scholarly journals A Cold-Sensitive Listeria monocytogenes Mutant Has a Transposon Insertion in a Gene Encoding a Putative Membrane Protein and Shows Altered (p)ppGpp Levels

2006 ◽  
Vol 72 (6) ◽  
pp. 3955-3959 ◽  
Author(s):  
Siqing Liu ◽  
Darrell O. Bayles ◽  
Tricia M. Mason ◽  
Brian J. Wilkinson
Keyword(s):  
Listeria Monocytogenes ◽  
Low Temperature ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
Critical Role ◽  
Peptide Sequence ◽  
Temperature Adaptation ◽  
Signal Peptide Sequence ◽  
C Terminus ◽  
Cold Sensitive

ABSTRACT A cold-sensitive Listeria monocytogenes mutant designated cld-14 was obtained by transposon Tn917 mutagenesis. The gene interrupted by Tn917 in cld-14 was the L. monocytogenes LMOf2365_1485 homolog, which exhibits 45.7% homology to the Bacillus subtilis yqfF locus. LMOf2365_1485, here designated pgpH, encodes a putative integral membrane protein with a predicted molecular mass of 81 kDa. PgpH is predicted to contain a conserved N-terminal signal peptide sequence, seven transmembrane helices, and a hydrophilic C terminus, which likely extends into the cytosol. The Tn917 insertion in pgpH is predicted to result in production of a premature polypeptide truncated at the fifth transmembrane domain. The C terminus of PgpH, which is probably absent in cld-14, contains a highly conserved HD domain that belongs to a metal-dependent phosphohydrolase family. Strain cld-14 accumulated higher levels of (p)ppGpp than the wild type accumulated, indicating that the function of PgpH may be to adjust cellular (p)ppGpp levels during low-temperature growth. The cld-14pgpH + complemented strain was able to grow at a low temperature, like the parent strain, providing direct evidence that the activity of PgpH is important in low-temperature adaptation. Because of its predicted membrane location, PgpH may play a critical role in sensing the environmental temperature and altering cellular (p)ppGpp levels to allow the organism to adapt to low temperatures.

Molecular cloning of a 47 kDa tissue-specific and differentiation-dependent urothelial cell surface glycoprotein

1993 ◽  
Vol 106 (1) ◽  
pp. 31-43 ◽  
Author(s):  
X.R. Wu ◽  
T.T. Sun
Keyword(s):  
Transmembrane Domain ◽  
Core Protein ◽  
Surface Glycoprotein ◽  
Peptide Sequence ◽  
Type I ◽  
Apical Surface ◽  
Bladder Epithelium ◽  
Cell Surface Glycoprotein ◽  
Signal Peptide Sequence ◽  
C Terminus

Despite the fact that bladder epithelium has many interesting biological features and is a frequent site of carcinoma formation, relatively little is known about its biochemical differentiation. We have shown recently that a 47 kDa glycoprotein, uroplakin III (UPIII), in conjunction with uroplakins I (27 kDa) and II (15 kDa), forms the asymmetric unit membrane (AUM)--a highly specialized biomembrane characteristic of the apical surface of bladder epithelium. Deglycosylation and cDNA sequencing revealed that UPIII contains up to 20 kDa of N-linked sugars attached to a core protein of 28.9 kDa. The presence of an N-terminal signal peptide sequence and a single transmembrane domain located near the C terminus, plus the N-terminal location of all the potential N-glycosylation sites, points to a type I (N-exo/C-cyto) configuration. Thus the mass of the extracellular domain (20 kDa plus up to 20 kDa of sugar) of UPIII greatly exceeds that of its intracellular domain (5 kDa). Such an asymmetrical mass distribution, a feature shared by the other two major uroplakins, provides a molecular explanation as to why the luminal leaflet of AUM is almost twice as thick as the cytoplasmic one. The fact that of the three major proteins of AUM only UPIII has a significant cytoplasmic domain suggests that this molecule may play an important role in AUM-cytoskeleton interaction in terminally differentiated urothelial cells.

Cryptopain-1, a cysteine protease of Cryptosporidium parvum, does not require the pro-domain for folding

Parasitology ◽  
2008 ◽  
Vol 136 (2) ◽  
pp. 149-157 ◽  
Author(s):  
B.-K. NA ◽  
J.-M. KANG ◽  
H.-I. CHEUN ◽  
S.-H. CHO ◽  
S.-U. MOON ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Cysteine Protease ◽  
Transmembrane Domain ◽  
Cathepsin L ◽  
Active Form ◽  
Protozoan Parasite ◽  
Biochemical Properties ◽  
Peptide Sequence ◽  
Signal Peptide Sequence ◽  
Mature Domain

SUMMARYCryptosporidium parvum is an intracellular protozoan parasite that causes cryptosporidiosis in mammals including humans. In the current study, the gene encoding the cysteine protease of C. parvum (cryptopain-1) was identified and the biochemical properties of the recombinant enzyme were characterized. Cryptopain-1 shared common structural properties with cathepsin L-like papain family enzymes, but lacked a typical signal peptide sequence and contained a possible transmembrane domain near the amino terminus and a unique insert in the front of the mature domain. The recombinant cryptopain-1 expressed in Escherichia coli and refolded to the active form showed typical biochemical properties of cathepsin L-like enzymes. The folding determinant of cryptopain-1 was characterized through multiple constructs with or without different lengths of the pro-domain of the enzyme expressed in E. coli and assessment of their refolding abilities. All constructs, except one that did not contain the full-length mature domain, successfully refolded into the active enzymes, suggesting that cryptopain-1 did not require the pro-domain for folding. Western blot analysis showed that cryptopain-1 was expressed in the sporozoites and the enzyme preferentially degraded proteins, including collagen and fibronectin, but not globular proteins. This suggested a probable role for cryptopain-1 in host cell invasion and/or egression by the parasite.

scholarly journals Structure of APP-C991-99 and Implications for Role of Extra-Membrane Domains in Function and Oligomerization

2018 ◽  
Author(s):  
George A. Pantelopulos ◽  
John E. Straub ◽  
D. Thirumalai ◽  
Yuji Sugita
Keyword(s):  
Transmembrane Domain ◽  
Chemical Shifts ◽  
Membrane Surface ◽  
Critical Role ◽  
Amyloid Formation ◽  
Cytoplasmic Proteins ◽  
C Terminus ◽  
N Terminus ◽  
Thin Membranes

AbstractThe 99 amino acid C-terminal fragment of Amyloid Precursor Protein APP-C99 (C99) is cleaved by γ-secretase to form Aβ peptide, which plays a critical role in the etiology of Alzheimer’s Disease (AD). The structure of C99 consists of a single transmembrane domain flanked by intra and intercellular domains. While the structure of the transmembrane domain has been well characterized, little is known about the structure of the flanking domains and their role in C99 processing by γ-secretase. To gain insight into the structure of full-length C99, REMD simulations were performed for monomeric C99 in model membranes of varying thickness. We find equilibrium ensembles of C99 from simulation agree with experimentally-inferred residue insertion depths and protein backbone chemical shifts. In thin membranes, the transmembrane domain structure is correlated with extra-membrane structural states. Mean and variance of the transmembrane and G37G38 hinge angles are found to increase with thinning membrane. The N-terminus of C99 forms β-strands that may seed aggregation of Aβ on the membrane surface, promoting amyloid formation. The N-terminus, which forms α-helices that interact with the nicastrin domain of γ-secretase. The C-terminus of C99 becomes more α-helical as the membrane thickens, forming structures that may be suitable for binding by cytoplasmic proteins, while C-terminal residues essential to cytotoxic function become α-helical as the membrane thins. The heterogeneous but discrete extra-membrane domain states analyzed here open the path to new investigations of the role of C99 structure and membrane in amyloidogenesis.

scholarly journals Exogenous Isoleucine and Fatty Acid Shortening Ensure the High Content of Anteiso-C15:0 Fatty Acid Required for Low-Temperature Growth of Listeria monocytogenes

2005 ◽  
Vol 71 (12) ◽  
pp. 8002-8007 ◽  
Author(s):  
Kun Zhu ◽  
Xiang Ding ◽  
Mudcharee Julotok ◽  
Brian J. Wilkinson
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Listeria Monocytogenes ◽  
Low Temperature ◽  
Fatty Acid Profile ◽  
Acyl Carrier Protein ◽  
Critical Role ◽  
Chain Fatty Acid ◽  
Fatty Acid Chain ◽  
Branched Chain

ABSTRACT Previous studies have demonstrated that the branched-chain fatty acid anteiso-C15:0 plays a critical role in the growth of Listeria monocytogenes at low temperatures by ensuring sufficient membrane fluidity. Studies utilizing a chemically defined minimal medium revealed that the anteiso fatty acid precursor isoleucine largely determined the fatty acid profile and fatty acid response of the organism to lowered growth temperature. When isoleucine was sufficient, the fatty acid profile was very uniform, with anteiso fatty acids comprising up to 95% of total fatty acid, and the major fatty acid adjustment to low temperature was fatty acid chain shortening, which resulted in an increase of anteiso-C15:0 solely at the expense of anteiso-C17:0. When isoleucine was not supplied, the fatty acid profile became more complex and was readily modified by leucine, which resulted in a significant increase of corresponding iso fatty acids and an inability to grow at 10°C. Under this condition, the increase of anteiso-C15:0 at low temperature resulted from the combined effect of increasing the anteiso:iso ratio and chain shortening. A branched-chain α-keto acid dehydrogenase-defective strain largely lost the ability to increase the anteiso:iso ratio. Cerulenin, an inhibitor of β-ketoacyl-acyl carrier protein synthase (FabF), induced a similar fatty acid chain shortening as low temperature did. We propose that the anteiso precursor preferences of enzymes in the branched-chain fatty acid biosynthesis pathway ensure a high production of anteiso fatty acids, and cold-regulated chain shortening results in a further increase of anteiso-C15:0 at the expense of anteiso-C17:0.

scholarly journals Influence of the Transposition of the Thermostabilizing Domain of Clostridium thermocellum Xylanase (XynX) on Xylan Binding and Thermostabilization

2002 ◽  
Vol 68 (7) ◽  
pp. 3496-3501 ◽  
Author(s):  
Eun-Sun Shin ◽  
Mi-Jeong Yang ◽  
Kyung Hwa Jung ◽  
Eun-Ju Kwon ◽  
Jae Sung Jung ◽  
...  
Keyword(s):  
Clostridium Thermocellum ◽  
Catalytic Domain ◽  
Binding Capacity ◽  
Residual Activity ◽  
Peptide Sequence ◽  
Optimum Temperature ◽  
Xylanase Gene ◽  
Signal Peptide Sequence ◽  
C Terminus ◽  
The One

ABSTRACT A xylanase gene, xynX, of Clostridium thermocellum had one thermostabilizing domain (TSD) between the signal peptide sequence and the catalytic domain (CD). The TSD of a truncated xylanase gene, xynX′TSD-CD, was transpositioned from the N terminus to the C terminus of the CD by overlapping PCRs, and a modified product, xynX′CD-TSD, was constructed. XynX′TSD-CD had a higher optimum temperature (70°C versus 65°C) and was more thermostable (residual activity of 68% versus 46% after a 20-min preincubation at 70°C) than the one without the TSD, XynX′CD. However, the domain-transpositioned enzyme, XynX′CD-TSD, showed a lower optimum temperature (30°C) and thermostability (20%) than XynX′CD. Both XynX′TSD-CD and XynX′CD-TSD showed significantly higher binding capacity toward xylan than XynX′CD, and the domain transposition did not cause any change in the binding ability. XynX′TSD-CD and XynX′CD-TSD also showed considerable binding to lichenan but not to carboxymethyl cellulose and laminarin. XynX′TSD-CD and XynX′CD-TSD had higher activities for insoluble xylan than XynX′CD, while XynX′CD was more active against soluble xylan than XynX′TSD-CD and XynX′CD-TSD. These results indicate that the TSD of XynX has dual functions, xylan binding and thermostabilization, and the domain should also be classified as a xylan-binding domain (XBD). The binding capacity of the XBD was not affected by domain transpositioning within the gene.

scholarly journals Characterisation and functional analysis of canine TLR5

2020 ◽  
Vol 26 (6) ◽  
pp. 451-458
Author(s):  
Aihua Zhu ◽  
Lingling Wei ◽  
Sujuan Hu ◽  
Cheng Yang ◽  
Caifa Chen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Salmonella Enteritidis ◽  
Transmembrane Domain ◽  
Immunofluorescence Assay ◽  
Peptide Sequence ◽  
Signal Peptide Sequence ◽  
Reading Frame ◽  
Serovar Typhimurium ◽  
Receptor Domain

In this study, we characterised the single exon TLR5 gene of the Chinese rural dog. Sequence analysis revealed a 2577 nucleotide-long open reading frame of canine TLR5, encoding an 858 amino acid-long protein. The putative amino acid sequence of canine TLR5 consisted of a signal peptide sequence, 15 LRR domains, a LRR C-terminal domain, a transmembrane domain and an intracellular Toll-IL-1 receptor domain. The amino acid sequence of the canine TLR5 protein shared 95.4% identity with vulpine, 72.2% with feline and 64.7% with human TLR5. Plasmids expressing canine TLR5 and NF-κB-luciferase were constructed and transfected into HEK293T cells. Expression was confirmed by indirect immunofluorescence assay. These HEK293T cells transfected with the canine TLR5- and NF-κB-luciferase plasmids significantly responded to flagellin from Salmonella enteritidis serovar Typhimurium, indicating that it is a functional TLR5 homolog. In response to stimulation with Salmonella enteritidis, the level of TLR5 mRNA significantly increased over the control in PBMCs at 4 h. The levels of IL-8, IL-6 and IL-1β also increased after exposure. The highest levels of TLR5, IL-8 and IL-1β expression were detected at 8, 4 and 12 h after stimulation, respectively. These results imply that the expression of canine TLR5 may participate in the immune response against bacterial pathogens.

scholarly journals Biosynthesis of the dystonia-associated AAA+ ATPase torsinA at the endoplasmic reticulum

2006 ◽  
Vol 401 (2) ◽  
pp. 607-612 ◽  
Author(s):  
Anna C. Callan ◽  
Sandra Bunning ◽  
Owen T. Jones ◽  
Stephen High ◽  
Eileithyia Swanton
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Integral Membrane Protein ◽  
Signal Peptidase ◽  
Aaa Atpase ◽  
C Terminus ◽  
Membrane Spanning ◽  
Aaa Atpases

TorsinA is a widely expressed AAA+ (ATPases associated with various cellular activities) ATPase of unknown function. Previous studies have described torsinA as a type II protein with a cleavable signal sequence, a single membrane spanning domain, and its C-terminus located in the ER (endoplasmic reticulum) lumen. However, in the present study we show that torsinA is not in fact an integral membrane protein. Instead we find that the mature protein associates peripherally with the ER membrane, most likely through an interaction with an integral membrane protein. Consistent with this model, we provide evidence that the signal peptidase complex cleaves the signal sequence of torsinA, and we show that the region previously suggested to form a transmembrane domain is translocated into the lumen of the ER. The finding that torsinA is a peripheral, and not an integral membrane protein as previously thought, has important implications for understanding the function of this novel ATPase.

scholarly journals ANKRD22 is an N-myristoylated hairpin-like monotopic membrane protein specifically localized to lipid droplets

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Toshihiko Utsumi ◽  
Takuro Hosokawa ◽  
Mayu Shichita ◽  
Misato Nishiue ◽  
Natsuko Iwamoto ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Lipid Droplets ◽  
Transmembrane Domain ◽  
Intracellular Localization ◽  
Membrane Topology ◽  
Intracellular Targeting ◽  
C Terminus ◽  
Charged Residues ◽  
Necrosis Factor ◽  
Positively Charged

AbstractThe membrane topology and intracellular localization of ANKRD22, a novel human N-myristoylated protein with a predicted single-pass transmembrane domain that was recently reported to be overexpressed in cancer, were examined. Immunofluorescence staining of COS-1 cells transfected with cDNA encoding ANKRD22 coupled with organelle markers revealed that ANKRD22 localized specifically to lipid droplets (LD). Analysis of the intracellular localization of ANKRD22 mutants C-terminally fused to glycosylatable tumor necrosis factor (GLCTNF) and assessment of their susceptibility to protein N-glycosylation revealed that ANKRD22 is synthesized on the endoplasmic reticulum (ER) membrane as an N-myristoylated hairpin-like monotopic membrane protein with the amino- and carboxyl termini facing the cytoplasm and then sorted to LD. Pro98 located at the center of the predicted membrane domain was found to be essential for the formation of the hairpin-like monotopic topology of ANKRD22. Moreover, the hairpin-like monotopic topology, and positively charged residues located near the C-terminus were demonstrated to be required for the sorting of ANKRD22 from ER to LD. Protein N-myristoylation was found to positively affect the LD localization. Thus, multiple factors, including hairpin-like monotopic membrane topology, C-terminal positively charged residues, and protein N-myristoylation cooperatively affected the intracellular targeting of ANKRD22 to LD.

scholarly journals Tail-anchored PEX26 targets peroxisomes via a PEX19-dependent and TRC40-independent class I pathway

2013 ◽  
Vol 200 (5) ◽  
pp. 651-666 ◽  
Author(s):  
Yuichi Yagita ◽  
Takahide Hiromasa ◽  
Yukio Fujiki
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Adenosine Triphosphatase ◽  
Transmembrane Domain ◽  
Peroxisomal Membrane ◽  
Basic Amino Acids ◽  
C Terminus ◽  
Peroxisomal Targeting ◽  
Ta Proteins

Tail-anchored (TA) proteins are anchored into cellular membranes by a single transmembrane domain (TMD) close to the C terminus. Although the targeting of TA proteins to peroxisomes is dependent on PEX19, the mechanistic details of PEX19-dependent targeting and the signal that directs TA proteins to peroxisomes have remained elusive, particularly in mammals. The present study shows that PEX19 formed a complex with the peroxisomal TA protein PEX26 in the cytosol and translocated it directly to peroxisomes by interacting with the peroxisomal membrane protein PEX3. Unlike in yeast, the adenosine triphosphatase TRC40, which delivers TA proteins to the endoplasmic reticulum, was dispensable for the peroxisomal targeting of PEX26. Moreover, the basic amino acids within the luminal domain of PEX26 were essential for binding to PEX19 and thereby for peroxisomal targeting. Finally, our results suggest that a TMD that escapes capture by TRC40 and is followed by a highly basic luminal domain directs TA proteins to peroxisomes via the PEX19-dependent route.

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