scholarly journals Complete maturation of the plastid protein translocation channel requires a type I signal peptidase

2005 ◽  
Vol 171 (3) ◽  
pp. 425-430 ◽  
Author(s):  
Kentaro Inoue ◽  
Amy J. Baldwin ◽  
Rebecca L. Shipman ◽  
Kyoko Matsui ◽  
Steven M. Theg ◽  
...  
Keyword(s):  
Protein Translocation ◽  
Biological Significance ◽  
Transit Peptide ◽  
Signal Peptidase ◽  
Type I ◽  
Gene Encoding ◽  
Severe Reduction ◽  
Plastid Protein ◽  
Outer Envelope Membrane

The protein translocation channel at the plastid outer envelope membrane, Toc75, is essential for the viability of plants from the embryonic stage. It is encoded in the nucleus and is synthesized with a bipartite transit peptide that is cleaved during maturation. Despite its important function, the molecular mechanism and the biological significance of the full maturation of Toc75 remain unclear. In this study, we show that a type I signal peptidase (SPase I) is responsible for this process. First, we demonstrate that a bacterial SPase I converted Toc75 precursor to its mature form in vitro. Next, we show that disruption of a gene encoding plastidic SPase I (Plsp1) resulted in the accumulation of immature forms of Toc75, severe reduction of plastid internal membrane development, and a seedling lethal phenotype. These phenotypes were rescued by the overexpression of Plsp1 complementary DNA. Plsp1 appeared to be targeted both to the envelope and to the thylakoidal membranes; thus, it may have multiple functions.

scholarly journals A Putative Bacterial ABC Transporter Circumvents the Essentiality of Signal Peptidase

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
J. Hiroshi Morisaki ◽  
Peter A. Smith ◽  
Shailesh V. Date ◽  
Kimberly K. Kajihara ◽  
Chau Linda Truong ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Abc Transporter ◽  
Signal Peptide ◽  
Transcriptional Repressor ◽  
Signal Peptidase ◽  
Clinical Strain ◽  
Type I ◽  
Atpase Gene ◽  
Associated Proteins

ABSTRACT The type I signal peptidase of Staphylococcus aureus , SpsB, is an attractive antibacterial target because it is essential for viability and extracellularly accessible. We synthesized compound 103, a novel arylomycin-derived inhibitor of SpsB with significant potency against various clinical S. aureus strains (MIC of ~1 µg/ml). The predominant clinical strain USA300 developed spontaneous resistance to compound 103 with high frequency, resulting from single point mutations inside or immediately upstream of cro / cI , a homolog of the lambda phage transcriptional repressor cro . These cro / cI mutations led to marked (>50-fold) overexpression of three genes encoding a putative ABC transporter. Overexpression of this ABC transporter was both necessary and sufficient for resistance and, notably, circumvented the essentiality of SpsB during in vitro culture. Mutation of its predicted ATPase gene abolished resistance, suggesting a possible role for active transport; in these bacteria, resistance to compound 103 occurred with low frequency and through mutations in spsB . Bacteria overexpressing the ABC transporter and lacking SpsB were capable of secreting a subset of proteins that are normally cleaved by SpsB and instead were cleaved at a site distinct from the canonical signal peptide. These bacteria secreted reduced levels of virulence-associated proteins and were unable to establish infection in mice. This study reveals the mechanism of resistance to a novel arylomycin derivative and demonstrates that the nominal essentiality of the S. aureus signal peptidase can be circumvented by the upregulation of a putative ABC transporter in vitro but not in vivo . IMPORTANCE The type I signal peptidase of Staphylococcus aureus (SpsB) enables the secretion of numerous proteins by cleavage of the signal peptide. We synthesized an SpsB inhibitor with potent activity against various clinical S. aureus strains. The predominant S. aureus strain USA300 develops resistance to this inhibitor by mutations in a novel transcriptional repressor ( cro / cI ), causing overexpression of a putative ABC transporter. This mechanism promotes the cleavage and secretion of various proteins independently of SpsB and compensates for the requirement of SpsB for viability in vitro . However, bacteria overexpressing the ABC transporter and lacking SpsB secrete reduced levels of virulence-associated proteins and are unable to infect mice. This study describes a bacterial resistance mechanism that provides novel insights into the biology of bacterial secretion.

scholarly journals A cytomegaloviral protein reveals a dual role for STAT2 in IFN-γ signaling and antiviral responses

2005 ◽  
Vol 201 (10) ◽  
pp. 1543-1553 ◽  
Author(s):  
Albert Zimmermann ◽  
Mirko Trilling ◽  
Markus Wagner ◽  
Manuel Wilborn ◽  
Ivan Bubic ◽  
...  
Keyword(s):  
Biological Significance ◽  
Receptor Expression ◽  
Type I ◽  
Antiviral Responses ◽  
Type I Ifns ◽  
New Strategy ◽  
Infected Cells ◽  
Ifn Γ

A mouse cytomegalovirus (MCMV) gene conferring interferon (IFN) resistance was identified. This gene, M27, encodes a 79-kD protein that selectively binds and down-regulates for signal transducer and activator of transcription (STAT)-2, but it has no effect on STAT1 activation and signaling. The absence of pM27 conferred MCMV susceptibility to type I IFNs (α/β), but it had a much more dramatic effect on type II IFNs (γ) in vitro and in vivo. A comparative analysis of M27+ and M27− MCMV revealed that the antiviral efficiency of IFN-γ was partially dependent on the synergistic action of type I IFNs that required STAT2. Moreover, STAT2 was directly activated by IFN-γ. This effect required IFN receptor expression and was independent of type I IFNs. IFN-γ induced increasing levels of tyrosine-phosphorylated STAT2 in M27− MCMV-infected cells that were essential for the antiviral potency of IFN-γ. pM27 represents a new strategy for simultaneous evasions from types I and II IFNs, and it documents an unknown biological significance for STAT2 in antiviral IFN-γ responses.

scholarly journals Molecular characterization and analysis of a gene encoding the acidic repeat protein (Arp) of Treponema pallidum

2007 ◽  
Vol 56 (6) ◽  
pp. 715-721 ◽  
Author(s):  
Hsi Liu ◽  
Berta Rodes ◽  
Robert George ◽  
Bret Steiner
Keyword(s):  
Synthetic Peptides ◽  
Treponema Pallidum ◽  
Signal Peptidase ◽  
Type I ◽  
Type Ii ◽  
Gene Encoding ◽  
Western Blot Assay ◽  
Repeat Protein ◽  
Signal Peptidase I ◽  
Cdc 2

The acidic repeat protein (arp) genes from three subspecies of the treponeme Treponema pallidum (T. pallidum subsp. pallidum, Nichols strain; T. pallidum subsp. pertenue, CDC-1 and CDC-2 strains; and T. pallidum subsp. endemicum, Bosnia A strain) were cloned and sequenced. The predicted protein sequence contained a high percentage of glutamic acid, hence the name acidic repeat protein, or Arp. The protein had a potential membrane-spanning domain and a signal peptidase I site. The gene from the Nichols strain of T. pallidum subsp. pallidum contained a set of 14 nearly identical repeats of a 60 bp sequence, which occupied ∼51 % of the length of the gene. Analyses of arp from laboratory strains showed that the 5′ and 3′ ends of the genes were conserved, but there was considerable heterogeneity in the number of repeats of this 60 bp sequence. Based on amino acid variations, the 14 sequence repeats could be classified into three types, which were named type I, type II and type III repeats. The type II repeat was the most common in the strains examined. The arp gene of the Nichols strain was subsequently cloned into the expression vector pBAD/TOPO ThioFusion. The expressed protein was detected in a Western blot assay using rabbit immune sera produced against T. pallidum, or synthetic peptides derived from the repeat sequences. Using an ELISA, rapid plasma reagin (RPR) test-positive sera reacted with synthetic peptides derived from the repeat region but not with peptides derived from N and C termini of the Arp protein. These results show that the Arp protein is immunogenic and could prove to be a useful target for serological diagnosis of T. pallidum infection.

scholarly journals Chloroplast chaperonin-mediated targeting of a thylakoid membrane protein

2020 ◽  
Author(s):  
Laura Klasek ◽  
Kentaro Inoue ◽  
Steven M. Theg
Keyword(s):  
Membrane Protein ◽  
Thylakoid Membrane ◽  
Protein Targeting ◽  
Transmembrane Proteins ◽  
Signal Peptidase ◽  
Aqueous Environment ◽  
Type I ◽  
Isolated Chloroplasts

AbstractPost-translational protein targeting requires chaperone assistance to direct insertion-competent proteins to integration pathways. Chloroplasts integrate nearly all thylakoid transmembrane proteins post-translationally, but mechanisms in the stroma that assist their insertion remain largely undefined. Here, we investigated how the chloroplast chaperonin (Cpn60) facilitated the thylakoid integration of Plastidic type I signal peptidase 1 (Plsp1) using in vitro targeting assays. Cpn60 bound Plsp1 in the stroma. In isolated chloroplasts, the membrane integration of imported Plsp1 correlated with its dissociation from Cpn60. When the Plsp1 residues that interacted with Cpn60 were removed, Plsp1 did not integrate into the membrane. These results suggested Cpn60 was an intermediate in Plsp1’s thylakoid targeting. In isolated thylakoids, the integration of Plsp1 decreased if Cpn60 was present in excess of cpSecA1, the stromal motor of the cpSec1 translocon which inserts unfolded Plsp1 into the thylakoid. An excess of cpSecA1 favored integration. Introducing Cpn60’s obligate substrate RbcL displaced Cpn60-bound Plsp1; then, the released Plsp1 exhibited increased accessibility to cpSec1. These in vitro targeting experiments support a model in which Cpn60 captures and then releases insertion-competent Plsp1, while cpSecA1 recognizes free Plsp1 for integration. Thylakoid transmembrane proteins transiting the stroma can interact with Cpn60 to shield from the aqueous environment.One-sentence summaryThe chloroplast chaperonin captures and releases Plastidic type I signal peptidase 1 during its targeting to the thylakoid membrane.

scholarly journals Broadening the Spectrum of β-Lactam Antibiotics through Inhibition of Signal Peptidase Type I

2012 ◽  
Vol 56 (9) ◽  
pp. 4662-4670 ◽  
Author(s):  
Alex G. Therien ◽  
Joann L. Huber ◽  
Kenneth E. Wilson ◽  
Patrick Beaulieu ◽  
Alexandre Caron ◽  
...  
Keyword(s):  
Treatment Options ◽  
Signal Peptidase ◽  
Type I ◽  
New Agents ◽  
Content Type ◽  
Cyclic Depsipeptide ◽  
Serious Infections ◽  
Synthetic Derivative

ABSTRACTThe resistance of methicillin-resistantStaphylococcus aureus(MRSA) to all β-lactam classes limits treatment options for serious infections involving this organism. Our goal is to discover new agents that restore the activity of β-lactams against MRSA, an approach that has led to the discovery of two classes of natural product antibiotics, a cyclic depsipeptide (krisynomycin) and a lipoglycopeptide (actinocarbasin), which potentiate the activity of imipenem against MRSA strain COL. We report here that these imipenem synergists are inhibitors of the bacterial type I signal peptidase SpsB, a serine protease that is required for the secretion of proteins that are exported through the Sec and Tat systems. A synthetic derivative of actinocarbasin, M131, synergized with imipenem bothin vitroandin vivowith potent efficacy. Thein vitroactivity of M131 extends to clinical isolates of MRSA but not to a methicillin-sensitive strain. Synergy is restricted to β-lactam antibiotics and is not observed with other antibiotic classes. We propose that the SpsB inhibitors synergize with β-lactams by preventing the signal peptidase-mediated secretion of proteins required for β-lactam resistance. Combinations of SpsB inhibitors and β-lactams may expand the utility of these widely prescribed antibiotics to treat MRSA infections, analogous to β-lactamase inhibitors which restored the utility of this antibiotic class for the treatment of resistant Gram-negative infections.

Presence of a plant-like proton-translocating pyrophosphatase in a scuticociliate parasite and its role as a possible drug target

Parasitology ◽  
2014 ◽  
Vol 142 (3) ◽  
pp. 449-462 ◽  
Author(s):  
NATALIA MALLO ◽  
JESÚS LAMAS ◽  
CARLA PIAZZON ◽  
JOSÉ M. LEIRO
Keyword(s):  
Functional Characterization ◽  
New Drugs ◽  
Type I ◽  
Sequence Motifs ◽  
Gene Encoding ◽  
Western Blots ◽  
Inorganic Pyrophosphate ◽  
Apparent Lack ◽  
Common Motif

SUMMARYThe proton-translocating inorganic pyrophosphatases (H+-PPases) are primary electrogenic H+ pumps that derive energy from the hydrolysis of inorganic pyrophosphate (PPi). They are widely distributed among most land plants and have also been found in several species of protozoan parasites. Here we describe, for the first time, the molecular cloning and functional characterization of a gene encoding an H+-pyrophosphatase in the protozoan scuticociliate parasite Philasterides dicentrarchi, which infects turbot. The predicted P. dicentrarchi PPase (PdPPase) consists of 587 amino acids of molecular mass 61·7 kDa and an isoelectric point of 5·0. Several motifs characteristic of plant vacuolar H+-PPases (V–H+-PPases) were also found in the PdPPase, which contains all the sequence motifs of the prototypical type I V–H+-PPase from Arabidopsis thaliana vacuolar pyrophosphatase type I (AVP1) plant. The PdPPase has a characteristic residue that determines strict K+-dependence, but unlike AVP1, PdPPase contains an N-terminal signal peptide (SP) sequence. Antibodies generated by vaccination of mice with a genetic or recombinant protein containing a partial sequence of the PdPPase and a common motif with the polyclonal antibody PABHK specific to AVP1 recognized a single band of about 62 kDa in western blots. These antibodies specifically stained both vacuole and the alveolar membranes of trophozoites of P. dicentrarchi. H+ transport was partially inhibited by the bisphosphonate pamidronate (PAM) and completely inhibited by NaF. The bisphosphonate PAM inhibited both H+-translocation and gene expression. PdPPase and PAM also inhibited in vitro growth of the ciliates. The apparent lack of V–H+-PPases in vertebrates and the parasite sensitivity to PPI analogues may provide a molecular target for developing new drugs to control scuticociliatosis.

Evaluation of the type I signal peptidase as antibacterial target for biofilm-associated infections of Staphylococcus epidermidis

Microbiology ◽  
2009 ◽  
Vol 155 (11) ◽  
pp. 3719-3729 ◽  
Author(s):  
Katrijn Bockstael ◽  
Nick Geukens ◽  
Lieve Van Mellaert ◽  
Piet Herdewijn ◽  
Jozef Anné ◽  
...  
Keyword(s):  
Staphylococcus Epidermidis ◽  
Functional Activity ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Antimicrobial Treatment ◽  
Signal Peptidase ◽  
Temperature Sensitive ◽  
Type I

The development of antibacterial resistance is inevitable and is a major concern in hospitals and communities. Moreover, biofilm-grown bacteria are less sensitive to antimicrobial treatment. In this respect, the Gram-positive Staphylococcus epidermidis is an important source of nosocomial biofilm-associated infections. In the search for new antibacterial therapies, the type I signal peptidase (SPase I) serves as a potential target for development of antibacterials with a novel mode of action. This enzyme cleaves off the signal peptide from secreted proteins, making it essential for protein secretion, and hence for bacterial cell viability. S. epidermidis encodes three putative SPases I (denoted Sip1, Sip2 and Sip3), of which Sip1 lacks the catalytic lysine. In this report, we investigated the active S. epidermidis SPases I in more detail. Sip2 and Sip3 were found to complement a temperature-sensitive Escherichia coli lepB mutant, demonstrating their in vivo functional activity. In vitro functional activity of purified Sip2 and Sip3 proteins and inhibition of their activity by the SPase I inhibitor arylomycin A2 were further illustrated using a fluorescence resonance energy transfer (FRET)-based assay. Furthermore, we demonstrated that SPase I not only is an attractive target for development of novel antibacterials against free-living bacteria, but also is a feasible target for biofilm-associated infections.

scholarly journals Molecular Analysis of Phr Peptide Processing in Bacillus subtilis

2003 ◽  
Vol 185 (16) ◽  
pp. 4861-4871 ◽  
Author(s):  
Sophie Stephenson ◽  
Christian Mueller ◽  
Min Jiang ◽  
Marta Perego
Keyword(s):  
Bacillus Subtilis ◽  
Response Regulator ◽  
Signal Peptidase ◽  
Type I ◽  
Peptidase Activity ◽  
Amino Terminal ◽  
Processing Event ◽  
Signal Peptidases

ABSTRACT In Bacillus subtilis, an export-import pathway regulates production of the Phr pentapeptide inhibitors of Rap proteins. Processing of the Phr precursor proteins into the active pentapeptide form is a key event in the initiation of sporulation and competence development. The PhrA (ARNQT) and PhrE (SRNVT) peptides inhibit the RapA and RapE phosphatases, respectively, whose activity is directed toward the Spo0F∼P intermediate response regulator of the sporulation phosphorelay. The PhrC (ERGMT) peptide inhibits the RapC protein acting on the ComA response regulator for competence with regard to DNA transformation. The structural organization of PhrA, PhrE, and PhrC suggested a role for type I signal peptidases in the processing of the Phr preinhibitor, encoded by the phr genes, into the proinhibitor form. The proinhibitor was then postulated to be cleaved to the active pentapeptide inhibitor by an additional enzyme. In this report, we provide evidence that Phr preinhibitor proteins are subject to only one processing event at the peptide bond on the amino-terminal end of the pentapeptide. This processing event is most likely independent of type I signal peptidase activity. In vivo and in vitro analyses indicate that none of the five signal peptidases of B. subtilis (SipS, SipT, SipU, SipV, and SipW) are indispensable for Phr processing. However, we show that SipV and SipT have a previously undescribed role in sporulation, competence, and cell growth.

scholarly journals A Novel N-Tetrasaccharide in Patients with Congenital Disorders of Glycosylation, Including Asparagine-Linked Glycosylation Protein 1, Phosphomannomutase 2, and Mannose Phosphate Isomerase Deficiencies

2016 ◽  
Vol 62 (1) ◽  
pp. 208-217 ◽  
Author(s):  
Wenyue Zhang ◽  
Philip M James ◽  
Bobby G Ng ◽  
Xueli Li ◽  
Baoyun Xia ◽  
...  
Keyword(s):  
Diagnostic Marker ◽  
Alternative Pathway ◽  
Biological Significance ◽  
Congenital Disorders ◽  
Type I ◽  
Congenital Disorders Of Glycosylation ◽  
Cell Lysate ◽  
Cultured Skin ◽  
Phosphomannomutase 2

Abstract BACKGROUND Primary deficiencies in mannosylation of N-glycans are seen in a majority of patients with congenital disorders of glycosylation (CDG). We report the discovery of a series of novel N-glycans in sera, plasma, and cultured skin fibroblasts from patients with CDG having deficient mannosylation. METHOD We used LC-MS/MS and MALDI-TOF-MS analysis to identify and quantify a novel N-linked tetrasaccharide linked to the protein core, an N-tetrasaccharide (Neu5Acα2,6Galβ1,4-GlcNAcβ1,4GlcNAc) in plasma, serum glycoproteins, and a fibroblast lysate from patients with CDG caused by ALG1 [ALG1 (asparagine-linked glycosylation protein 1), chitobiosyldiphosphodolichol β-mannosyltransferase], PMM2 (phosphomannomutase 2), and MPI (mannose phosphate isomerase). RESULTS Glycoproteins in sera, plasma, or cell lysate from ALG1-CDG, PMM2-CDG, and MPI-CDG patients had substantially more N-tetrasaccharide than unaffected controls. We observed a >80% decline in relative concentrations of the N-tetrasaccharide in MPI-CDG plasma after mannose therapy in 1 patient and in ALG1-CDG fibroblasts in vitro supplemented with mannose. CONCLUSIONS This novel N-tetrasaccharide could serve as a diagnostic marker of ALG1-, PMM2-, or MPI-CDG for screening of these 3 common CDG subtypes that comprise >70% of CDG type I patients. Its quantification by LC-MS/MS may be useful for monitoring therapeutic efficacy of mannose. The discovery of these small N-glycans also indicates the presence of an alternative pathway in N-glycosylation not recognized previously, but its biological significance remains to be studied.

Bcl-XL is up-regulated by HTLV-I and HTLV-II in vitro and in ex vivo ATLL samples

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 275-281 ◽  
Author(s):  
Christophe Nicot ◽  
Renaud Mahieux ◽  
Shigeki Takemoto ◽  
Genoveffa Franchini
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Biological Significance ◽  
Lymphocytic Leukemia ◽  
Type I ◽  
Peripheral Blood Mononuclear ◽  
Term Survival

Human T lymphotropic virus type I (HTLV-I) is the etiological agent of adult T-cell lymphocytic leukemia (ATLL), whereas HTLV-II has not been associated with hematopoietic malignancies. The control of apoptotic pathways has emerged as a critical step in the development of many cancer types. As a result, the underlying mechanism of long-term survival of HTLV-I and HTLV-II was studied in infected T cells in vitro and in ex vivo ATLL samples. Results indicate that HTLV-I– and HTLV-II–infected T cells in vitro express high levels of the antiapoptotic protein Bcl compared with other human leukemic T cell lines or uninfected peripheral blood mononuclear cells. The levels of proapoptotic proteins Bax, BAD, and Bak were not significantly altered. HTLV-I and HTLV-II viral transactivators, Tax1 and Tax2, are known to increase expression of cellular genes. These proteins were tested for increased transcription from the human Bcl2 and Bcl-XL promoters. Whereas no effect was observed on the Bcl2 promoter, both Tax1 and Tax2 increased transcription of the Bcl-XL promoter in T cells, although Tax1 appeared to be more efficient than Tax2. The biological significance of these observations was validated by the finding of an increased expression of Bcl-XL in ex vivo ATLL cells, especially from patients unresponsive to various chemotherapy regimens. Altogether, these data suggest that overexpression of Bcl-XL in vivomay be in part responsible for the resistance of ATLL cells to chemotherapy. In addition, inefficient activation of the Bcl-XL promoter by Tax2 may result in a shorter survival time of HTLV-II–infected cells in vivo and a diminished risk of leukemia development.

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