scholarly journals Subcellular Localization and Functional Characterization of GII.4 Norovirus-Encoded NTPase

2017 ◽  
Vol 92 (5) ◽  
Author(s):  
Ju-Bei Yen ◽  
Ling-Huei Wei ◽  
Lee-Wen Chen ◽  
Li-Yu Chen ◽  
Chien-Hui Hung ◽  
...  
Keyword(s):  
Viral Replication ◽  
Self Assembly ◽  
Functional Characterization ◽  
Terminal Region ◽  
Vesicle Formation ◽  
Nonstructural Proteins ◽  
Efficient Cell Culture System ◽  
Norovirus Gii ◽  
In Cells

ABSTRACTThe genotype II.4 (GII.4) variants of human noroviruses (HuNVs) are recognized as the major agent of global gastroenteritis outbreaks. Due to the lack of an efficient cell culture system for HuNV propagation, the exact roles of HuNV-encoded nonstructural proteins (including Nterm, NTPase, P22, VPg, Pro, and RdRp) in viral replication or pathogenesis have not yet been fully understood. Here, we report the molecular characterization of the GII.4 HuNV-encoded NTPase (designated GII-NTPase). Results from our studies showed that GII-NTPase forms vesicular or nonvesicular textures in the cell cytoplasm, and the nonvesicular fraction of GII-NTPase significantly localizes to the endoplasmic reticulum (ER) or mitochondria. Deletion analysis revealed that the N-terminal 179-amino-acid (aa) region of GII-NTPase is required for vesicle formation and for ER colocalization, whereas the C-terminal region is involved in mitochondrial colocalization. In particular, two mitochondrion-targeting domains were identified in the C-terminal region of GII-NTPase which perfectly colocalized with mitochondria when the N-terminal region of GII-NTPase was deleted. However, the corresponding C-terminal portions of NTPase derived from the GI HuNV did not show mitochondrial colocalization. We also found that GII-NTPase physically interacts with itself as well as with Nterm and P22, but not VPg, Pro, and RdRp, in cells. The Nterm- and P22-interacting region was mapped to the N-terminal 179-aa region of GII-NTPase, whereas the self-assembly of GII-NTPase could be achieved via a head-to-head, tail-to-tail, or head-to-tail configuration. More importantly, we demonstrate that GII-NTPase possesses a proapoptotic activity, which can be further enhanced by coexpression with Nterm or P22.IMPORTANCEDespite the importance of human norovirus GII.4 variants in global gastroenteritis outbreaks, the basic biological functions of the viral nonstructural proteins in cells remain rarely investigated. In this report, we focus our studies on characteristics of the GII.4 norovirus-encoded NTPase (GII-NTPase). We unexpectedly find that GII-NTPase can perfectly colocalize with mitochondria after its N-terminal region is deleted. However, such a phenomenon is not observed for NTPase encoded by a GI strain. We further reveal that the N-terminal 179-aa region of GII-NTPase is sufficient to mediate (i) vesicle formation, (ii) ER colocalization, (iii) the interaction with two other nonstructural proteins, including Nterm and P22, (iv) the formation of homodimers or homo-oligomers, and (v) the induction of cell apoptosis. Taken together, our findings emphasize that the virus-encoded NTPase must have multiple activities during viral replication or pathogenesis; however, these activities may vary somewhat among different genogroups.

scholarly journals Human xylosyltransferase I and N-terminal truncated forms: functional characterization of the core enzyme

2006 ◽  
Vol 394 (1) ◽  
pp. 163-171 ◽  
Author(s):  
Sandra Müller ◽  
Jennifer Disse ◽  
Manuela Schöttler ◽  
Sylvia Schön ◽  
Christian Prante ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Binding Capacity ◽  
Functional Characterization ◽  
Terminal Region ◽  
Binding Motif ◽  
Heparin Binding ◽  
Loss Of Function ◽  
The Impact

Human XT-I (xylosyltransferase I; EC 2.4.2.26) initiates the biosynthesis of the glycosaminoglycan linkage region and is a diagnostic marker of an enhanced proteoglycan biosynthesis. In the present study, we have investigated mutant enzymes of human XT-I and assessed the impact of the N-terminal region on the enzymatic activity. Soluble mutant enzymes of human XT-I with deletions at the N-terminal domain were expressed in insect cells and analysed for catalytic activity. As many as 260 amino acids could be truncated at the N-terminal region of the enzyme without affecting its catalytic activity. However, truncation of 266, 272 and 273 amino acids resulted in a 70, 90 and >98% loss in catalytic activity. Interestingly, deletion of the single 12 amino acid motif G261KEAISALSRAK272 leads to a loss-of-function XT-I mutant. This is in agreement with our findings analysing the importance of the Cys residues where we have shown that C276A mutation resulted in a nearly inactive XT-I enzyme. Moreover, we investigated the location of the heparin-binding site of human XT-I using the truncated mutants. Heparin binding was observed to be slightly altered in mutants lacking 289 or 568 amino acids, but deletion of the potential heparin-binding motif P721KKVFKI727 did not lead to a loss of heparin binding capacity. The effect of heparin or UDP on the XT-I activity of all mutants was not significantly different from that of the wild-type. Our study demonstrates that over 80% of the nucleotide sequence of the XT-I-cDNA is necessary for expressing a recombinant enzyme with full catalytic activity.

scholarly journals Functional Characterization of an Amino-terminal Region of HDAC4 That Possesses MEF2 Binding and Transcriptional Repressive Activity

2003 ◽  
Vol 278 (26) ◽  
pp. 23515-23521 ◽  
Author(s):  
Jonathan K. L. Chan ◽  
Luguo Sun ◽  
Xiang-Jiao Yang ◽  
Guang Zhu ◽  
Zhenguo Wu
Keyword(s):  
Functional Characterization ◽  
Terminal Region ◽  
Amino Terminal

Structural and functional characterization of an epitope in the conserved C-terminal region of HIV-1 gp120

1999 ◽  
Vol 54 (1) ◽  
pp. 32-42 ◽  
Author(s):  
M. Ferrer ◽  
B.J. Sullivan ◽  
K.L. Godbout ◽  
E. Burke ◽  
H.S. Stump ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Terminal Region ◽  
Hiv 1

scholarly journals Functional Characterization of the N-terminal Region of Myosin-2

2006 ◽  
Vol 281 (47) ◽  
pp. 36102-36109 ◽  
Author(s):  
Setsuko Fujita-Becker ◽  
Georgios Tsiavaliaris ◽  
Reiko Ohkura ◽  
Takashi Shimada ◽  
Dietmar J. Manstein ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Terminal Region

scholarly journals Analysis of 2.1 million SARS-CoV-2 genomes identifies mutations associated with transmissibility

2021 ◽  
Author(s):  
Fritz Heinrich Obermeyer ◽  
Stephen F. Schaffner ◽  
Martin Jankowiak ◽  
Nikolaos Barkas ◽  
Jesse D. Pyle ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Logistic Regression Model ◽  
Multinomial Logistic Regression ◽  
Functional Characterization ◽  
Health Concern ◽  
Nonstructural Proteins ◽  
Multinomial Logistic Regression Model ◽  
Public Health Concern ◽  
Geographic Regions

Repeated emergence of SARS-CoV-2 variants with increased transmissibility necessitates rapid detection and characterization of new lineages. To address this need, we developed PyR0, a hierarchical Bayesian multinomial logistic regression model that infers relative transmissibility of all viral lineages across geographic regions, detects lineages increasing in prevalence, and identifies mutations relevant to transmissibility. Applying PyR0 to all publicly available SARS-CoV-2 genomes, we identify numerous substitutions that increase transmissibility, including previously identified spike mutations and many non-spike mutations within the nucleocapsid and nonstructural proteins. PyR0 forecasts growth of new lineages from their mutational profile, identifies viral lineages of concern as they emerge, and prioritizes mutations of biological and public health concern for functional characterization.

Functional characterization of retromer in GLUT4 storage vesicle formation and adipocyte differentiation

2015 ◽  
Vol 30 (3) ◽  
pp. 1037-1050 ◽  
Author(s):  
Zhe Yang ◽  
Lee Kian Hong ◽  
Jordan Follett ◽  
Martin Wabitsch ◽  
Nicholas A. Hamilton ◽  
...  
Keyword(s):  
Adipocyte Differentiation ◽  
Functional Characterization ◽  
Vesicle Formation ◽  
Storage Vesicle

scholarly journals Enzymatic Characterization of a Prokaryotic Urea Carboxylase

2004 ◽  
Vol 186 (9) ◽  
pp. 2532-2539 ◽  
Author(s):  
Takeshi Kanamori ◽  
Norihisa Kanou ◽  
Haruyuki Atomi ◽  
Tadayuki Imanaka
Keyword(s):  
Enzyme Reaction ◽  
Nitrogen Sources ◽  
Structural Similarity ◽  
Terminal Region ◽  
Enzymatic Characterization ◽  
Acetyl Coa ◽  
Carboxylase Activity ◽  
Bona Fide ◽  
In Cells

ABSTRACT We identified the first prokaryotic urea carboxylase (UCA) from a member of the alpha subclass of the class Proteobacteria, Oleomonas sagaranensis. This enzyme (O. sagaranensis Uca) was composed of 1,171 amino acids, and its N-terminal region resembled the biotin carboxylase domains of various biotin-dependent carboxylases. The C-terminal region of the enzyme harbored the Met-Lys-Met motif found in biotin carboxyl carrier proteins. The primary structure of the enzyme was 45% identical to that of the urea carboxylase domain of urea amidolyase from Saccharomyces cerevisiae. O. sagaranensis Uca did not harbor the allophanate hydrolase domain found in the yeast enzyme, but a separate gene with structural similarity was found to be adjacent to the uca gene. Purified recombinant O. sagaranensis Uca displayed ATP-dependent carboxylase activity towards urea (V max = 21.2 μmol mg−1 min−1) but not towards acetyl coenzyme A (acetyl-CoA) and propionyl-CoA, indicating that the gene encoded a bona fide UCA and not an acetyl-CoA or propionyl-CoA carboxylase. The enzyme also exhibited high levels of activity towards acetamide and formamide. Kinetic parameters of the enzyme reaction were determined with ATP, urea, acetamide, and formamide. O. sagaranensis could grow on urea, acetamide, and formamide as sole nitrogen sources; moreover, ATP-dependent urea-degrading activity was found in cells grown with urea but not in cells grown with ammonia. The results suggest that the UCA of this organism may be involved in the assimilation of these compounds as nitrogen sources. Furthermore, orthologues of the O. sagaranensis uca gene were found to be widely distributed among Bacteria. This implies that there are two systems of urea degradation in Bacteria, a pathway catalyzed by the previously described ureases and the UCA-allophanate hydrolase pathway identified in this study.

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