scholarly journals N-Terminal Presequence-Independent Import of Phosphofructokinase into Hydrogenosomes of Trichomonas vaginalis

2015 ◽  
Vol 14 (12) ◽  
pp. 1264-1275 ◽  
Author(s):  
Petr Rada ◽  
Abhijith Radhakrishna Makki ◽  
Verena Zimorski ◽  
Sriram Garg ◽  
Vladimír Hampl ◽  
...  
Keyword(s):  
Trichomonas Vaginalis ◽  
Cellular Localization ◽  
Protein Import ◽  
Catalytic Domain ◽  
Matrix Proteins ◽  
Evolutionary Perspective ◽  
Content Type ◽  
Organelle Evolution ◽  
Reduced Forms ◽  
Early Phases

ABSTRACT Mitochondrial evolution entailed the origin of protein import machinery that allows nuclear-encoded proteins to be targeted to the organelle, as well as the origin of cleavable N-terminal targeting sequences (NTS) that allow efficient sorting and import of matrix proteins. In hydrogenosomes and mitosomes, reduced forms of mitochondria with reduced proteomes, NTS-independent targeting of matrix proteins is known. Here, we studied the cellular localization of two glycolytic enzymes in the anaerobic pathogen Trichomonas vaginalis : PP i -dependent phosphofructokinase ( Tv PP i -PFK), which is the main glycolytic PFK activity of the protist, and ATP-dependent PFK ( Tv ATP-PFK), the function of which is less clear. Tv PP i -PFK was detected predominantly in the cytosol, as expected, while all four Tv ATP-PFK paralogues were imported into T. vaginalis hydrogenosomes, although none of them possesses an NTS. The heterologous expression of Tv ATP-PFK in Saccharomyces cerevisiae revealed an intrinsic capability of the protein to be recognized and imported into yeast mitochondria, whereas yeast ATP-PFK resides in the cytosol. Tv ATP-PFK consists of only a catalytic domain, similarly to “short” bacterial enzymes, while Sc ATP-PFK includes an N-terminal extension, a catalytic domain, and a C-terminal regulatory domain. Expression of the catalytic domain of Sc ATP-PFK and short Escherichia coli ATP-PFK in T. vaginalis resulted in their partial delivery to hydrogenosomes. These results indicate that Tv ATP-PFK and the homologous ATP-PFKs possess internal structural targeting information that is recognized by the hydrogenosomal import machinery. From an evolutionary perspective, the predisposition of ancient ATP-PFK to be recognized and imported into hydrogenosomes might be a relict from the early phases of organelle evolution.

scholarly journals S-Layer Homology Domain Proteins Csac_0678 and Csac_2722 Are Implicated in Plant Polysaccharide Deconstruction by the Extremely Thermophilic Bacterium Caldicellulosiruptor saccharolyticus

2011 ◽  
Vol 78 (3) ◽  
pp. 768-777 ◽  
Author(s):  
Inci Ozdemir ◽  
Sara E. Blumer-Schuette ◽  
Robert M. Kelly
Keyword(s):  
Cellular Localization ◽  
Catalytic Domain ◽  
Plant Biomass ◽  
Thermophilic Bacteria ◽  
Biochemical Properties ◽  
Crystalline Cellulose ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Caldicellulosiruptor Saccharolyticus ◽  
Content Type

ABSTRACTThe genusCaldicellulosiruptorcontains extremely thermophilic bacteria that grow on plant polysaccharides. The genomes ofCaldicellulosiruptorspecies reveal certain surface layer homology (SLH) domain proteins that have distinguishing features, pointing to a role in lignocellulose deconstruction. Two of these proteins inCaldicellulosiruptor saccharolyticus(Csac_0678 and Csac_2722) were examined from this perspective. In addition to three contiguous SLH domains, the Csac_0678 gene encodes a glycoside hydrolase family 5 (GH5) catalytic domain and a family 28 carbohydrate-binding module (CBM); orthologs to Csac_0678 could be identified in all genome-sequencedCaldicellulosiruptorspecies. Recombinant Csac_0678 was optimally active at 75°C and pH 5.0, exhibiting both endoglucanase and xylanase activities. SLH domain removal did not impact Csac_0678 GH activity, but deletion of the CBM28 domain eliminated binding to crystalline cellulose and rendered the enzyme inactive on this substrate. Csac_2722 is the largest open reading frame (ORF) in theC. saccharolyticusgenome (predicted molecular mass of 286,516 kDa) and contains two putative sugar-binding domains, two Big4 domains (bacterial domains with an immunoglobulin [Ig]-like fold), and a cadherin-like (Cd) domain. Recombinant Csac_2722, lacking the SLH and Cd domains, bound to cellulose and had detectable carboxymethylcellulose (CMC) hydrolytic activity. Antibodies directed against Csac_0678 and Csac_2722 confirmed that these proteins bound to theC. saccharolyticusS-layer. Their cellular localization and functional biochemical properties indicate roles for Csac_0678 and Csac_2722 in recruitment and hydrolysis of complex polysaccharides and the deconstruction of lignocellulosic biomass. Furthermore, these results suggest that related SLH domain proteins in otherCaldicellulosiruptorgenomes may also be important contributors to plant biomass utilization.

scholarly journals Protein Import into Hydrogenosomes of Trichomonas vaginalis Involves both N-Terminal and Internal Targeting Signals: a Case Study of Thioredoxin Reductases

2008 ◽  
Vol 7 (10) ◽  
pp. 1750-1757 ◽  
Author(s):  
Marek Mentel ◽  
Verena Zimorski ◽  
Patrick Haferkamp ◽  
William Martin ◽  
Katrin Henze
Keyword(s):  
Trichomonas Vaginalis ◽  
Protein Import ◽  
Atp Synthesis ◽  
Antioxidant Systems ◽  
Low Oxygen ◽  
Oxygen Sensitive ◽  
Targeting Signal ◽  
Targeting Signals ◽  
Thioredoxin Reductases

ABSTRACT The parabasalian flagellate Trichomonas vaginalis harbors mitochondrion-related and H2-producing organelles of anaerobic ATP synthesis, called hydrogenosomes, which harbor oxygen-sensitive enzymes essential to its pyruvate metabolism. In the human urogenital tract, however, T. vaginalis is regularly exposed to low oxygen concentrations and therefore must possess antioxidant systems protecting the organellar environment against the detrimental effects of molecular oxygen and reactive oxygen species. We have identified two closely related hydrogenosomal thioredoxin reductases (TrxRs), the hitherto-missing component of a thioredoxin-linked hydrogenosomal antioxidant system. One of the two hydrogenosomal TrxR isoforms, TrxRh1, carried an N-terminal extension resembling known hydrogenosomal targeting signals. Expression of hemagglutinin-tagged TrxRh1 in transfected T. vaginalis cells revealed that its N-terminal extension was necessary to import the protein into the organelles. The second hydrogenosomal TrxR isoform, TrxRh2, had no N-terminal targeting signal but was nonetheless efficiently targeted to hydrogenosomes. N-terminal presequences from hydrogenosomal proteins with known processing sites, i.e., the alpha subunit of succinyl coenzyme A synthetase (SCSα) and pyruvate:ferredoxin oxidoreductase A, were investigated for their ability to direct mature TrxRh1 to hydrogenosomes. Neither presequence directed TrxRh1 to hydrogenosomes, indicating that neither extension is, by itself, sufficient for hydrogenosomal targeting. Moreover, SCSα lacking its N-terminal extension was efficiently imported into hydrogenosomes, indicating that this extension is not required for import of this major hydrogenosomal protein. The finding that some hydrogenosomal enzymes require N-terminal signals for import but that in others the N-terminal extension is not necessary for targeting indicates the presence of additional targeting signals within the mature subunits of several hydrogenosome-localized proteins.

Benefits and challenges to applying IPD: experiences from a Norwegian mega-project

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Monique Rieger Rodrigues ◽  
Søren Munch Lindhard
Keyword(s):  
Early Stage ◽  
Lessons Learned ◽  
Structured Interviews ◽  
Main Concept ◽  
Content Type ◽  
Positive Effects ◽  
Collaboration And Communication ◽  
Traditional Construction ◽  
Supporting Element ◽  
Early Phases

Purpose The traditional construction delivery method is challenged by low trust and collaboration issues, resulting in increased project costs. The integrated project delivery (IPD) method is developed, through a contractual agreement, to overcome these challenges by creating a common set of terms, expectations and project goals. Design/methodology/approach A singular construction case was followed during a four-month period. Data collection consisted of contract documents and a series of semi-structured interviews with representatives from the owner, design-group and contractors. Findings The IPD contract was found to have a number of positive effects; it improved project behavior (e.g. trust, collaboration and communication), increased ownership among project participants and improved buildability of the design, leading to fewer surprises and interruptions in the construction phase. The study also revealed a number of challenges including contractual and legal challenges and involving too many participants in the early phases. Moreover, co-location was identified as a particular important supporting element, to build relations and improve collaboration. Originality/value This research identified lessons learned from the application, as well as initial barriers and persistent barriers for implementing IPD. To improve IPD application the top three lessons were as follows: 1) the contractual documents should be adapted and signed at an early stage as this increases financial transparency, 2) cost estimates should be carried as an iterative process and project main concept be freezed at an early stage to increase understanding and minimize risks, 3) only the most important project developers should be involved in the early phases, to avoid going into detailed design issues before the main concept is completed.

scholarly journals Insights into the Maturation of Pernisine, a Subtilisin-Like Protease from the Hyperthermophilic Archaeon Aeropyrum pernix

2020 ◽  
Vol 86 (17) ◽  
Author(s):  
Miha Bahun ◽  
Marko Šnajder ◽  
Dušan Turk ◽  
Nataša Poklar Ulrih
Keyword(s):  
High Temperature ◽  
High Temperatures ◽  
Catalytic Domain ◽  
Industrial Applications ◽  
Binding Motif ◽  
Content Type ◽  
Hyperthermophilic Archaeon ◽  
Aeropyrum Pernix ◽  
Autocatalytic Process ◽  
Exceptional Stability

ABSTRACT Pernisine is a subtilisin-like protease that was originally identified in the hyperthermophilic archaeon Aeropyrum pernix, which lives in extreme marine environments. Pernisine shows exceptional stability and activity due to the high-temperature conditions experienced by A. pernix. Pernisine is of interest for industrial purposes, as it is one of the few proteases that has demonstrated prion-degrading activity. Like other extracellular subtilisins, pernisine is synthesized in its inactive pro-form (pro-pernisine), which needs to undergo maturation to become proteolytically active. The maturation processes of mesophilic subtilisins have been investigated in detail; however, less is known about the maturation of their thermophilic homologs, such as pernisine. Here, we show that the structure of pro-pernisine is disordered in the absence of Ca2+ ions. In contrast to the mesophilic subtilisins, pro-pernisine requires Ca2+ ions to adopt the conformation suitable for its subsequent maturation. In addition to several Ca2+-binding sites that have been conserved from the thermostable Tk-subtilisin, pernisine has an additional insertion sequence with a Ca2+-binding motif. We demonstrate the importance of this insertion for efficient folding and stabilization of pernisine during its maturation. Moreover, analysis of the pernisine propeptide explains the high-temperature requirement for pro-pernisine maturation. Of note, the propeptide inhibits the pernisine catalytic domain more potently at high temperatures. After dissociation, the propeptide is destabilized at high temperatures only, which leads to its degradation and finally to pernisine activation. Our data provide new insights into and understanding of the thermostable subtilisin autoactivation mechanism. IMPORTANCE Enzymes from thermophilic organisms are of particular importance for use in industrial applications, due to their exceptional stability and activity. Pernisine, from the hyperthermophilic archaeon Aeropyrum pernix, is a proteolytic enzyme that can degrade infective prion proteins and thus has a potential use for disinfection of prion-contaminated surfaces. Like other subtilisin-like proteases, pernisine needs to mature through an autocatalytic process to become an active protease. In the present study, we address the maturation of pernisine and show that the process is regulated specifically at high temperatures by the propeptide. Furthermore, we demonstrate the importance of a unique Ca2+-binding insertion for stabilization of mature pernisine. Our results provide a novel understanding of thermostable subtilisin autoactivation, which might advance the development of these enzymes for commercial use.

scholarly journals Autophagy Inhibitors Do Not Restore Peroxisomal Functions in Cells With the Most Common Peroxisome Biogenesis Defect

2021 ◽  
Vol 9 ◽  
Author(s):  
Femke C. C. Klouwer ◽  
Kim D. Falkenberg ◽  
Rob Ofman ◽  
Janet Koster ◽  
Démi van Gent ◽  
...  
Keyword(s):  
Matrix Protein ◽  
Protein Import ◽  
Therapeutic Option ◽  
Cell Types ◽  
Matrix Proteins ◽  
Peroxisome Biogenesis ◽  
Metabolic Functions ◽  
Minimal Improvement ◽  
Different Cell Types ◽  
Autophagy Inhibition

Peroxisome biogenesis disorders within the Zellweger spectrum (PBD-ZSDs) are most frequently associated with the c.2528G>A (p.G843D) mutation in the PEX1 gene (PEX1-G843D), which results in impaired import of peroxisomal matrix proteins and, consequently, defective peroxisomal functions. A recent study suggested that treatment with autophagy inhibitors, in particular hydroxychloroquine, would be a potential therapeutic option for PBD-ZSD patients carrying the PEX1-G843D mutation. Here, we studied whether autophagy inhibition by chloroquine, hydroxychloroquine and 3-methyladenine indeed can improve peroxisomal functions in four different cell types with the PEX1-G843D mutation, including primary patient cells. Furthermore, we studied whether autophagy inhibition may be the mechanism underlying the previously reported improvement of peroxisomal functions by L-arginine in PEX1-G843D cells. In contrast to L-arginine, we observed no improvement but a worsening of peroxisomal metabolic functions and peroxisomal matrix protein import by the autophagy inhibitors, while genetic knock-down of ATG5 and NBR1 in primary patient cells resulted in only a minimal improvement. Our results do not support the use of autophagy inhibitors as potential treatment for PBD-ZSD patients, whereas L-arginine remains a therapeutically promising compound.

Import of microinjected proteins bearing the SKL peroxisomal targeting sequence into the peroxisomes of a human fibroblast cell line: evidence that virtually all peroxisomes are import-competent

1995 ◽  
Vol 108 (4) ◽  
pp. 1469-1476
Author(s):  
P.E. Hill ◽  
P.A. Walton
Keyword(s):  
Cell Line ◽  
Human Fibroblast ◽  
Mammalian Cells ◽  
Protein Import ◽  
Fibroblast Cell ◽  
Matrix Proteins ◽  
Fibroblast Cell Line ◽  
Peroxisomal Protein ◽  
Human Fibroblast Cell ◽  
Peroxisomal Targeting

Peroxisomes import virtually all of their membrane and matrix proteins post-translationally. It is presently unknown whether, in mammalian cells, their exists a pool of mature peroxisomes which have received their complement of proteins and are import-incompetent. Previous work has shown that fibroblasts are capable of importing microinjected peroxisomal proteins into peroxisomes. This report describes the import of a hybrid peroxisomal protein into virtually all peroxisomes of the microinjected cell. The peroxisomal import was uniform in both short and long incubations. Pretreatment of the cells with cycloheximide did not affect the import of the peroxisomal protein, nor was there any difference in the distribution of the imported protein. Sequential microinjection experiments demonstrated that peroxisomes that had imported luciferase were capable of importing another peroxisomal protein injected 24 hours later. These results suggest that, in fibroblasts, all peroxisomes have associated protein-import machinery; this evidence does not support the hypothesis that there exists a pool of import-incompetent peroxisomes.

Cyclic GMP-dependent protein kinase is required for thrombospondin and tenascin mediated focal adhesion disassembly

1996 ◽  
Vol 109 (10) ◽  
pp. 2499-2508 ◽  
Author(s):  
J.E. Murphy-Ullrich ◽  
M.A. Pallero ◽  
N. Boerth ◽  
J.A. Greenwood ◽  
T.M. Lincoln ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Focal Adhesion ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Focal Adhesions ◽  
Matrix Proteins ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein

Focal adhesions are specialized regions of cell membranes that are foci for the transmission of signals between the outside and the inside of the cell. Intracellular signaling events are important in the organization and stability of these structures. In previous work, we showed that the counter-adhesive extracellular matrix proteins, thrombospondin, tenascin, and SPARC, induce the disassembly of focal adhesion plaques and we identified the active regions of these proteins. In order to determine the mechanisms whereby the anti-adhesive matrix proteins modulate cytoskeletal organization and focal adhesion integrity, we examined the role of protein kinases in mediating the loss of focal adhesions by these proteins. Data from these studies show that cGMP-dependent protein kinase is necessary to mediate focal adhesion disassembly triggered by either thrombospondin or tenascin, but not by SPARC. In experiments using various protein kinase inhibitors, we observed that selective inhibitors of cyclic GMP-dependent protein kinase, KT5823 and Rp-8-Br-cGMPS, blocked the effects of both the active sequence of thrombospondin 1 (hep I) and the alternatively-spliced segment (TNfnA-D) of tenascin-C on focal adhesion disassembly. Moreover, early passage rat aortic smooth muscle cells which have high levels of cGMP-dependent protein kinase were sensitive to hep I treatment, in contrast to passaged cGMP-dependent protein kinase deficient cells which were refractory to hep I or TNfnA-D treatment, but were sensitive to SPARC. Transfection of passaged smooth muscle cells with the catalytic domain of PKG I alpha restored responsiveness to hep I and TNfnA-D. While these studies show that cGMP-dependent protein kinase activity is necessary for thrombospondin and tenascin-mediated focal adhesion disassembly, kinase activity alone is not sufficient to induce disassembly as transfection of the catalytic domain of the kinase in the absence of additional stimuli does not result in loss of focal adhesions.

scholarly journals Plasmodium falciparum Clag9-Associated PfRhopH Complex Is Involved in Merozoite Binding to Human Erythrocytes

2019 ◽  
Vol 88 (2) ◽  
Author(s):  
Bishwanath Kumar Chourasia ◽  
Arunaditya Deshmukh ◽  
Inderjeet Kaur ◽  
Gourab Paul ◽  
Ashutosh Panda ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Cellular Localization ◽  
Natural Infection ◽  
Human Erythrocytes ◽  
Spectrometric Analysis ◽  
Endothelial Lining ◽  
Content Type ◽  
Protein Protein Interaction ◽  
Associated Proteins ◽  
Human Rbcs

ABSTRACT Cytoadherence-linked asexual gene 9 (Clag9), a conserved Plasmodium protein expressed during the asexual blood stages, is involved in the cytoadherence of infected red blood cells (RBCs) to the endothelial lining of blood vessels. Here, we show that Plasmodium falciparum Clag9 (PfClag9) is a component of the PfClag9-RhopH complex that is involved in merozoite binding to human erythrocytes. To characterize PfClag9, we expressed four fragments of PfClag9, encompassing the entire protein. Immunostaining analysis using anti-PfClag9 antibodies showed expression and localization of PfClag9 at the apical end of the merozoites. Mass spectrometric analysis of merozoite extracts after immunoprecipitation using anti-PfClag9 antibody identified P. falciparum rhoptry-associated protein 1 (PfRAP1), PfRAP2, PfRAP3, PfRhopH2, and PfRhopH3 as associated proteins. The identified rhoptry proteins were expressed, and their association with PfClag9 domains was assessed by using protein-protein interaction tools. We further showed that PfClag9 binds human RBCs by interacting with the glycophorin A-band 3 receptor-coreceptor complex. In agreement with its cellular localization, PfClag9 was strongly recognized by antibodies generated during natural infection. Mice immunized with the C-terminal domain of PfClag9 were partially protected against a subsequent challenge infection with Plasmodium berghei, further supporting a biological role of PfClag9 during natural infection. Taken together, these results provide direct evidence for the existence of a PfRhopH-Clag9 complex on the Plasmodium merozoite surface that binds to human RBCs.

Conceptualizing and contextualizing overtourism: the dynamics of accelerating urban tourism

2020 ◽  
Vol 6 (4) ◽  
pp. 657-671 ◽  
Author(s):  
Jan Henrik Nilsson
Keyword(s):  
Scientific Literature ◽  
Driving Forces ◽  
Urban Tourism ◽  
Structural Development ◽  
Evolutionary Perspective ◽  
Holistic View ◽  
Content Type ◽  
Inner Cities ◽  
Starting Point

Purpose From the background of the dramatic increase of urban tourism, framed by the concept of overtourism, the purpose of this paper is to analyze and discuss current dynamic processes of urban tourism growth, as presented in the scientific literature. With the help of a literature review, this paper aims to discuss current definitions and conceptualizations of overtourism and discuss the driving forces for the growth of urban tourism, thereby situating overtourism in relational to general structural change. Design/methodology/approach This paper builds on a non-exhaustive review of the scientific literature about overtourism and related topics, supplemented by a review of a few central policy documents. Findings Conceptually, overtourism relates to two different, but related, perspectives. The first one concern (negative) experiences of resident population and visitors, whereas the second relates to thresholds for the carrying capacity of destinations. Most of the reviewed literature focuses on three aspects of overtourism: localized problems in inner cities, the supply of unregulated accommodation through Airbnb and Airbnbs as a driving force of gentrification. Important perspectives are missing from the literature, mainly related to the development of driving forces of urban tourism growth in time and space. This observation is the starting point for a discussion on driving forces in an evolutionary perspective with the ambition of relating the growth of urban tourism to long waves of structural development. Research limitations/implications The paper focuses on overtourism in urban contexts, rural tourism is not discussed. Practical implications In identifying the importance of driving forces for understanding the dynamics of urban tourism growth, a holistic view on managing mitigation might be possible. Originality/value The paper adds an evolutionary perspective to the discussion about overtourism and its causes. Thereby, it answers to a need to take tourism seriously in social science, as a major economic, social and ecologic force. In emphasizing the relationship between driving forces on different geographic scales and levels, power relations are highlighted. The paper discusses the role of driving forces for mitigating overtourism. An understanding of the dynamics of driving forces is essential for the development of urban sustainable tourism.

scholarly journals A comparison of hotel ratings between verified and non-verified online review platforms

2020 ◽  
Vol 14 (2) ◽  
pp. 157-171
Author(s):  
Paolo Figini ◽  
Laura Vici ◽  
Giampaolo Viglia
Keyword(s):  
Mobile Apps ◽  
Time Windows ◽  
Structural Difference ◽  
Online Review ◽  
Review System ◽  
Content Type ◽  
Platform Comparison ◽  
Fake Reviews ◽  
Early Phases

Purpose This study aims to compare the rating dynamics of the same hotels in two online review platforms (Booking.com and Trip Advisor), which mainly differ in requiring or not requiring proof of prior reservation before posting a review (respectively, a verified vs a non-verified platform). Design/methodology/approach A verified system, by definition, cannot host fake reviews. Should also the non-verified system be free from “ambiguous” reviews, the structure of ratings (valence, variability, dynamics) for the same items should also be similar. Any detected structural difference, on the contrary, might be linked to a possible review bias. Findings Travelers’ scores in the non-verified platform are higher and much more volatile than ratings in the verified platform. Additionally, the verified review system presents a faster convergence of ratings towards the long-term scores of individual hotels, whereas the non-verified system shows much more discordance in the early phases of the review window. Research limitations/implications The paper offers insights into how to detect suspicious reviews. Non-verified platforms should add indices of scores’ dispersion to existing information available in websites and mobile apps. Moreover, they can use time windows to delete older (and more likely biased) reviews. Findings also ring a warning bell to tourists about the reliability of ratings, particularly when only a few reviews are posted online. Originality/value The across-platform comparison of single items (in terms of ratings’ dynamics and speed of convergence) is a novel contribution that calls for extending the analysis to different destinations and types of platform.

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