV treatment. It is known that nine substrate cleavage site peptides bind to wild type (WT) HIV-1 protease in a conserved pattern. However, how the multidrug-resistant (MDR) HIV-1 protease binds to the substrate cleavage site peptides is yet to be determined. MDR769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, and 90) was selected for present study to understand the binding to its natural substrates. MDR769 HIV-1 protease was co-crystallized with nine substrate cleavage site hepta-peptides. Crystallographic studies show that MDR769 HIV-1 protease has an expanded substrate envelope with wide open flaps. Furthermore, ligand binding energy calculations indicate weaker binding in MDR769 HIV-1 protease-substrate complexes. These results help in designing the next generation of HIV-1 protease inhibitors by targeting the MDR HIV-1 protease.

  12. Cysteine Proteases: Modes of Activation and Future Prospects as Pharmacological Targets

    PubMed Central

    Verma, Sonia; Dixit, Rajnikant; Pandey, Kailash C.

    2016-01-01

    Proteolytic enzymes are crucial for a variety of biological processes in organisms ranging from lower (virus, bacteria, and parasite) to the higher organisms (mammals). Proteases cleave proteins into smaller fragments by catalyzing peptide bonds hydrolysis. Proteases are classified according to their catalytic site, and distributed into four major classes: cysteine proteases, serine proteases, aspartic proteases, and metalloproteases. This review will cover only cysteine proteases, papain family enzymes which are involved in multiple functions such as extracellular matrix turnover, antigen presentation, processing events, digestion, immune invasion, hemoglobin hydrolysis, parasite invasion, parasite egress, and processing surface proteins. Therefore, they are promising drug targets for various diseases. For preventing unwanted digestion, cysteine proteases are synthesized as zymogens, and contain a prodomain (regulatory) and a mature domain (catalytic). The prodomain acts as an endogenous inhibitor of the mature enzyme. For activation of the mature enzyme, removal of the prodomain is necessary and achieved by different modes. The pro-mature domain interaction can be categorized as protein–protein interactions (PPIs) and may be targeted in a range of diseases. Cysteine protease inhibitors are available that can block the active site but no such inhibitor available yet that can be targeted to block the pro-mature domain interactions and prevent it activation. This review specifically highlights the modes of activation (processing) of papain family enzymes, which involve auto-activation, trans-activation and also clarifies the future aspects of targeting PPIs to prevent the activation of cysteine proteases. PMID:27199750

  13. Cysteine Proteases: Modes of Activation and Future Prospects as Pharmacological Targets.

    PubMed

    Verma, Sonia; Dixit, Rajnikant; Pandey, Kailash C

    2016-01-01

    Proteolytic enzymes are crucial for a variety of biological processes in organisms ranging from lower (virus, bacteria, and parasite) to the higher organisms (mammals). Proteases cleave proteins into smaller fragments by catalyzing peptide bonds hydrolysis. Proteases are classified according to their catalytic site, and distributed into four major classes: cysteine proteases, serine proteases, aspartic proteases, and metalloproteases. This review will cover only cysteine proteases, papain family enzymes which are involved in multiple functions such as extracellular matrix turnover, antigen presentation, processing events, digestion, immune invasion, hemoglobin hydrolysis, parasite invasion, parasite egress, and processing surface proteins. Therefore, they are promising drug targets for various diseases. For preventing unwanted digestion, cysteine proteases are synthesized as zymogens, and contain a prodomain (regulatory) and a mature domain (catalytic). The prodomain acts as an endogenous inhibitor of the mature enzyme. For activation of the mature enzyme, removal of the prodomain is necessary and achieved by different modes. The pro-mature domain interaction can be categorized as protein-protein interactions (PPIs) and may be targeted in a range of diseases. Cysteine protease inhibitors are available that can block the active site but no such inhibitor available yet that can be targeted to block the pro-mature domain interactions and prevent it activation. This review specifically highlights the modes of activation (processing) of papain family enzymes, which involve auto-activation, trans-activation and also clarifies the future aspects of targeting PPIs to prevent the activation of cysteine proteases. PMID:27199750

  14. Comparative analysis on the distribution of protease activities among fruits and vegetable resources.

    PubMed

    Sun, Qian; Zhang, Bin; Yan, Qiao-Juan; Jiang, Zheng-Qiang

    2016-12-15

    In this study, a comparative analysis on the distribution of protease activities among 90 plant resources, including fruits and vegetables, has been performed. Protease activities of plant extracts were assayed at different pH values (pH 3.0, pH 7.5 and pH 10.5) using casein as a substrate. Ten fruits and thirteen vegetables show protease activities above 10U/g. Pineapple, fig and papaya, which are used for commercial protease production, exhibited high protease activities. Additionally, high protease activities were detected in kiwifruit (28.8U/g), broccoli (16.9U/g), ginger (16.6U/g), leek (32.7U/g) and red pepper (15.8U/g) at different pH values. SDS-PAGE and zymograms confirmed that various types of proteases existed in the five plant extracts and might be explored. Furthermore, five plant extracts were treated by different protease inhibitors. These results show that there are still many plant resources unexplored, which may be promising candidates for plant-derived protease production.

  15. Comparative analysis on the distribution of protease activities among fruits and vegetable resources.

    PubMed

    Sun, Qian; Zhang, Bin; Yan, Qiao-Juan; Jiang, Zheng-Qiang

    2016-12-15

    In this study, a comparative analysis on the distribution of protease activities among 90 plant resources, including fruits and vegetables, has been performed. Protease activities of plant extracts were assayed at different pH values (pH 3.0, pH 7.5 and pH 10.5) using casein as a substrate. Ten fruits and thirteen vegetables show protease activities above 10U/g. Pineapple, fig and papaya, which are used for commercial protease production, exhibited high protease activities. Additionally, high protease activities were detected in kiwifruit (28.8U/g), broccoli (16.9U/g), ginger (16.6U/g), leek (32.7U/g) and red pepper (15.8U/g) at different pH values. SDS-PAGE and zymograms confirmed that various types of proteases existed in the five plant extracts and might be explored. Furthermore, five plant extracts were treated by different protease inhibitors. These results show that there are still many plant resources unexplored, which may be promising candidates for plant-derived protease production. PMID:27451238

  16. Synthesis and herbicidal evaluation of novel benzothiazole derivatives as potential inhibitors of D1 protease.

    PubMed

    Huang, Tonghui; Sun, Jie; An, Lin; Zhang, Lixian; Han, Cuiping

    2016-04-01

    D1 protease is a C-terminal processing protease that has been predicted to be an ideal herbicidal target. Three novel series of benzothiazole derivatives were synthesized and evaluated for their herbicidal activities against Brassica napus (rape) and Echinochloa crusgalli (barnyard grass). The preliminary bioassay indicated that most of the synthesized compounds possess promising D1 protease inhibitory activities and considerable herbicidal activities. Molecular docking was performed to position representative compounds into the active site of D1 protease to determine a probable binding model. PMID:26905829

  17. Structural and thermodynamic basis of resistance to HIV-1 protease inhibition: implications for inhibitor design.

    PubMed

    Velazquez-Campoy, Adrian; Muzammil, Salman; Ohtaka, Hiroyasu; Schön, Arne; Vega, Sonia; Freire, Ernesto

    2003-12-01

    One of the most serious side effects associated with the therapy of HIV-1 infection is the appearance of viral strains that exhibit resistance to protease inhibitors. At the molecular level, resistance to protease inhibition predominantly takes the form of mutations within the protease molecule that preferentially lower the affinity of protease inhibitors with respect to protease substrates, while still maintaining a viable catalytic activity. Mutations associated with drug resistance occur within the active site cavity as well as distal sites. Active site mutations affect directly inhibitor/protease interactions while non-active site mutations affect inhibitor binding through long range cooperative perturbations. The effects of mutations associated with drug resistance are compounded by the presence of naturally occurring polymorphisms, especially those observed in non-B subtypes of HIV-1. The binding thermodynamics of all clinical inhibitors against the wild type protease, drug resistant mutations and non-B subtype HIV-1 proteases has been determined by high sensitivity isothermal titration calorimetry. In conjunction with structural information, these data have provided a precise characterization of the binding mechanism of different inhibitors and their response to mutations. Inhibitors that exhibit extremely high affinity and low susceptibility to the effects of mutations share common features and binding determinants even if they belong to different chemical scaffolds. These binding determinants define a set of rules and constraints for the design of better HIV-1 protease inhibitors.

  18. Potent inhibition of feline coronaviruses with peptidyl compounds targeting coronavirus 3C-like protease.

    PubMed

    Kim, Yunjeong; Mandadapu, Sivakoteswara Rao; Groutas, William C; Chang, Kyeong-Ok

    2013-02-01

    Feline coronavirus infection is common among domestic and exotic felid species and usually associated with mild or asymptomatic enteritis; however, feline infectious peritonitis (FIP) is a fatal disease of cats that is caused by systemic infection with a feline infectious peritonitis virus (FIPV), a variant of feline enteric coronavirus (FECV). Currently, there is no specific treatment approved for FIP despite the importance of FIP as the leading infectious cause of death in young cats. During the replication process, coronavirus produces viral polyproteins that are processed into mature proteins by viral proteases, the main protease (3C-like [3CL] protease) and the papain-like protease. Since the cleavages of viral polyproteins are an essential step for virus replication, blockage of viral protease is an attractive target for therapeutic intervention. Previously, we reported the generation of broad-spectrum peptidyl inhibitors against viruses that possess a 3C or 3CL protease. In this study, we further evaluated the antiviral effects of the peptidyl inhibitors against feline coronaviruses, and investigated the interaction between our protease inhibitor and a cathepsin B inhibitor, an entry blocker, against a feline coronavirus in cell culture. Herein we report that our compounds behave as reversible, competitive inhibitors of 3CL protease, potently inhibited the replication of feline coronaviruses (EC(50) in a nanomolar range) and, furthermore, combination of cathepsin B and 3CL protease inhibitors led to a strong synergistic interaction against feline coronaviruses in a cell culture system.

  19. Immobilized protease on the magnetic nanoparticles used for the hydrolysis of rapeseed meals

    NASA Astrophysics Data System (ADS)

    Jin, Xin; Li, Ju-Fang; Huang, Ping-Ying; Dong, Xu-Yan; Guo, Lu-Lu; Yang, Liang; Cao, Yuan-Cheng; Wei, Fang; Zhao, Yuan-Di; Chen, Hong

    2010-07-01

    (3-aminopropl) triethoxysilaneand modified magnetic nanoparticles with the average diameter of 25.4 nm were synthesized in water-phase co-precipitation method. And then these nanoparticles were covalently coupled with alkaline protease as enzyme carrier by using 1,4-phenylene diisothlocyanate as coupling agent. Experiments showed that the immobilized protease can keep the catalytic bioactivity, which can reach to 47.8% when casein was served as substrate. Results showed that the catalytic activity of immobilized protease on these magnetic nanoparticles could retain 98.63±2.37% after 60 days. And it is more stable than the free protease during the shelf-life test. The enzyme reaction conditions such as optimum reaction temperature and pH are the same as free protease. Furthermore, mix-and-separate experiments showed that the immobilized protease could be recycled through the magnetic nanoparticles after the biocatalysis process. When the rapeseed meals were used as substrate, the degree of hydrolysis of immobilized alkaline protease achieved 9.86%, while it was 10.41% for the free protease. The macromolecular proteins of rapeseed meals were hydrolyzed by immobilized protease into small molecules such as polypeptides or amino acids. Thus, a novel efficient and economic way for the recycling of enzymes in the application of continuous production of active peptides was provided based on these magnetic nanoparticles.

  20. A biochemical comparison of proteases from pathogenic naegleria fowleri and non-pathogenic Naegleria gruberi.

    PubMed

    Serrano-Luna, Jesús; Cervantes-Sandoval, Isaac; Tsutsumi, Victor; Shibayama, Mineko

    2007-01-01

    Naegleria fowleri is the etiologic agent of primary amoebic meningoencephalitis (PAM). Proteases have been suggested to be involved in tissue invasion and destruction during infection. We analyzed and compared the complete protease profiles of total crude extract and conditioned medium of both pathogenic N. fowleri and non-pathogenic Naegleria gruberi trophozoites. Using SDS-PAGE, we found differences in the number and molecular weight of proteolytic bands between the two strains. The proteases showed optimal activity at pH 7.0 and 35 degrees C for both strains. Inhibition assays showed that the main proteolytic activity in both strains is due to cysteine proteases although serine proteases were also detected. Both N. fowleri and N. gruberi have a variety of different protease activities at different pH levels and temperatures. These proteases may allow the amoebae to acquire nutrients from different sources, including those from the host. Although, the role of the amoebic proteases in the pathogenesis of PAM is not clearly defined, it seems that proteases and other molecules of the parasite as well as those from the host, could be participating in the damage to the human central nervous system.

  1. Scouring Potential of Mesophile Acidic Proteases of Pseudomonas aeruginosa for Grey Cotton Fabrics

    NASA Astrophysics Data System (ADS)

    Saravanan, D.

    2013-04-01

    Mesophile, acidic proteases were produced using the microbial source, Pseudomonas aeruginosa, with wider thermal tolerances. Process conditions of scouring treatment were optimized using Taguchi method for optimum temperature, time, pH and concentration of protease. Treatment with the protease lower weight loss values compared to the alkali scouring, however, significant improvement in the absorbency compared to the grey samples was observed. Large amounts of pectin left out in the samples resulted in higher extractable impurities, substantiated by the FTIR results. Relatively, lower reduction in the tear strengths was observed in both warp and weft directions after protease treatment of the cotton fabrics.

  2. Determination of the protease cleavage site repertoire—The RNase H but not the RT domain is essential for foamy viral protease activity

    SciTech Connect

    Spannaus, Ralf; Bodem, Jochen

    2014-04-15

    In contrast to orthoretroviruses, the foamy virus protease is only active as a protease-reverse transcriptase fusion protein and requires viral RNA for activation. Maturation of foamy viral proteins seems to be restricted to a single cleavage site in Gag and Pol. We provide evidence that unprocessed Gag is required for optimal infectivity, which is unique among retroviruses. Analyses of the cleavage site sequences of the Gag and Pol cleavage sites revealed a high similarity compared to those of Lentiviruses. We show that positions P2' and P2 are invariant and that Gag and Pol cleavage sites are processed with similar efficiencies. The RNase H domain is essential for protease activity, but can functionally be substituted by RNase H domains of other retroviruses. Thus, the RNase H domain might be involved in the stabilization of the protease dimer, while the RT domain is essential for RNA dependent protease activation. - Highlights: • Unprocessed Gag is required for optimal infectivity of foamy viruses. • Positions P2 and P2' are invariant in the foamy viral cleavage sites. • The RNaseH domain is essential for protease activity. • The RNaseH domains of other retroviruses support foamy viral protease activity.

  3. HreP, an In Vivo-Expressed Protease of Yersinia enterocolitica, Is a New Member of the Family of Subtilisin/Kexin-Like Proteases

    PubMed Central

    Heusipp, Gerhard; Young, Glenn M.; Miller, Virginia L.

    2001-01-01

    The role of proteases in pathogenesis is well established for several microorganisms but has not been described for Yersinia enterocolitica. Previously, we identified a gene, hreP, which showed significant similarity to proteases in a screen for chromosomal genes of Y. enterocolitica that were exclusively expressed during an infection of mice. We cloned this gene by chromosome capture and subsequently determined its nucleotide sequence. Like inv, the gene encoding the invasin protein of Y. enterocolitica, hreP is located in a cluster of flagellum biosynthesis and chemotaxis genes. The genomic organization of this chromosomal region is different in Escherichia coli, Salmonella, and Yersinia pestis than in Y. enterocolitica. Analysis of the distribution of hreP between different Yersinia isolates and the relatively low G+C content of the gene suggests acquisition by horizontal gene transfer. Sequence analysis also revealed that HreP belongs to a family of eukaryotic subtilisin/kexin-like proteases. Together with the calcium-dependent protease PrcA of Anabaena variabilis, HreP forms a new subfamily of bacterial subtilisin/kexin-like proteases which might have originated from a common eukaryotic ancestor. Like other proteases of this family, HreP is expressed with an N-terminal prosequence. Expression of an HreP-His6 tag fusion protein in E. coli revealed that HreP undergoes autocatalytic processing at a consensus cleavage site of subtilisin/kexin-like proteases, thereby releasing the proprotein. PMID:11371518

  4. Decomposing the energetic impact of drug-resistant mutations: the example of HIV-1 protease-DRV binding.

    PubMed

    Cai, Yufeng; Schiffer, Celia

    2012-01-01

    HIV-1 protease is a major drug target for AIDS therapy. With the appearance of drug-resistant HIV-1 protease variants, understanding the mechanism of drug resistance becomes critical for rational drug design. Computational methods can provide more details about inhibitor-protease binding than crystallography and isothermal titration calorimetry. The latest FDA-approved HIV-1 protease inhibitor is Darunavir (DRV). Herein, each DRV atom is evaluated by free energy component analysis for its contribution to the binding affinity with wild-type protease and ACT, a drug-resistant variant. This information can contribute to the rational design of new HIV-1 protease inhibitors.

  5. Endothelial Progenitor Cells in Sprouting Angiogenesis: Proteases Pave the Way.

    PubMed

    Laurenzana, A; Fibbi, G; Margheri, F; Biagioni, A; Luciani, C; Del Rosso, M; Chillà, A

    2015-01-01

    Sprouting angiogenesis consists of the expansion and remodelling of existing vessels, where the vascular sprouts connect each other to form new vascular loops. Endothelial Progenitor Cells (EPCs) are a subtype of stem cells, with high proliferative potential, able to differentiate into mature Endothelial Cells (ECs) during the neovascularization process. In addition to this direct structural role EPCs improve neovascularization, also secreting numerous pro-angiogenic factors able to enhance the proliferation, survival and function of mature ECs, and other surrounding progenitor cells. While sprouting angiogenesis by mature ECs involves resident ECs, the vasculogenic contribution of EPCs is a high hurdle race. Bone marrowmobilized EPCs have to detach from the stem cell niche, intravasate into bone marrow vessels, reach the hypoxic area or tumour site, extravasate and incorporate into the new vessel lumen, thus complementing the resident mature ECs in sprouting angiogenesis. The goal of this review is to highlight the role of the main protease systems able to control each of these steps. The pivotal protease systems here described, involved in vascular patterning in sprouting angiogenesis, are the matrix-metalloproteinases (MMPs), the serineproteinases urokinase-type plasminogen activator (uPA) associated with its receptor (uPAR) and receptorassociated plasminogen/plasmin, the neutrophil elastase and the cathepsins. Since angiogenesis plays a critical role not only in physiological but also in pathological processes, such as in tumours, controlling the contribution of EPCs to the angiogenic process, through the regulation of the protease systems involved, could yield new opportunities for the therapeutic prospect of efficient control of pathological angiogenesis. PMID:26321757

  6. Phytochelatin synthase: of a protease a peptide polymerase made.

    PubMed

    Rea, Philip A

    2012-05-01

    Of the mechanisms known to protect vascular plants and some algae, fungi and invertebrates from the toxic effects of non-essential heavy metals such as As, Cd or Hg, one of the most sophisticated is the enzyme-catalyzed synthesis of phytochelatins (PCs). PCs, (γ-Glu-Cys)(n) Gly polymers, which serve as high-affinity, thiol-rich cellular chelators and contribute to the detoxification of heavy metal ions, are derived from glutathione (GSH; γ-Glu-Cys-Gly) and related thiols in a reaction catalyzed by phytochelatin synthases (PC synthases, EC 2.3.2.15). Using the enzyme from Arabidopsis thaliana (AtPCS1) as a model, the reasoning and experiments behind the conclusion that PC synthases are novel papain-like Cys protease superfamily members are presented. The status of S-substituted GSH derivatives as generic PC synthase substrates and the sufficiency of the N-terminal domain of the enzyme from eukaryotic and its half-size equivalents from prokaryotic sources, for net PC synthesis and deglycylation of GSH and its derivatives, respectively, are emphasized. The question of the common need or needs met by PC synthases and their homologs is discussed. Of the schemes proposed to account for the combined protease and peptide polymerase capabilities of the eukaryotic enzymes vs the limited protease capabilities of the prokaryotic enzymes, two that will be considered are the storage and homeostasis of essential heavy metals in eukaryotes and the metabolism of S-substituted GSH derivatives in both eukaryotes and prokaryotes.

  7. Activity based chemical proteomics: profiling proteases as drug targets.

    PubMed

    Heal, William Percy; Wickramasinghe, Sasala Roshinie; Tate, Edward William

    2008-09-01

    The pivotal role of proteases in many diseases has generated considerable interest in their basic biology, and in the potential to target them for chemotherapy. Although fundamental to the initiation and progression of diseases such as cancer, diabetes, arthritis and malaria, in many cases their precise role remains unknown. Activity-based chemical proteomics-an emerging field involving a combination of organic synthesis, biochemistry, cell biology, biophysics and bioinformatics-allows the detection, visualisation and activity quantification of whole families or selected sub-sets of proteases based upon their substrate specificity. This approach can be applied for drug target/lead identification and validation, the fundamentals of drug discovery. The activity-based probes discussed in this review contain three key features; a 'warhead' (binds irreversibly but selectively to the active site), a 'tag' (allowing enzyme 'handling', with a combination of fluorescent, affinity and/or radio labels), and a linker region between warhead and tag. From the design and synthesis of the linker arise some of the latest developments discussed here; not only can the physical properties (e.g., solubility, localisation) of the probe be tuned, but the inclusion of a cleavable moiety allows selective removal of tagged enzyme from affinity beads etc. The design and synthesis of recently reported probes is discussed, including modular assembly of highly versatile probes via solid phase synthesis. Recent applications of activity-based protein profiling to specific proteases (serine, threonine, cysteine and metalloproteases) are reviewed as are demonstrations of their use in the study of disease function in cancer and malaria.

  8. Binding to the Open Conformation of HIV-1 Protease

    PubMed Central

    Lexa, Katrina W.; Carlson, Heather A.

    2011-01-01

    A recent crystal structure of HIV-1 protease (HIVp) was the first to experimentally observe a ligand targeting an open-flap conformation. Researchers studying a symmetric pyrrolidine inhibitor found that two ligands co-crystallized with the protease, forcing an unusual configuration and unique crystallographic contacts. One molecule is centered in the traditional binding site (α pose), and the other binds between the flaps (β pose). The ligands stack against each other in a region termed the “eye” site. Ligands bound to the eye site should prevent flap closure, but it is unclear if the pyrrolidine inhibitors or the crystal packing are causing the open state. Molecular dynamics simulations were used to examine the solution-state behavior of three possible binding modes: the ternary complex of HIVp+αβ and the binary complexes, HIVp+α and HIVp+β. We show that HIVp+α is the most stable of the three states. During conformational sampling, α takes an asymmetric binding pose, with one naphthyl ring occupying the eye site and the other reoriented down to occupy positions seen with traditional inhibitors. This finding supports previous studies that reveal a requirement for asymmetric binding at the eye site. In fact, if the α pose is modified to splay both naphthyl rings across the binding site like traditional inhibitors, one ring consistently flips to occupy the eye site. Our simulations reveal that interactions to the eye site encourage a conformationally-restrained state, and understanding those contacts may aid the design of ligands to specifically target alternate conformations of the protease. PMID:21604303

  9. Structural basis of substrate specificity in the serine proteases.

    PubMed Central

    Perona, J. J.; Craik, C. S.

    1995-01-01

    Structure-based mutational analysis of serine protease specificity has produced a large database of information useful in addressing biological function and in establishing a basis for targeted design efforts. Critical issues examined include the function of water molecules in providing strength and specificity of binding, the extent to which binding subsites are interdependent, and the roles of polypeptide chain flexibility and distal structural elements in contributing to specificity profiles. The studies also provide a foundation for exploring why specificity modification can be either straightforward or complex, depending on the particular system. PMID:7795518

  10. Lung vascular injury with protease infusion. Relationship to plasma fibronectin.

    PubMed Central

    Cohler, L F; Saba, T M; Lewis, E P

    1985-01-01

    Fibronectin exists in a soluble form in plasma and in an insoluble form in tissues. Plasma fibronectin can modulate phagocytic function as well as incorporate into the tissue matrix where it is believed to influence microvascular integrity and tissue repair. The temporal alterations in plasma and lung lymph fibronectin were studied in relation to increased pulmonary vascular permeability induced by protease infusion. The acute sheep lung lymph fistula model was used. A 39% decrease in plasma fibronectin (control = 421 +/- 67 micrograms/ml) was observed 2.5 hours (255 +/- 43 micrograms/ml) after protease infusion. There was an elevation of lymph fibronectin early after protease infusion, followed by a progressive decline. Concomitant with the decrease in plasma fibronectin, an increase in lymph flow (QL) of greater than 200% (from a control of 6.7 +/- 1.0 ml/hr to 13.9 +/- 1.4 ml/hr) was observed within 2.5 hours. Also, there was a sustained elevation in the total protein lymph/plasma concentration (L/P) ratio, which was maximal at 2.5 hours. The transvascular protein clearance (TVPC = QL X L/P) was 4.5 +/- 0.7 ml/hr at the control period and 13.1 +/- 2.0 ml/hr by 2.5 hours. This was indicative of increased flux of protein-rich fluid across the pulmonary endothelial barrier. Lung vascular permeability stabilized after 2.5 hours as manifested by a slowly declining L/P ratio. Thus, plasma fibronectin deficiency may contribute to the etiology of increased lung vascular permeability with protease infusion. Since the progressive decline in plasma fibronectin was not reflected in a proportional increase in lymph fibronectin, plasma fibronectin may have sequestered in tissues such as the lung, or perhaps in reticuloendothelial cells during the injury phase. Whether the progressive decrease in plasma fibronectin reflects its incorporation into the endothelial barrier matrix where it may mediate stabilization of the pulmonary microvascular barrier remains to be determined

  11. Protease activity, localization and inhibition in the human hair follicle

    PubMed Central

    Bhogal, R K; Mouser, P E; Higgins, C A; Turner, G A

    2014-01-01

    Synopsis Objective In humans, the process of hair shedding, referred to as exogen, is believed to occur independently of the other hair cycle phases. Although the actual mechanisms involved in hair shedding are not fully known, it has been hypothesized that the processes leading to the final step of hair shedding may be driven by proteases and/or protease inhibitor activity. In this study, we investigated the presence of proteases and protease activity in naturally shed human hairs and assessed enzyme inhibition activity of test materials. Methods We measured enzyme activity using a fluorescence-based assay and protein localization by indirect immunohistochemistry (IHC). We also developed an ex vivo skin model for measuring the force required to pull hair fibres from skin. Results Our data demonstrate the presence of protease activity in the tissue material surrounding club roots. We also demonstrated the localization of specific serine protease protein expression in human hair follicle by IHC. These data provide evidence demonstrating the presence of proteases around the hair club roots, which may play a role during exogen. We further tested the hypothesis that a novel protease inhibitor system (combination of Trichogen® and climbazole) could inhibit protease activity in hair fibre club root extracts collected from a range of ethnic groups (UK, Brazil, China, first-generation Mexicans in the USA, Thailand and Turkey) in both males and females. Furthermore, we demonstrated that this combination is capable of increasing the force required to remove hair in an ex vivo skin model system. Conclusion These studies indicate the presence of proteolytic activity in the tissue surrounding the human hair club root and show that it is possible to inhibit this activity with a combination of Trichogen® and climbazole. This technology may have potential to reduce excessive hair shedding. Résumé Objectif Chez l'homme, le processus de perte de cheveux, désigné comme exog

  12. Expression and activation of proteases in co-cultures.

    PubMed

    Paduch, Roman; Kandefer-Szerszeń, Martyna

    2011-01-01

    The present study concerned the expression and activation of metalloproteinase-2 (MMP-2), metalloproteinase-9 (MMP-9) and the urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) system in co-cultures of human colon carcinoma cell spheroids (HT29, LS180, SW948) with human normal colon epithelium (CCD 841 CoTr), myofibroblasts (CCD-18Co) and endothelial cells (HUVEC). Additionally, the influence of monensin on the production and function of the proteases was tested. Tumor cells expressed small amounts of MMP-2, MMP-9 and uPA. Normal cells generally produced proportionally higher concentrations of these proteases (especially MMP-2, compared with significantly smaller yields of MMP-9 and significantly lower amounts of uPAR than tumors. In co-cultures of tumor spheroids with normal cell monolayers, the concentration of the proteases was equal to the sum of the enzymes produced in monocultures of both types of cells. The highest activity of uPA, measured as the reduction of the chromogenic substrate (S-2444), was detected in supernatants and lysates of endothelial cells. Interestingly, in normal cells, the higher expression of proteases, mainly uPA, measured as the level of protein concentration, was closely linked with their lower activity and inversely, in tumor cells, the low level of the expression of the enzymes correlated with their high enzymatic activity. In zymography analysis, mainly pro-MMPs were detected both in culture supernatants and cell lysates. The highest amounts of active forms of the MMPs were detected in tumor spheroids co-cultured with endothelial cells. Monensin inhibited MMPs and uPA secretion but significantly increased uPAR release, mainly from normal cells. In conclusion, during direct interactions of tumor cells with normal cells, MMPs and the uPA/uPAR system play an important role in the degradation of ECM and tumor development, but as we found, there is a reverse relationship between the concentration and the

  13. An acidic amino acid-specific protease from germinating soybeans.

    PubMed

    Tan-Wilson, A L; Liu, X; Chen, R; Qi, X; Wilson, K A

    1996-05-01

    The degradation of the beta-conglycinin protein reserves in soybean seeds during germination and early growth begins with the proteolysis of its alpha and alpha' subunits by an enzyme called Protease C1. In the pathway, a number of proteolytic intermediates are produced and subsequently degraded. Determination of the N-terminal sequences of these intermediates provides insight regarding the requirements of the cleavage sites. The N-terminal sequence of three such proteolytic intermediates has been determined. The sequence has been located in the published sequences of the beta-conglycinin subunits. Comparing these cleavage sites, plus those of two others previously delineated, shows that the P1' and P4' positions always bear either a Glu or an Asp residue while the P1 position always bears either a Glu or a Gln residue. In addition, other sites from P3 to P7' are also rich in either Glu or Asp, and the whole region is predicted to be in a alpha-helix. Consistent with the observation, synthetic poly-L-Glu inhibits the Protease C1-catalysed degradation of the alpha and alpha' subunits of beta-conglycinin. Poly-L-Glu (av. M(r) = 1000) at 12.5 mM was more effective at inhibiting the reaction than poly-L-Glu (av. M(r) = 600) or poly-L-Glu (av. M(r) = 14,300) at the same concentration. Comparing large synthetic polypeptides at 12.5mM, inhibition by poly-L-Asp (av. M(r) = 15,000) is as effective as poly-L-Glu (av. M(r) = 14,300), while poly-L-Ser (av. M(r) = 15,000) had no effect at all. Poly-D-Glu (av. M(r) = 15,000) is a better inhibitor than poly-L-Glu of the same size. A serine protease of similar molecular weight as Protease C1 and also capable of catalysing the proteolysis of the alpha and alpha' subunits of beta-conglycinin to generate proteolytic intermediates of the same size has been found in mung bean.

  14. Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

    PubMed

    Guerra, Yasel; Valiente, Pedro A; Pons, Tirso; Berry, Colin; Rudiño-Piñera, Enrique

    2016-08-01

    Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical β-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop.

  15. Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

    PubMed

    Guerra, Yasel; Valiente, Pedro A; Pons, Tirso; Berry, Colin; Rudiño-Piñera, Enrique

    2016-08-01

    Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical β-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop. PMID:27329566

  16. Pneumococcal IgA1 protease subverts specific protection by human IgA1.

    PubMed

    Janoff, E N; Rubins, J B; Fasching, C; Charboneau, D; Rahkola, J T; Plaut, A G; Weiser, J N

    2014-03-01

    Bacterial immunoglobulin A1 (IgA1) proteases may sabotage the protective effects of IgA. In vitro, both exogenous and endogenously produced IgA1 protease inhibited phagocytic killing of Streptococcus pneumoniae by capsule-specific IgA1 human monoclonal antibodies (hMAbs) but not IgA2. These IgA1 proteases cleaved and reduced binding of the the effector Fcα1 heavy chain but not the antigen-binding F(ab)/light chain to pneumococcal surfaces. In vivo, IgA1 protease-resistant IgA2, but not IgA1 protease-sensitive IgA1, supported 60% survival in mice infected with wild-type S. pneumoniae. IgA1 hMAbs protected mice against IgA1 protease-deficient but not -producing pneumococci. Parallel mouse sera with human IgA2 showed more efficient complement-mediated reductions in pneumococci with neutrophils than did IgA1, particularly with protease-producing organisms. After natural human pneumococcal bacteremia, purified serum IgG inhibited IgA1 protease activity in 7 of 11 patients (64%). These observations provide the first evidence in vivo that IgA1 protease can circumvent killing of S. pneumoniae by human IgA. Acquisition of IgA1 protease-neutralizing IgG after infection directs attention to IgA1 protease both as a determinant of successful colonization and infection and as a potential vaccine candidate.

  17. A cysteine protease encoded by the baculovirus Bombyx mori nuclear polyhedrosis virus.

    PubMed Central

    Ohkawa, T; Majima, K; Maeda, S

    1994-01-01

    Sequence analysis of the BamHI F fragment of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) revealed an open reading frame whose deduced amino acid sequence had homology to those of cysteine proteases of the papain superfamily. The putative cysteine protease sequence (BmNPV-CP) was 323 amino acids long and showed 35% identity to a cysteine proteinase precursor from Trypanosoma brucei. Of 36 residues conserved among cathepsins B, H, L, and S and papain, 31 were identical in BmNPV-CP. In order to determine the activity and function of the putative cysteine protease, a BmNPV mutant (BmCysPD) was constructed by homologous recombination of the protease gene with a beta-galactosidase gene cassette. BmCysPD-infected BmN cell extracts were significantly reduced in acid protease activity compared with wild-type virus-infected cell extracts. The cysteine protease inhibitor E-64 [trans-epoxysuccinylleucylamido-(4-guanidino)butane] inhibited wild-type virus-expressed protease activity. Deletion of the cysteine protease gene had no significant effect on viral growth or polyhedron production in BmN cells, indicating that the cysteine protease was not essential for viral replication in vitro. However, B. mori larvae infected with BmCysPD showed symptoms different from those of wild-type BmNPV-infected larvae, e.g., less degradation of the body, including fat body cells, white body surface color due presumably to undegraded epidermal cells, and an increase in the number of polyhedra released into the hemolymph. This is the first report of (i) a virus-encoded protease with activity on general substrates and (ii) evidence that a virus-encoded protease may play a role in degradation of infected larvae to facilitate horizontal transmission of the virus. Images PMID:8083997

  18. Kinetic Intermediates en Route to the Final Serpin-Protease Complex

    PubMed Central

    Maddur, Ashoka A.; Swanson, Richard; Izaguirre, Gonzalo; Gettins, Peter G. W.; Olson, Steven T.

    2013-01-01

    Serpin protein protease inhibitors inactivate their target proteases through a unique mechanism in which a major serpin conformational change, resulting in a 70-Å translocation of the protease from its initial reactive center loop docking site to the opposite pole of the serpin, kinetically traps the acyl-intermediate complex. Although the initial Michaelis and final trapped acyl-intermediate complexes have been well characterized structurally, the intermediate stages involved in this remarkable transformation are not well understood. To better characterize such intermediate steps, we undertook rapid kinetic studies of the FRET and fluorescence perturbation changes of site-specific fluorophore-labeled derivatives of the serpin, α1-protease inhibitor (α1PI), which report the serpin and protease conformational changes involved in transforming the Michaelis complex to the trapped acyl-intermediate complex in reactions with trypsin. Two kinetically resolvable conformational changes were observed in the reactions, ascribable to (i) serpin reactive center loop insertion into sheet A with full protease translocation but incomplete protease distortion followed by, (ii) full conformational distortion and movement of the protease and coupled serpin conformational changes involving the F helix-sheet A interface. Kinetic studies of calcium effects on the labeled α1PI-trypsin reactions demonstrated both inactive and low activity states of the distorted protease in the final complex that were distinct from the intermediate distorted state. These studies provide new insights into the nature of the serpin and protease conformational changes involved in trapping the acyl-intermediate complex in serpin-protease reactions and support a previously proposed role for helix F in the trapping mechanism. PMID:24047901

  19. Optimisation of the detection of bacterial proteases using adsorbed immunoglobulins as universal substrates.

    PubMed

    Abuknesha, Ram A; Jeganathan, Fiona; Wildeboer, Dirk; Price, Robert G

    2010-06-15

    Bacterial proteases, Type XXIV from Bacillus licheniformens and Type XIV from Streptomyces griseus, were used to investigate the utility and optimisation of a solid phase assay for proteases, using immunoglobulin proteins as substrates. Immunoglobulins IgA and IgG were adsorbed on to surfaces of ELISA plates and exposed to various levels of the bacterial proteases which led to digestion and desorption of proportional amounts of the immunoglobulins. The assay signal was developed by measuring the remaining proteins on the polystyrene surface with appropriate enzyme-labelled anti-immunoglobulin reagents. The assay was fully optimised in terms of substrate levels employing ELISA techniques to titrate levels of adsorbed substrates and protease analytes. The critical factor which influences assay sensitivity was found to be the substrate concentration, the levels of adsorbed immunoglobulins. The estimated detection limits for protease XXIV and XIV were 10micro units/test and 9micro units/test using IgA as a substrate. EC(50) values were calculated as 213 and 48micro units/test for each protease respectively. Using IgG as a substrate, the estimated detection limits were 104micro units/test for protease XXIV and 9micro units/test for protease XIV. EC(50) values were calculated at 529micro units/test and 28micro units/test for protease XXIV and XIV respectively. The solid phase protease assay required no modification of the substrates and the adsorption step is merely simple addition of immunoglobulins to ELISA plates. Adsorption of the immunoglobulins to polystyrene enabled straightforward separation of reaction mixtures prior to development of assay signal. The assay exploits the advantages of the technical facilities of ELISA technology and commercially available reagents enabling the detection and measurement of a wide range of proteases. However, the key issue was found to be that in order to achieve the potential performance of the simple assay, optimisation of the

  20. Intramolecular Interactions between the Protease and Structural Domains Are Important for the Functions of Serine Protease Autotransporters▿ †

    PubMed Central

    Tsang, Casey; Malik, Huma; Nassman, Deana; Huang, Antony; Tariq, Fayha; Oelschlaeger, Peter; Stathopoulos, Christos

    2010-01-01

    Autotransporter (AT) is a protein secretion pathway found in Gram-negative bacteria featuring a multidomain polypeptide with a signal sequence, a passenger domain, and a translocator domain. An AT subfamily named serine protease ATs of the family Enterobacteriaceae (SPATEs) is characterized by the presence of a conserved serine protease motif in the passenger domain which contributes to bacterial pathogenesis. The goal of the current study is to determine the importance of the passenger domain conserved residues in the SPATE proteolytic and adhesive functions using the temperature-sensitive hemagglutinin (Tsh) protein as our model. To begin, mutations of 21 fully conserved residues in the four passenger domain conserved motifs were constructed by PCR-based site-directed mutagenesis. Seventeen mutants exhibited a wild-type secretion level; among these mutants, eight displayed reduced proteolytic activities in Tsh-specific oligopeptide and mucin cleavage assays. These eight mutants also demonstrated lower affinities to extracellular matrix proteins, collagen IV, and fibronectin. These eight conserved residues were analyzed by molecular graphics modeling to demonstrate their intramolecular interactions with the catalytic triad and other key residues. Additional mutations were made to confirm the above interactions in order to demonstrate their significance to the SPATE functions. Altogether our data suggest that certain conserved residues in the SPATE passenger domain are important for both the proteolytic and adhesive activities of SPATE by maintaining the proper protein structure via intramolecular interactions between the protease and β-helical domains. Here, we provide new insight into the structure-function relationship of the SPATEs and the functional roles of their conserved residues. PMID:20479079

  1. Characterization of an Alphamesonivirus 3C-Like Protease Defines a Special Group of Nidovirus Main Proteases

    PubMed Central

    Blanck, Sandra; Stinn, Anne; Tsiklauri, Lali; Zirkel, Florian; Junglen, Sandra

    2014-01-01

    ABSTRACT Cavally virus (CavV) and related viruses in the family Mesoniviridae diverged profoundly from other nidovirus lineages but largely retained the characteristic set of replicative enzymes conserved in the Coronaviridae and Roniviridae. The expression of these enzymes in virus-infected cells requires the extensive proteolytic processing of two large replicase polyproteins, pp1a and pp1ab, by the viral 3C-like protease (3CLpro). Here, we show that CavV 3CLpro autoproteolytic cleavage occurs at two N-terminal (N1 and N2) and one C-terminal (C1) processing site(s). The mature form of 3CLpro was revealed to be a 314-residue protein produced by cleavage at FKNK1386|SAAS (N2) and YYNQ1700|SATI (C1). Site-directed mutagenesis data suggest that the mesonivirus 3CLpro employs a catalytic Cys-His dyad comprised of CavV pp1a/pp1ab residues Cys-1539 and His-1434. The study further suggests that mesonivirus 3CLpro substrate specificities differ from those of related nidovirus proteases. The presence of Gln (or Glu) at the P1 position was not required for cleavage, although residues that control Gln/Glu specificity in related viral proteases are retained in the CavV 3CLpro sequence. Asn at the P2 position was identified as a key determinant for mesonivirus 3CLpro substrate specificity. Other positions, including P4 and P1′, each are occupied by structurally related amino acids, indicating a supportive role in substrate binding. Together, the data identify a new subgroup of nidovirus main proteases and support previous conclusions on phylogenetic relationships between the main nidovirus lineages. IMPORTANCE Mesoniviruses have been suggested to provide an evolutionary link between nidovirus lineages with small (13 to 16 kb) and large (26 to 32 kb) RNA genome sizes, and it has been proposed that a specific set of enzymes, including a proofreading exoribonuclease and other replicase gene-encoded proteins, play a key role in the major genome expansion leading to the currently

  2. Prevalence, mutation patterns, and effects on protease inhibitor susceptibility of the L76V mutation in HIV-1 protease.

    PubMed

    Young, Thomas P; Parkin, Neil T; Stawiski, Eric; Pilot-Matias, Tami; Trinh, Roger; Kempf, Dale J; Norton, Michael

    2010-11-01

    Patterns of HIV-1 protease inhibitor (PI) resistance-associated mutations (RAMs) and effects on PI susceptibility associated with the L76V mutation were studied in a large database. Of 20,501 sequences with ≥1 PI RAM, 3.2% contained L76V; L76V was alone in 0.04%. Common partner mutations included M46I, I54V, V82A, I84V, and L90M. L76V was associated with a 2- to 6-fold decrease in susceptibility to lopinavir, darunavir, amprenavir, and indinavir and a 7- to 8-fold increase in susceptibility to atazanavir and saquinavir. PMID:20805393

  3. Moringa oleifera Lam.: Protease activity against blood coagulation cascade

    PubMed Central

    Satish, A; Sairam, Sudha; Ahmed, Faiyaz; Urooj, Asna

    2012-01-01

    Background: The present study evaluated the protease activity of aqueous extracts of Moringa oleifera (Moringaceae) leaf (MOL) and root (MOR). Materials and Methods: Protease activity was assayed using casein, human plasma clot and human fibrinogen as substrates. Results: Caseinolytic activity of MOL was significantly higher (P ≤ 0.05) than that of MOR. Similar observations were found in case of human plasma clot hydrolyzing activity, wherein MOL caused significantly higher (P ≤ 0.05) plasma clot hydrolysis than MOR. Zymographic techniques were used to detect proteolytic enzymes following electrophoretic separation in gels. Further, both the extracts exhibited significant procoagulant activity as reflected by a significant decrease (P ≤ 0.05) in recalcification time, accompanied by fibrinogenolytic and fibrinolytic activities; clotting time was decreased from 180 ± 10 sec to 119 ± 8 sec and 143 ± 10 sec by MOL and MOR, respectively, at a concentration of 2.5 mg/mL. Fibrinogenolytic (human fibrinogen) and fibrinolytic activity (human plasma clot) was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), plate method and colorimetric method. Zymographic profile indicated that both the extracts exerted their procoagulant activity by selectively hydrolyzing Aα and Bβ subunits of fibrinogen to form fibrin clot, thereby exhibiting fibrinogenolytic activity. However, prolonged incubation resulted in degradation of the formed fibrin clot, suggesting fibrinolytic like activity. Conclusions: These findings support the traditional usage of M. oleifera extracts for wound healing. PMID:22224061

  4. Design of HIV Protease Inhibitors Based on Inorganic Polyhedral Metallacarboranes

    SciTech Connect

    Rezacova, Pavlina; Pokorna, Jana; Brynda, Ji; Kozisek, Milan; Cigler, Petr; Lesik, Martin; Fanfrlik, Jindrich; Rezac, Jan; Saskova, Klara Grantz; Sieglova, Irena; Plesek, Jaromir; Sicha, Vaclav; Gruner, Bohumir; Oberwinkler, Heike; Sedlacek, Juraj; Krausslich, Hans-Georg; Hobza, Pavel; Kral, Vladimir; Konvalinka, Jan

    2010-04-19

    HIV protease (HIV PR) is a primary target for anti-HIV drug design. We have previously identified and characterized substituted metallacarboranes as a new class of HIV protease inhibitors. In a structure-guided drug design effort, we connected the two cobalt bis(dicarbollide) clusters with a linker to substituted ammonium group and obtained a set of compounds based on a lead formula [H{sub 2}N-(8-(C{sub 2}H{sub 4}O){sub 2}-1,2-C{sub 2}B{sub 9}H{sub 10})(1',2'-C{sub 2}B{sub 9}H{sub 11})-3,3'-Co){sub 2}]Na. We explored inhibition properties of these compounds with various substitutions, determined the HIV PR:inhibitor crystal structure, and computationally explored the conformational space of the linker. Our results prove the capacity of linker-substituted dual-cage cobalt bis(dicarbollides) as lead compounds for design of more potent inhibitors of HIV PR.

  5. Fullerene-based inhibitors of HIV-1 protease.

    PubMed

    Strom, T Amanda; Durdagi, Serdar; Ersoz, Suha Salih; Salmas, Ramin Ekhteiari; Supuran, Claudiu T; Barron, Andrew R

    2015-12-01

    A series of Fmoc-Phe(4-aza-C60)-OH of fullerene amino acid derived peptides have been prepared by solid phase peptide synthesis, in which the terminal amino acid, Phe(4-aza-C60)-OH, is derived from the dipolar addition to C60 of the Fmoc-Nα-protected azido amino acids derived from phenylalanine: Fmoc-Phe(4-aza-C60)-Lys3-OH (1), Fmoc-Phe(4-aza-C60)-Pro-Hyp-Lys-OH (2), and Fmoc-Phe(4-aza-C60)-Hyp-Hyp-Lys-OH (3). The inhibition constant of our fullerene aspartic protease PRIs utilized FRET-based assay to evaluate the enzyme kinetics of HIV-1 PR at various concentrations of inhibitors. Simulation of the docking of the peptide Fmoc-Phe-Pro-Hyp-Lys-OH overestimated the inhibition, while the amino acid PRIs were well estimated. The experimental results show that C60-based amino acids are a good base structure in the design of protease inhibitors and that their inhibition can be improved upon by the addition of designer peptide sequences.

  6. Dynamic properties of extremophilic subtilisin-like serine-proteases.

    PubMed

    Tiberti, Matteo; Papaleo, Elena

    2011-04-01

    The investigation of the structural determinants of enzymatic temperature adaptation is a crucial pre-requisite both in terms of fundamental research and industrial applications to develop new biocatalysts active at different temperature ranges. In several cases, the differences related to cold- or warm-adaptation are related to subtle structural and aminoacidic differences at the molecular level, often hard to detect. In this context, we present a comparative study of psychrophilic, mesophilic and thermophilic subtilisin-like serine proteases by all-atom molecular dynamics (MD) simulations in explicit solvent using a multiple-replica approach. Our results strongly enforce the current view on localized flexibility in crucial functional regions for cold-adapted serine proteases and point out a different optimization and usage of salt-bridge interactions and networks in cold- and warm-adapted enzymes. The analyses allow to identify a subset of structural and dynamic features strictly associated to cold adaptation and which change from cold- to heat-active subtilisins. In particular, the thermophilic subtilisin presents a high affinity calcium binding site which is not structurally conserved in the mesophilic and psychrophilic counterparts, which, as it turns out from the MD analyses, at the same position show a stable salt bridge network and no stabilizing intra-molecular interactions, respectively. These aspects, along with differential flexibility in regions close to the active site or substrate binding pocket, can be an indication of evolution at this protein site toward a lower stability moving from high to low temperature conditions.

  7. Protease activation in glycerol-based deep eutectic solvents

    PubMed Central

    Zhao, Hua; Baker, Gary A.; Holmes, Shaletha

    2011-01-01

    Deep eutectic solvents (DESs) consisting of mixtures of a choline salt (chloride or acetate form) and glycerol are prepared as easily accessible, biodegradable, and inexpensive alternatives to conventional aprotic cation-anion paired ionic liquids. These DES systems display excellent fluidity coupled with thermal stability to nearly 200 °C. In this work, the transesterification activities of cross-linked proteases (subtilisin and α-chymotrypsin), immobilized on chitosan, were individually examined in these novel DESs. In the 1:2 molar ratio mixture of choline chloride/glycerol containing 3% (v/v) water, cross-linked subtilisin exhibited an excellent activity (2.9 μmo l min−1 g−1) in conjunction with a selectivity of 98% in the transesterification reaction of N-acetyl-L-phenylalanine ethyl ester with 1-propanol. These highly encouraging results advocate more extensive exploration of DESs in protease-mediated biotransformations of additional polar substrates and use of DESs in biocatalysis more generally. PMID:21909232

  8. Targeting the AKT pathway: Repositioning HIV protease inhibitors as radiosensitizers

    PubMed Central

    Goda, Jayant S.; Pachpor, Tejaswini; Basu, Trinanjan; Chopra, Supriya; Gota, Vikram

    2016-01-01

    Cellular resistance in tumour cells to different therapeutic approaches has been a limiting factor in the curative treatment of cancer. Resistance to therapeutic radiation is a common phenomenon which significantly reduces treatment options and impacts survival. One of the mechanisms of acquiring resistance to ionizing radiation is the overexpression or activation of various oncogenes like the EGFR (epidermal growth factor receptor), RAS (rat sarcoma) oncogene or loss of PTEN (phosphatase and tensin homologue) which in turn activates the phosphatidyl inositol 3-kinase/protein kinase B (PI3-K)/AKT pathway responsible for radiation resistance in various tumours. Blocking the pathway enhances the radiation response both in vitro and in vivo. Due to the differential activation of this pathway (constitutively activated in tumour cells and not in the normal host cells), it is an excellent candidate target for molecular targeted therapy to enhance radiation sensitivity. In this regard, HIV protease inhibitors (HPIs) known to interfere with PI3-K/AKT signaling in tumour cells, have been shown to sensitize various tumour cells to radiation both in vitro and in vivo. As a result, HPIs are now being investigated as possible radiosensitizers along with various chemotherapeutic drugs. This review describes the mechanisms by which PI3-K/AKT pathway causes radioresistance and the role of HIV protease inhibitors especially nelfinavir as a potential candidate drug to target the AKT pathway for overcoming radioresistance and its use in various clinical trials for different malignancies. PMID:27121513

  9. Targeting the AKT pathway: Repositioning HIV protease inhibitors as radiosensitizers.

    PubMed

    Goda, Jayant S; Pachpor, Tejaswini; Basu, Trinanjan; Chopra, Supriya; Gota, Vikram

    2016-02-01

    Cellular resistance in tumour cells to different therapeutic approaches has been a limiting factor in the curative treatment of cancer. Resistance to therapeutic radiation is a common phenomenon which significantly reduces treatment options and impacts survival. One of the mechanisms of acquiring resistance to ionizing radiation is the overexpression or activation of various oncogenes like the EGFR (epidermal growth factor receptor), RAS (rat sarcoma) oncogene or loss of PTEN (phosphatase and tensin homologue) which in turn activates the phosphatidyl inositol 3-kinase/protein kinase B (PI3-K)/AKT pathway responsible for radiation resistance in various tumours. Blocking the pathway enhances the radiation response both in vitro and in vivo. Due to the differential activation of this pathway (constitutively activated in tumour cells and not in the normal host cells), it is an excellent candidate target for molecular targeted therapy to enhance radiation sensitivity. In this regard, HIV protease inhibitors (HPIs) known to interfere with PI3-K/AKT signaling in tumour cells, have been shown to sensitize various tumour cells to radiation both in vitro and in vivo. As a result, HPIs are now being investigated as possible radiosensitizers along with various chemotherapeutic drugs. This review describes the mechanisms by which PI3-K/AKT pathway causes radioresistance and the role of HIV protease inhibitors especially nelfinavir as a potential candidate drug to target the AKT pathway for overcoming radioresistance and its use in various clinical trials for different malignancies.

  10. Site-1 protease is required for cartilage development in zebrafish.

    PubMed

    Schlombs, Kornelia; Wagner, Thomas; Scheel, Jochen

    2003-11-25

    gonzo (goz) is a zebrafish mutant with defects in cartilage formation. The goz phenotype comprises cartilage matrix defects and irregular chondrocyte morphology. Expression of endoderm, mesoderm, and cartilage marker genes is, however, normal, indicating a defect in chondrocyte morphogenesis. The mutated gene responsible for the goz phenotype, identified by positional cloning and confirmed by phosphomorpholino knockdown, encodes zebrafish site-1 protease (s1p). S1P has been shown to process and activate sterol regulatory element-binding proteins (SREBPs), which regulate expression of key enzymes of lipid biosynthesis or transport. This finding is consistent with the abnormal distribution of lipids in goz embryos. Knockdown of site-2 protease, which is also involved in activation of SREBPs, results in similar lipid and cartilage phenotypes as S1P knockdown. However, knockdown of SREBP cleavage-activating protein, which forms a complex with SREBP and is essential for S1P cleavage, results only in lipid phenotypes, whereas cartilage appears normal. This indicates that the cartilage phenoptypes of goz are caused independently of the lipid defects. PMID:14612568

  11. Site-1 protease is required for cartilage development in zebrafish

    PubMed Central

    Schlombs, Kornelia; Wagner, Thomas; Scheel, Jochen

    2003-01-01

    gonzo (goz) is a zebrafish mutant with defects in cartilage formation. The goz phenotype comprises cartilage matrix defects and irregular chondrocyte morphology. Expression of endoderm, mesoderm, and cartilage marker genes is, however, normal, indicating a defect in chondrocyte morphogenesis. The mutated gene responsible for the goz phenotype, identified by positional cloning and confirmed by phosphomorpholino knockdown, encodes zebrafish site-1 protease (s1p). S1P has been shown to process and activate sterol regulatory element-binding proteins (SREBPs), which regulate expression of key enzymes of lipid biosynthesis or transport. This finding is consistent with the abnormal distribution of lipids in goz embryos. Knockdown of site-2 protease, which is also involved in activation of SREBPs, results in similar lipid and cartilage phenotypes as S1P knockdown. However, knockdown of SREBP cleavage-activating protein, which forms a complex with SREBP and is essential for S1P cleavage, results only in lipid phenotypes, whereas cartilage appears normal. This indicates that the cartilage phenoptypes of goz are caused independently of the lipid defects. PMID:14612568

  12. Sphingosine-induced apoptosis is dependent on lysosomal proteases.

    PubMed Central

    Kågedal, K; Zhao, M; Svensson, I; Brunk, U T

    2001-01-01

    We propose a new mechanism for sphingosine-induced apoptosis, involving relocation of lysosomal hydrolases to the cytosol. Owing to its lysosomotropic properties, sphingosine, which is also a detergent, especially when protonated, accumulates by proton trapping within the acidic vacuolar apparatus, where most of its action as a detergent would be exerted. When sphingosine was added in low-to-moderate concentrations to Jurkat and J774 cells, partial lysosomal rupture occurred dose-dependently, starting within a few minutes. This phenomenon preceded caspase activation, as well as changes of mitochondrial membrane potential. High sphingosine doses rapidly caused extensive lysosomal rupture and ensuing necrosis, without antecedent apoptosis or caspase activation. The sphingosine effect was prevented by pre-treatment with another, non-toxic, lysosomotropic base, ammonium chloride, at 10 mM. The lysosomal protease inhibitors, pepstatin A and epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl ester ('E-64d'), inhibited markedly sphingosine-induced caspase activity to almost the same degree as the general caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone ('Z-VAD-FMK'), although they did not by themselves inhibit caspases. We conclude that cathepsin D and one or more cysteine proteases, such as cathepsins B or L, are important mediators of sphingosine-induced apoptosis, working upstream of the caspase cascade and mitochondrial membrane-potential changes. PMID:11583579

  13. Engineering fluorescent protein substrates for the AAA+ Lon protease.

    PubMed

    Wohlever, Matthew L; Nager, Andrew R; Baker, Tania A; Sauer, Robert T

    2013-04-01

    AAA+ proteases, such as Escherichia coli Lon, recognize protein substrates by binding to specific peptide degrons and then unfold and translocate the protein into an internal degradation chamber for proteolysis. For some AAA+ proteases, attaching specific degrons to the N- or C-terminus of green fluorescent protein (GFP) generates useful substrates, whose unfolding and degradation can be monitored by loss of fluorescence, but Lon fails to degrade appropriately tagged GFP variants at a significant rate. Here, we demonstrate that Lon catalyzes robust unfolding and degradation of circularly permuted variants of GFP with a β20 degron appended to the N terminus or a sul20 degron appended to the C terminus. Lon degradation of non-permuted GFP-sul20 is very slow, in part because the enzyme cannot efficiently extract the degron-proximal C-terminal β-strand to initiate denaturation. The circularly permuted GFP substrates described here allow convenient high-throughput assays of the kinetics of Lon degradation in vitro and also permit assays of Lon proteolysis in vivo.

  14. Preliminary crystallographic analysis of avian infectious bronchitis virus main protease

    SciTech Connect

    Li, Jun; Shen, Wei; Liao, Ming; Bartlam, Mark

    2007-01-01

    The avian infectious bronchitis virus main protease has been crystallized; crystals diffract to 2.7 Å resolution. Infectious bronchitis virus (IBV) is the prototype of the genus Coronavirus. It causes a highly contagious disease which affects the respiratory, reproductive, neurological and renal systems of chickens, resulting great economic losses in the poultry industry worldwide. The coronavirus (CoV) main protease (M{sup pro}), which plays a pivotal role in viral gene expression and replication through a highly complex cascade involving the proteolytic processing of replicase polyproteins, is an attractive target for antiviral drug design. In this study, IBV M{sup pro} was overexpressed in Escherichia coli. Crystals suitable for X-ray crystallography have been obtained using microseeding techniques and belong to space group P6{sub 1}22. X-ray diffraction data were collected in-house to 2.7 Å resolution from a single crystal. The unit-cell parameters were a = b = 119.1, c = 270.7 Å, α = β = 90, γ = 120°. Three molecules were predicted to be present in the asymmetric unit from a calculated self-rotation function.

  15. Factor V activation and inactivation by venom proteases.

    PubMed

    Rosing, J; Govers-Riemslag, J W; Yukelson, L; Tans, G

    2001-01-01

    Blood coagulation factor V is a single-chain glycoprotein with M(r) = 330,000 which plays an important role in the procoagulant and anticoagulant pathways. Thrombin activates factor V into factor Va, a two-chain molecule which is composed of a heavy (M(r) = 105,000) and a light chain (M(r) = 71,000/74,000). Factor Va accelerates factor Xa-catalysed prothrombin activation more than 1,000-fold and under physiological conditions the cofactor activity of factor Va in prothrombin activation is down-regulated by activated protein C. Factor V can also be activated by a wide variety of snake venoms (e.g. from Vipera species, Naja naja oxiana, Bothrops atrox) and by proteases present in the bristles of a South American caterpillar (Lonomia achelous). Some venoms, notably of Vipera lebetina turanica and Lonomia achelous, contain proteases that are able to inactivate factor V or factor Va. Venom factor V activators are excellent tools in studying the structure-function relationship of factor V(a) and they are also used in diagnostic tests for quantification of plasma factor V levels and for the screening of defects in the protein C pathway. In this review, the structural and functional properties of animal venom factor V activators and inactivators is described. PMID:11910191

  16. Recent patents on microbial proteases for the dairy industry.

    PubMed

    Feijoo-Siota, Lucía; Blasco, Lucía; Rodríguez-Rama, José Luis; Barros-Velázquez, Jorge; Miguel, Trinidad de; Sánchez-Pérez, Angeles; Villa, Tomás G

    2014-01-01

    This paper reviews the general characteristics of exo and endopeptidases of microbial origin currently used in the milk industry. It also includes recent patents developed either to potentiate the enzymatic activity or to improve the resulting milk derivatives. The main application of these proteases is in the cheese-making industry. Although this industry preferentially uses animal rennets, and in particular genetically engineered chymosins, it also utilizes milk coagulants of microbial origin. Enzymes derived from Rhizomucor miehei, Rhizomucor pusillus and Cryphonectria parasitica are currently used to replace the conventional milk-clotting enzymes. In addition, the dairy industry uses microbial endo and exoproteases for relatively new applications, such as debittering and flavor generation in cheese, accelerated cheese ripening, manufacture of protein hydrolysates with improved functional properties, and production of enzyme-modified cheeses. Lactic acid bacteria play an essential role in these processes, hence these bacteria and the proteases they produce are currently being investigated by the dairy industry and are the subject of many of their patent applications.

  17. Regulation of Adrenal Aldosterone Production by Serine Protease Prostasin

    PubMed Central

    Ko, Takehiro; Kakizoe, Yutaka; Wakida, Naoki; Hayata, Manabu; Uchimura, Kohei; Shiraishi, Naoki; Miyoshi, Taku; Adachi, Masataka; Aritomi, Shizuka; Konda, Tomoyuki; Tomita, Kimio; Kitamura, Kenichiro

    2010-01-01

    A serine protease prostasin has been demonstrated to have a pivotal role in the activation of the epithelial sodium channel. Systemic administration of adenovirus carrying human prostasin gene in rats resulted in an increase in plasma prostasin and aldosterone levels. However, the mechanism by which the elevation of prostasin levels in the systemic circulation stimulated the plasma aldosterone levels remains unknown. Therefore, we examined if prostasin increases the aldosterone synthesis in a human adrenocortical cell line (H295R cells). Luciferase assay using CYP11B2 promoter revealed that prostasin significantly increased the transcriptional activity of CYP11B2. Prostasin significantly increased both CYP11B2 mRNA expression and aldosterone production in a dose-dependent manner. Surprisingly, treatment with camostat mesilate, a potent prostasin inhibitor, had no effect on the aldosterone synthesis by prostasin and also a protease-dead mutant of prostasin significantly stimulated the aldosterone production. A T-type/L-type calcium channel blocker and a protein kinase C (PKC) inhibitor significantly reduced the aldosterone synthesis by prostasin. Our findings suggest a stimulatory effect of prostasin on the aldosterone synthesis by adrenal gland through the nonproteolytic action and indicate a new role of prostasin in the systemic circulation. PMID:20204133

  18. Antimalarial activity of HIV-1 protease inhibitor in chromone series.

    PubMed

    Lerdsirisuk, Pradith; Maicheen, Chirattikan; Ungwitayatorn, Jiraporn

    2014-12-01

    Increasing parasite resistance to nearly all available antimalarial drugs becomes a serious problem to human health and necessitates the need to continue the search for new effective drugs. Recent studies have shown that clinically utilized HIV-1 protease (HIV-1 PR) inhibitors can inhibit the in vitro and in vivo growth of Plasmodium falciparum. In this study, a series of chromone derivatives possessing HIV-1 PR inhibitory activity has been tested for antimalarial activity against P. falciparum (K1 multi-drug resistant strain). Chromone 15, the potent HIV-1 PR inhibitor (IC50=0.65μM), was found to be the most potent antimalarial compound with IC50=0.95μM while primaquine and tafenoquine showed IC50=2.41 and 1.95μM, respectively. Molecular docking study of chromone compounds against plasmepsin II, an aspartic protease enzyme important in hemoglobin degradation, revealed that chromone 15 exhibited the higher binding affinity (binding energy=-13.24kcal/mol) than the known PM II inhibitors. Thus, HIV-1 PR inhibitor in chromone series has the potential to be a new class of antimalarial agent. PMID:25462990

  19. Advances in the development of SUMO specific protease (SENP) inhibitors

    PubMed Central

    Kumar, Ashutosh; Zhang, Kam Y.J.

    2015-01-01

    Sumoylation is a reversible post-translational modification that involves the covalent attachment of small ubiquitin-like modifier (SUMO) proteins to their substrate proteins. Prior to their conjugation, SUMO proteins need to be proteolytically processed from its precursor form to mature or active form. SUMO specific proteases (SENPs) are cysteine proteases that cleave the pro or inactive form of SUMO at C-terminus using its hydrolase activity to expose two glycine residues. SENPs also catalyze the de-conjugation of SUMO proteins using their isopeptidase activity, which is crucial for recycling of SUMO from substrate proteins. SENPs are important for maintaining the balance between sumoylated and unsumoylated proteins required for normal cellular physiology. Several studies reported the overexpression of SENPs in disease conditions and highlighted their role in the development of various diseases, especially cancer. In this review, we will address the current biological understanding of various SENP isoforms and their role in the pathogenesis of different cancers and other diseases. We will then discuss the advances in the development of protein-based, peptidyl and small molecule inhibitors of various SENP isoforms. Finally, we will summarize successful examples of computational screening that allowed the identification of SENP inhibitors with therapeutic potential. PMID:25893082

  20. Heterogeneity of heat-resistant proteases from milk Pseudomonas species.

    PubMed

    Marchand, Sophie; Vandriesche, Gonzalez; Coorevits, An; Coudijzer, Katleen; De Jonghe, Valerie; Dewettinck, Koen; De Vos, Paul; Devreese, Bart; Heyndrickx, Marc; De Block, Jan

    2009-07-31

    Pseudomonas fragi, Pseudomonas lundensis and members of the Pseudomonas fluorescens group may spoil Ultra High Temperature (UHT) treated milk and dairy products, due to the production of heat-stable proteases in the cold chain of raw milk. Since the aprX gene codes for a heat-resistant protease in P. fluorescens, the presence of this gene has also been investigated in other members of the genus. For this purpose an aprX-screening PCR test has been developed. Twenty-nine representatives of important milk Pseudomonas species and thirty-five reference strains were screened. In 42 out of 55 investigated Pseudomonas strains, the aprX gene was detected, which proves the potential of the aprX-PCR test as a screening tool for potentially proteolytic Pseudomonas strains in milk samples. An extensive study of the obtained aprX-sequences on the DNA and the amino acid level, however, revealed a large heterogeneity within the investigated milk isolates. Although this heterogeneity sets limitations to a general detection method for all proteolytic Pseudomonas strains in milk, it offers a great potential for the development of a multiplex PCR screening test targeting individual aprX-genes. Furthermore, our data illustrated the potential use of the aprX gene as a taxonomic marker, which may help in resolving the current taxonomic deadlock in the P. fluorescens group.