A topological study of the human γ-glutamyl carboxylase

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 973-978 ◽  
Author(s):  
Jianke Tie ◽  
Sheue-Mei Wu ◽  
Dayun Jin ◽  
Christopher V. Nicchitta ◽  
Darrel W. Stafford
Keyword(s):  
Transmembrane Domain ◽  
Membrane Topology ◽  
Proteinase K ◽  
In Vitro Translation ◽  
C Terminus ◽  
293 Cells ◽  
Leader Peptidase ◽  
Polytopic Membrane Protein

γ-Glutamyl carboxylase (GC), a polytopic membrane protein found in the endoplasmic reticulum (ER), catalyzes vitamin K–dependent posttranslational modification of glutamate to γ-carboxyl glutamate. In an attempt to delineate the structure of this important enzyme, in vitro translation and in vivo mapping were used to study its membrane topology. Using terminus-tagged full-length carboxylase, expressed in 293 cells, it was demonstrated that the amino-terminus of the GC is on the cytoplasmic side of the ER, while the carboxyl-terminus is on the lumenal side. In addition, a series of fusions were made to encode each predicted transmembrane domain (TMD) followed by a leader peptidase (Lep) reporter tag, as analyzed by the computer algorithm TOPPRED II. Following in vitro translation of each fusion in the presence of canine microsomes, the topological orientation of the Lep tag was determined by proteinase K digestion and endoglycosidase H (Endo H) cleavage. From the topological orientation of the Lep tag in each fusion, the GC spans the ER membrane at least 5 times, with its N-terminus in the cytoplasm and its C-terminus in the lumen.

A topological study of the human γ-glutamyl carboxylase

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 973-978 ◽  
Author(s):  
Jianke Tie ◽  
Sheue-Mei Wu ◽  
Dayun Jin ◽  
Christopher V. Nicchitta ◽  
Darrel W. Stafford
Keyword(s):  
Transmembrane Domain ◽  
Membrane Topology ◽  
Proteinase K ◽  
In Vitro Translation ◽  
C Terminus ◽  
293 Cells ◽  
Leader Peptidase ◽  
Polytopic Membrane Protein

Abstract γ-Glutamyl carboxylase (GC), a polytopic membrane protein found in the endoplasmic reticulum (ER), catalyzes vitamin K–dependent posttranslational modification of glutamate to γ-carboxyl glutamate. In an attempt to delineate the structure of this important enzyme, in vitro translation and in vivo mapping were used to study its membrane topology. Using terminus-tagged full-length carboxylase, expressed in 293 cells, it was demonstrated that the amino-terminus of the GC is on the cytoplasmic side of the ER, while the carboxyl-terminus is on the lumenal side. In addition, a series of fusions were made to encode each predicted transmembrane domain (TMD) followed by a leader peptidase (Lep) reporter tag, as analyzed by the computer algorithm TOPPRED II. Following in vitro translation of each fusion in the presence of canine microsomes, the topological orientation of the Lep tag was determined by proteinase K digestion and endoglycosidase H (Endo H) cleavage. From the topological orientation of the Lep tag in each fusion, the GC spans the ER membrane at least 5 times, with its N-terminus in the cytoplasm and its C-terminus in the lumen.

scholarly journals Topogenesis in membranes of the NTB–VPg protein of Tomato ringspot nepovirus: definition of the C-terminal transmembrane domain

2004 ◽  
Vol 85 (2) ◽  
pp. 535-545 ◽  
Author(s):  
Aiming Wang ◽  
Sumin Han ◽  
Hélène Sanfaçon
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Proteinase K ◽  
Hydrophobic Region ◽  
C Terminus ◽  
Microsomal Membranes ◽  
Hydrophobic Amino Acids ◽  
Definition Of

The putative NTP-binding protein (NTB) of Tomato ringspot nepovirus (ToRSV) contains a hydrophobic region at its C terminus consisting of two adjacent stretches of hydrophobic amino acids separated by a few amino acids. In infected plants, the NTB–VPg polyprotein (containing the domain for the genome-linked protein) is associated with endoplasmic reticulum-derived membranes that are active in ToRSV replication. Recent results from proteinase K protection assays suggested a luminal location for the VPg domain in infected plants, providing support for the presence of a transmembrane domain at the C terminus of NTB. In this study, we have shown that NTB–VPg associates with canine microsomal membranes in the absence of other viral proteins in vitro and adopts a topology similar to that observed in vivo in that the VPg is present in the lumen. Truncated proteins containing 60 amino acids at the C terminus of NTB and the entire VPg exhibited a similar topology, confirming that this region of the protein contains a functional transmembrane domain. Deletion of portions of the C-terminal hydrophobic region of NTB by mutagenesis and introduction of glycosylation sites to map the luminal regions of the protein revealed that only the first stretch of hydrophobic amino acids traverses the membrane, while the second stretch of hydrophobic amino acids is located in the lumen. Our results provide additional evidence supporting the hypothesis that the NTB–VPg polyprotein acts as a membrane-anchor for the replication complex.

scholarly journals Membrane topology of the high-affinity L-glutamate transporter (GLAST-1) of the central nervous system.

1996 ◽  
Vol 135 (6) ◽  
pp. 1867-1877 ◽  
Author(s):  
S Wahle ◽  
W Stoffel
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Transmembrane Domain ◽  
Glutamate Transporter ◽  
Membrane Topology ◽  
High Affinity ◽  
Transporter Family ◽  
The Central Nervous System

The membrane topology of the high affinity, Na(+)-coupled L-glutamate/L-aspartate transporter (GLAST-1) of the central nervous system has been determined. Truncated GLAST-1 cDNA constructs encoding protein fragments with an increasing number of hydrophobic regions were fused to a cDNA encoding a reporter peptide with two N-glycosylation sites. The respective cRNA chimeras were translated in vitro and in vivo in Xenopus oocytes. Posttranslational N-glycosylation of the two reporter consensus sites monitors the number, size, and orientation of membrane-spanning domains. The results of our experiments suggest a novel 10-transmembrane domain topology of GLAST-1, a representative of the L-glutamate neurotransmitter transporter family, with its NH2 and COOH termini on the cytoplasmic side, six NH2-terminal hydrophobic transmembrane alpha-helices, and four COOH-terminal short hydrophobic domains spanning the bilayer predicted as beta-sheets.

scholarly journals Targeting of a Tail-anchored Protein to Endoplasmic Reticulum and Mitochondrial Outer Membrane by Independent but Competing Pathways

2001 ◽  
Vol 12 (8) ◽  
pp. 2482-2496 ◽  
Author(s):  
Nica Borgese ◽  
Ilaria Gazzoni ◽  
Massimo Barberi ◽  
Sara Colombo ◽  
Emanuela Pedrazzini
Keyword(s):  
Endoplasmic Reticulum ◽  
Outer Membrane ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Mitochondrial Outer Membrane ◽  
C Terminus ◽  
Green Fluorescent ◽  
Ta Proteins

Many mitochondrial outer membrane (MOM) proteins have a transmembrane domain near the C terminus and an N-terminal cytosolic moiety. It is not clear how these tail-anchored (TA) proteins posttranslationally select their target, but C-terminal charged residues play an important role. To investigate how discrimination between MOM and endoplasmic reticulum (ER) occurs, we used mammalian cytochrome b 5, a TA protein existing in two, MOM or ER localized, versions. Substitution of the seven C-terminal residues of the ER isoform or of green fluorescent protein reporter constructs with one or two arginines resulted in MOM-targeted proteins, whereas a single C-terminal threonine caused promiscuous localization. To investigate whether targeting to MOM occurs from the cytosol or after transit through the ER, we tagged a MOM-directed construct with a C-terminal N-glycosylation sequence. Although in vitro this construct was efficiently glycosylated by microsomes, the protein expressed in vivo localized almost exclusively to MOM, and was nearly completely unglycosylated. The small fraction of glycosylated protein was in the ER and was not a precursor to the unglycosylated form. Thus, targeting occurs directly from the cytosol. Moreover, ER and MOM compete for the same polypeptide, explaining the dual localization of some TA proteins.

scholarly journals A Peptide Found in Human Serum, Derived from the C-Terminus of Albumin, Shows Antifungal Activity In Vitro and In Vivo

2020 ◽  
Vol 8 (10) ◽  
pp. 1627
Author(s):  
Tecla Ciociola ◽  
Pier Paolo Zanello ◽  
Tiziana D’Adda ◽  
Serena Galati ◽  
Stefania Conti ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Human Serum ◽  
Fungicidal Activity ◽  
Galleria Mellonella ◽  
Serum Proteins ◽  
Morphological Alterations ◽  
C Terminus ◽  
Different Sources

The growing problem of antimicrobial resistance highlights the need for alternative strategies to combat infections. From this perspective, there is a considerable interest in natural molecules obtained from different sources, which are shown to be active against microorganisms, either alone or in association with conventional drugs. In this paper, peptides with the same sequence of fragments, found in human serum, derived from physiological proteins, were evaluated for their antifungal activity. A 13-residue peptide, representing the 597–609 fragment within the albumin C-terminus, was proved to exert a fungicidal activity in vitro against pathogenic yeasts and a therapeutic effect in vivo in the experimental model of candidal infection in Galleria mellonella. Studies by confocal microscopy and transmission and scanning electron microscopy demonstrated that the peptide penetrates and accumulates in Candida albicans cells, causing gross morphological alterations in cellular structure. These findings add albumin to the group of proteins, which already includes hemoglobin and antibodies, that could give rise to cryptic antimicrobial fragments, and could suggest their role in anti-infective homeostasis. The study of bioactive fragments from serum proteins could open interesting perspectives for the development of new antimicrobial molecules derived by natural sources.

Dementia with Lewy bodies—associated ß-synuclein mutations V70M and P123H cause mutation-specific neuropathological lesions

2021 ◽  
Author(s):  
Maryna Psol ◽  
Sofia Guerin Darvas ◽  
Kristian Leite ◽  
Sameehan U Mahajani ◽  
Mathias Bähr ◽  
...  
Keyword(s):  
Neuronal Network ◽  
Dementia With Lewy Bodies ◽  
Lewy Bodies ◽  
Sporadic Case ◽  
Network Activity ◽  
Proteinase K ◽  
Neuronal Network Activity ◽  
Distinct Disease

Abstract ß-Synuclein (ß-Syn) has long been considered to be an attenuator for the neuropathological effects caused by the Parkinson’s disease-related α-Synuclein (α-Syn) protein. However, recent studies demonstrated that overabundant ß-Syn can form aggregates and induce neurodegeneration in CNS neurons in vitro and in vivo, albeit at a slower pace as compared to α-Syn. Here we demonstrate that ß-Syn mutants V70M, detected in a sporadic case of Dementia with Lewy Bodies (DLB), and P123H, detected in a familial case of DLB, robustly aggravate the neurotoxic potential of ß-Syn. Intriguingly, the two mutations trigger mutually exclusive pathways. ß-Syn V70M enhances morphological mitochondrial deterioration and degeneration of dopaminergic and non-dopaminergic neurons, but has no influence on neuronal network activity. Conversely, ß-Syn P123H silences neuronal network activity, but does not aggravate neurodegeneration. ß-Syn WT, V70M and P123H formed proteinase K (PK) resistant intracellular fibrils within neurons, albeit with less stable C-termini as compared to α-Syn. Under cell free conditions, ß-Syn V70M demonstrated a much slower pace of fibril formation as compared to WT ß-Syn, and P123H fibrils present with a unique phenotype characterized by large numbers of short, truncated fibrils. Thus, it is possible that V70M and P123H cause structural alterations in ß-Syn, that are linked to their distinct neuropathological profiles. The extent of the lesions caused by these neuropathological profiles is almost identical to that of overabundant α-Syn, and thus likely to be directly involved into etiology of DLB. Over all, this study provides insights into distinct disease mechanisms caused by mutations of ß-Syn.

scholarly journals C-Terminal Deletions Can Suppress Temperature-Sensitive Mutations and Change Dominance in the Phage Mu Repressor

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 661-672 ◽  
Author(s):  
Jodi L Vogel ◽  
Vincent Geuskens ◽  
Lucie Desmet ◽  
N Patrick Higgins ◽  
Ariane Toussaint
Keyword(s):  
Binding Activity ◽  
Temperature Sensitive ◽  
Dna Binding Activity ◽  
Heat Stable ◽  
C Terminus ◽  
Acid Domain ◽  
Mutant Forms ◽  
Amino Acid Domain

Abstract Mutations in an N-terminal 70-amino acid domain of bacteriophage Mu's repressor cause temperature-sensitive DNA-binding activity. Surprisingly, amber mutations can conditionally correct the heat-sensitive defect in three mutant forms of the repressor gene, cts25 (D43-G), cts62 (R47-Q and cts71 (M28-I), and in the appropriate bacterial host produce a heat-stable Sts phenotype (for survival of temperature shifts). Sts repressor mutants are heat sensitive when in supE or supF hosts and heat resistant when in Sup° hosts. Mutants with an Sts phenotype have amber mutations at one of three codons, Q179, Q187, or Q190. The Sts phenotype relates to the repressor size: in Sup° hosts sts repressors are shorter by seven, 10, or 18 amino acids compared to repressors in supE or supF hosts. The truncated form of the sts62-1 repressor, which lacks 18 residues (Q179–V196), binds Mu operator DNA more stably at 42° in vitro compared to its full-length counterpart (cts62 repressor). In addition to influencing temperature sensitivity, the C-terminus appears to control the susceptibility to in vivo Clp proteolysis by influencing the multimeric structure of repressor.

scholarly journals TMOD-24. GENERATION AND CHARACTERIZATION OF A NOVEL TRANSGENIC IDO REPORTER MOUSE FOR IDO POSTTRANSLATIONAL MODIFICATION ANALYSIS IN SITU

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii233-ii233
Author(s):  
April Bell ◽  
Lijie Zhai ◽  
Erik Ladomersky ◽  
Kristen Lauing ◽  
Lakshmi Bollu ◽  
...  
Keyword(s):  
Mouse Model ◽  
Tumor Cell ◽  
Central Nervous System Tumor ◽  
Post Translational Modifications ◽  
Reporter Mouse ◽  
C Terminus ◽  
Human Gbm

Abstract Glioblastoma (GBM) is the most common and aggressive primary central nervous system tumor in adults with a median survival of 14.6 months. GBM is a potently immunosuppressive cancer due in-part to the prolific expression of immunosuppressive indoleamine 2,3 dioxygenase 1 (IDO). Tumor cell IDO facilitates the intratumoral accumulation of regulatory T cells (Tregs; CD4+CD25+FoxP3+). Although immunosuppressive IDO activity is canonically characterized by the conversion of tryptophan into kynurenine, we have utilized transgenic and syngeneic mouse models and mutant glioma lines to demonstrate that tumor cell IDO increases Treg accumulation independent of tryptophan metabolism. Here, we address the gap in our understanding of IDO signaling activity in vivo. Subcutaneously-engrafted human GBM expressing human IDO-GFP cDNA was isolated from immunodeficient humanized NSG-SGM3 mice. The tumor was immunoprecipitated for the GFP tag using GFP-TRAP followed by mass spectrometry which revealed a novel methylation site on a lysine residue at amino acid 373 in the IDO C-terminus region. Western blot analysis of IDO protein also revealed the presence of tyrosine phosphorylation. Additionally, we recently created a new transgenic IDO reporter mouse model whereby endogenous IDO is fused to GFP via a T2A linker (IDO→GFP). This model allows for the isolation of IDO+ cells in real-time and without causing cell death, thereby creating the opportunity for downstream molecular analysis of in situ-isolated GFP+ cells. Collectively, our work suggests that IDO non-enzyme activity may involve the post-translational modifications we recently identified. As IDO activity may differ between in vitro and in vivo modeling systems, we will use the new IDO→GFP reporter mouse model for an improved mechanistic understanding of how immunosuppressive IDO facilitates Treg accumulation in vivo.

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