scholarly journals Rv2969c, essential for optimal growth inMycobacterium tuberculosis, is a DsbA-like enzyme that interacts with VKOR-derived peptides and has atypical features of DsbA-like disulfide oxidases

2013 ◽  
Vol 69 (10) ◽  
pp. 1981-1994 ◽  
Author(s):  
Lakshmanane Premkumar ◽  
Begoña Heras ◽  
Wilko Duprez ◽  
Patricia Walden ◽  
Maria Halili ◽  
...  
Keyword(s):  
Functional Unit ◽  
Active Form ◽  
Protein A ◽  
Optimal Growth ◽  
Partner Protein ◽  
Binding Surface ◽  
Catalytic Cysteine ◽  
Hydrophobic Binding ◽  
Peptide Segments ◽  
Redox Features

The bacterial disulfide machinery is an attractive molecular target for developing new antibacterials because it is required for the production of multiple virulence factors. The archetypal disulfide oxidase proteins inEscherichia coli(Ec) are DsbA and DsbB, which together form a functional unit: DsbA introduces disulfides into folding proteins and DsbB reoxidizes DsbA to maintain it in the active form. InMycobacterium tuberculosis(Mtb), no DsbB homologue is encoded but a functionally similar but structurally divergent protein, MtbVKOR, has been identified. Here, the Mtb protein Rv2969c is investigated and it is shown that it is the DsbA-like partner protein of MtbVKOR. It is found that it has the characteristic redox features of a DsbA-like protein: a highly acidic catalytic cysteine, a highly oxidizing potential and a destabilizing active-site disulfide bond. Rv2969c also has peptide-oxidizing activity and recognizes peptide segments derived from the periplasmic loops of MtbVKOR. Unlike the archetypal EcDsbA enzyme, Rv2969c has little or no activity in disulfide-reducing and disulfide-isomerase assays. The crystal structure of Rv2969c reveals a canonical DsbA fold comprising a thioredoxin domain with an embedded helical domain. However, Rv2969c diverges considerably from other DsbAs, including having an additional C-terminal helix (H8) that may restrain the mobility of the catalytic helix H1. The enzyme is also characterized by a very shallow hydrophobic binding surface and a negative electrostatic surface potential surrounding the catalytic cysteine. The structure of Rv2969c was also used to model the structure of a paralogous DsbA-like domain of the Ser/Thr protein kinase PknE. Together, these results show that Rv2969c is a DsbA-like protein with unique properties and a limited substrate-binding specificity.

scholarly journals Crystal structure of an active form of RAS protein, a complex of a GTP analog and the HRAS p21 catalytic domain.

1990 ◽  
Vol 87 (12) ◽  
pp. 4849-4853 ◽  
Author(s):  
A. T. Brunger ◽  
M. V. Milburn ◽  
L. Tong ◽  
A. M. deVos ◽  
J. Jancarik ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Catalytic Domain ◽  
Active Form ◽  
Protein A ◽  
Ras Protein

scholarly journals Expressed in the Yeast Saccharomyces cerevisiae, Human ERK5 Is a Client of the Hsp90 Chaperone That Complements Loss of the Slt2p (Mpk1p) Cell Integrity Stress-Activated Protein Kinase

2006 ◽  
Vol 5 (11) ◽  
pp. 1914-1924 ◽  
Author(s):  
Andrew W. Truman ◽  
Stefan H. Millson ◽  
James M. Nuttall ◽  
Victoria King ◽  
Mehdi Mollapour ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Map Kinase ◽  
Protein Kinases ◽  
Strong Association ◽  
Active Form ◽  
Cell Integrity ◽  
Binding Studies ◽  
Hsp90 Inhibition ◽  
Binding Surface ◽  
Hsp90 Chaperone

ABSTRACT ERK5 is a mitogen-activated protein (MAP) kinase regulated in human cells by diverse mitogens and stresses but also suspected of mediating the effects of a number of oncogenes. Its expression in the slt2Δ Saccharomyces cerevisiae mutant rescued several of the phenotypes caused by the lack of Slt2p (Mpk1p) cell integrity MAP kinase. ERK5 is able to provide this cell integrity MAP kinase function in yeast, as it is activated by the cell integrity signaling cascade that normally activates Slt2p and, in its active form, able to stimulate at least one key Slt2p target (Rlm1p, the major transcriptional regulator of cell wall genes). In vitro ERK5 kinase activity was abolished by Hsp90 inhibition. ERK5 activity in vivo was also lost in a strain that expresses a mutant Hsp90 chaperone. Therefore, human ERK5 expressed in yeast is an Hsp90 client, despite the widely held belief that the protein kinases of the MAP kinase class are non-Hsp90-dependent activities. Two-hybrid and protein binding studies revealed that strong association of Hsp90 with ERK5 requires the dual phosphorylation of the TEY motif in the MAP kinase activation loop. These phosphorylations, at positions adjacent to the Hsp90-binding surface recently identified for a number of protein kinases, may cause a localized rearrangement of this MAP kinase region that leads to creation of the Hsp90-binding surface. Complementation of the slt2Δ yeast defect by ERK5 expression establishes a new tool with which to screen for novel agonists and antagonists of ERK5 signaling as well as for isolating mutant forms of ERK5.

Epithelial lining fluid free IGF-I-to-PAPP-A ratio is associated with bronchopulmonary dysplasia in preterm infants

2007 ◽  
Vol 292 (1) ◽  
pp. E308-E313 ◽  
Author(s):  
Ettore Capoluongo ◽  
Franco Ameglio ◽  
Paola Lulli ◽  
Angelo Minucci ◽  
Concetta Santonocito ◽  
...  
Keyword(s):  
Bronchopulmonary Dysplasia ◽  
Active Form ◽  
Protein A ◽  
Epithelial Lining ◽  
Epithelial Lining Fluid ◽  
Oxygen Administration ◽  
Severity Scores ◽  
Igf I ◽  
Postmenstrual Age ◽  
Preterm Newborns

Preterm newborns developing retinopathy of prematurity (ROP) and bronchopulmonary dysplasia (BPD) show persistently low levels of insulin-like growth factor-I (IGF-I) in sera. They also present higher free IGF-I concentrations in epithelial lining fluids (ELFs) and lung tissues. Pregnancy-associated plasma protein-A (PAPP-A) is a metalloproteinase that dissociates three binding proteins from the active form of IGF-I, namely free IGF-I. The present study analyzes the ELF concentrations of free IGF-I, PAPP-A, and their ratios in preterm newborns developing or not BPD, defined as O2 dependence at 36 wk postmenstrual age. Bronchoalveolar lavage fluids of 41 infants (34 without and 7 with BPD) were analyzed on the 2nd and 4th day after birth. Infants developing BPD showed increased ELF free IGF-I and decreased PAPP-A concentrations on both days 2 and 4 compared with newborns without BPD. A nonsignificant trend between these 2 days was observed for free IGF-I (increasing) and PAPP-A (decreasing). On the same days, the free IGF-I-to-PAPP-A ratio was always significantly higher in patients developing BPD. These differences were more significant than those of IGF-I or PAPP-A when individually evaluated. A multivariate analysis confirmed the significance for free IGF-I on day 4, whereas the ratio was confirmed on both days 2 and 4. The same ratio was significantly correlated with some indexes of disease severity, such as hours of oxygen administration, days of hospitalization, and ROP severity scores. Finally, the ratio between ELF free IGF-I and PAPP-A appears to be a useful marker for lung injury of premature newborns.

scholarly journals Diaphanous formin mDia2 regulates CENP-A levels at centromeres

2016 ◽  
Vol 213 (4) ◽  
pp. 415-424 ◽  
Author(s):  
Chenshu Liu ◽  
Yinghui Mao
Keyword(s):  
Quantitative Imaging ◽  
Active Form ◽  
Protein A ◽  
Small Gtpase ◽  
Genome Replication ◽  
Rac Gtpase ◽  
Epigenetic Marker ◽  
Centromere Protein A ◽  
Histone H3 Variant ◽  
Recognition Protein

Centromeres of higher eukaryotes are epigenetically defined by centromere protein A (CENP-A), a centromere-specific histone H3 variant. The incorporation of new CENP-A into centromeres to maintain the epigenetic marker after genome replication in S phase occurs in G1 phase; however, how new CENP-A is loaded and stabilized remains poorly understood. Here, we identify the formin mDia2 as essential for stable replenishment of new CENP-A at centromeres. Quantitative imaging, pulse-chase analysis, and high-resolution ratiometric live-cell studies demonstrate that mDia2 and its nuclear localization are required to maintain CENP-A levels at centromeres. Depletion of mDia2 results in a prolonged centromere association of holiday junction recognition protein (HJURP), the chaperone required for CENP-A loading. A constitutively active form of mDia2 rescues the defect in new CENP-A loading caused by depletion of male germ cell Rac GTPase-activating protein (MgcRacGAP), a component of the small GTPase pathway essential for CENP-A maintenance. Thus, the formin mDia2 functions downstream of the MgcRacGAP-dependent pathway in regulating assembly of new CENP-A containing nucleosomes at centromeres.

scholarly journals Role of the Twin-Arginine Translocation Pathway in Staphylococcus

2009 ◽  
Vol 191 (19) ◽  
pp. 5921-5929 ◽  
Author(s):  
Lalitha Biswas ◽  
Raja Biswas ◽  
Christiane Nerz ◽  
Knut Ohlsen ◽  
Martin Schlag ◽  
...  
Keyword(s):  
Functional Unit ◽  
Signal Sequence ◽  
Protein A ◽  
Mouse Kidney ◽  
Deletion Mutants ◽  
Twin Arginine Translocation ◽  
High Affinity ◽  
Tat Pathway ◽  
First Time

ABSTRACT In Staphylococcus, the twin-arginine translocation (Tat) pathway is present only in some species and is composed of TatA and TatC. The tatAC operon is associated with the fepABC operon, which encodes homologs to an iron-binding lipoprotein, an iron-dependent peroxidase (FepB), and a high-affinity iron permease. The FepB protein has a typical twin-arginine (RR) signal peptide. The tat and fep operons constitute an entity that is not present in all staphylococcal species. Our analysis was focused on Staphylococcus aureus and S. carnosus strains. Tat deletion mutants (ΔtatAC) were unable to export active FepB, indicating that this enzyme is a Tat substrate. When the RR signal sequence from FepB was fused to prolipase and protein A, their export became Tat dependent. Since no other protein with a Tat signal could be detected, the fepABC-tatAC genes comprise not only a genetic but also a functional unit. We demonstrated that FepABC drives iron import, and in a mouse kidney abscess model, the bacterial loads of ΔtatAC and Δtat-fep mutants were decreased. For the first time, we show that the Tat pathway in S. aureus is functional and serves to translocate the iron-dependent peroxidase FepB.

scholarly journals The induction of secondary cytolytic T lymphocytes by solubilized membrane proteins.

1978 ◽  
Vol 147 (3) ◽  
pp. 946-951 ◽  
Author(s):  
M Mescher ◽  
L Sherman ◽  
F Lemonnier ◽  
S Burakoff
Keyword(s):  
Plasma Membranes ◽  
Active Form ◽  
Protein A ◽  
Gel Filtration ◽  
Biologically Active ◽  
Cytolytic T Lymphocytes ◽  
Histocompatibility Complex ◽  
Cell Plasma ◽  
Immunological Specificity ◽  
Membrane Bound

Membrane-bound antigens responsible for induction of a secondary allogeneic murine cytolytic T-cell (CTL) response have been obtained in a soluble, biologically active form by deoxycholate solubilization of tumor cell plasma membranes. The active proteins are soluble by the criteria of both ultracentrifugation and gel filtration. The immunological specificity of the induced CTL and removal of the activity from solution by treatment with B6 anti-P815 (anti-H-2d) antiserum and Protein A-Sepharose demonstrate that the CTL-inducing activity is dependent upon solubilized major histocompatibility complex antigens.

scholarly journals Proteinase-Polymerase Precursor as the Active Form of Feline Calicivirus RNA-Dependent RNA Polymerase

2001 ◽  
Vol 75 (3) ◽  
pp. 1211-1219 ◽  
Author(s):  
Lai Wei ◽  
Jason S. Huhn ◽  
Aaron Mory ◽  
Harsh B. Pathak ◽  
Stanislav V. Sosnovtsev ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rna Polymerase ◽  
Active Form ◽  
Polymerase Activity ◽  
Feline Calicivirus ◽  
Amino Terminus ◽  
Rna Dependent Rna Polymerase ◽  
Reading Frame ◽  
Catalytically Active ◽  
Catalytic Cysteine

ABSTRACT The objective of this study was to identify the active form of the feline calicivirus (FCV) RNA-dependent RNA polymerase (RdRP). Multiple active forms of the FCV RdRP were identified. The most active enzyme was the full-length proteinase-polymerase (Pro-Pol) precursor protein, corresponding to amino acids 1072 to 1763 of the FCV polyprotein encoded by open reading frame 1 of the genome. Deletion of 163 amino acids from the amino terminus of Pro-Pol (the Val-1235 amino terminus) caused a threefold reduction in polymerase activity. Deletion of an additional one (the Thr-1236 amino terminus) or two (the Ala-1237 amino terminus) amino acids produced derivatives that were 7- and 175-fold, respectively, less active than Pro-Pol. FCV proteinase-dependent processing of Pro-Pol in the interdomain region preceding Val-1235 was not observed in the presence of a catalytically active proteinase; however, processing within the polymerase domain was observed. Inactivation of proteinase activity by changing the catalytic cysteine-1193 to glycine permitted the production and purification of intact Pro-Pol. Biochemical analysis of Pro-Pol showed that this enzyme has properties expected of a replicative polymerase, suggesting that Pro-Pol is an active form of the FCV RdRP.

scholarly journals Complementation of the Lactococcus lactis Secretion Machinery with Bacillus subtilis SecDF Improves Secretion of Staphylococcal Nuclease

2006 ◽  
Vol 72 (3) ◽  
pp. 2272-2279 ◽  
Author(s):  
S. Nouaille ◽  
E. Morello ◽  
N. Cortez-Peres ◽  
Y. Le Loir ◽  
J. Commissaire ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Lactococcus Lactis ◽  
Protein A ◽  
High Capacity ◽  
Optimal Growth ◽  
Staphylococcal Nuclease ◽  
Low Levels ◽  
Encoding Gene ◽  
Positive Effect ◽  
Different Levels

ABSTRACT Unlike Bacillus subtilis and Escherichia coli, the gram-positive lactic acid bacterium Lactococcus lactis does not possess the SecDF protein, a component of the secretion (Sec) machinery involved in late secretion stages and required for the high-capacity protein secretion in B. subtilis. In this study, we complemented the L. lactis Sec machinery with SecDF from B. subtilis and evaluated the effect on the secretion of two forms of staphylococcal nuclease, NucB and NucT, which are efficiently and poorly secreted, respectively. The B. subtilis SecDF-encoding gene was tested in L. lactis at different levels. Increased quantities of the precursor and mature forms were observed only at low levels of SecDF and at high NucT production levels. This SecDF secretion enhancement was observed at the optimal growth temperature (30°C) and was even greater at 15°C. Furthermore, the introduction of B. subtilis SecDF into L. lactis was shown to have a positive effect on a secreted form of Brucella abortus L7/L12 antigen.

scholarly journals Optimization of Expression, Purification and Secretion of Functional Recombinant Human Growth Hormone in Prokaryotic Hosts Using Modified Staphylococcal Protein A Signal Peptide

2021 ◽  
Author(s):  
Garshasb Rigi ◽  
Amin Rostami ◽  
Habib Ghomi ◽  
Gholamreza Ahmadian ◽  
Vasiqe Sadat Mirbagheri ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Western Blot ◽  
Signal Peptide ◽  
Extracellular Space ◽  
Culture Medium ◽  
Active Form ◽  
Protein A ◽  
Human Growth ◽  
E Coli ◽  
Sds Page

Abstract Background: Human Growth Hormone (hGH) is a glycoprotein released from the pituitary gland. Due to the wide range of effects in humans, any disruption in hGH secretion could have serious consequences. This highlights the clinical importance of hGH production in the treatment of different diseases associated with a deficiency of this hormone. The production of recombinant mature hormone in suitable hosts and secretion of this therapeutic protein into the extracellular space can be considered as one of the best cost-effective approaches not only to obtain the active form of the protein but also endotoxin-free preparation. Since the natural growth hormone signal peptide is of eukaryotic origin and is not detectable by any of the E. coli secretory systems, including Sec and Tat, and is therefore unable to secrete hGH in the prokaryotic systems, designing a new and efficient signal peptide is essential to direct hGh to the extracellular space. Results: In this study, using a combination of the bioinformatics design and molecular genetics, the protein A signal peptide from Staphylococcus aureus was modified, redesigned and then fused to the mature hGH coding region. The recombinant hGH was then expressed in E. coli and successfully secreted to the medium through the Sec pathway. Secretion of the hGH into the medium was verified using SDS-PAGE and western blot analysis. Recombinant hGH was then expressed in E. coli and successfully secreted into cell culture medium via the Sec pathway. The secretion of hGH into the extracellular medium was confirmed by SDS-PAGE and Western blot analysis. Furthermore, the addition of glycine was shown to improve hGH secretion onto the culture medium. Equations for determining the optimal conditions were also determined. Functional hGH analysis using an ELISA-based method confirmed that the ratio of the active form of secreted hGH to the inactive form in the periplasm is higher than this ratio in the cytoplasm.Conclusions: Since the native signal protein peptide of S. aureus protein A was not able to deliver hGH to the extracellular space, it was modified using bioinformatics tools and fused to the n-terminal region of hGh to show that the redesigned signal peptide was functional.

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