scholarly journals In Vivo Destabilization and Functional Defects of the Xeroderma Pigmentosum C Protein Caused by a Pathogenic Missense Mutation

2007 ◽  
Vol 27 (19) ◽  
pp. 6606-6614 ◽  
Author(s):  
Gentaro Yasuda ◽  
Ryotaro Nishi ◽  
Eriko Watanabe ◽  
Toshio Mori ◽  
Shigenori Iwai ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Amino Acid Substitution ◽  
Xeroderma Pigmentosum ◽  
Excision Repair ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Dependent Manner ◽  
Damaged Dna

ABSTRACT Xeroderma pigmentosum group C (XPC) protein plays an essential role in DNA damage recognition in mammalian global genome nucleotide excision repair (NER). Here, we analyze the functional basis of NER inactivation caused by a single amino acid substitution (Trp to Ser at position 690) in XPC, previously identified in the XPC patient XP13PV. The Trp690Ser change dramatically affects the in vivo stability of the XPC protein, thereby causing a significant reduction of its steady-state level in XP13PV fibroblasts. Despite normal heterotrimeric complex formation and physical interactions with other NER factors, the mutant XPC protein lacks binding affinity for both undamaged and damaged DNA. Thus, this single amino acid substitution is sufficient to compromise XPC function through both quantitative and qualitative alterations of the protein. Although the mutant XPC fails to recognize damaged DNA, it is still capable of accumulating in a UV-damaged DNA-binding protein (UV-DDB)-dependent manner to UV-damaged subnuclear domains. However, the NER factors transcription factor IIH and XPA failed to colocalize stably with the mutant XPC. As well as highlighting the importance of UV-DDB in recruiting XPC to UV-damaged sites, these findings demonstrate the role of DNA binding by XPC in the assembly of subsequent NER intermediate complexes.

scholarly journals Infection of Human Dendritic Cells by a Sindbis Virus Replicon Vector Is Determined by a Single Amino Acid Substitution in the E2 Glycoprotein

2000 ◽  
Vol 74 (24) ◽  
pp. 11849-11857 ◽  
Author(s):  
Jason P. Gardner ◽  
Ilya Frolov ◽  
Silvia Perri ◽  
Yaying Ji ◽  
Mary Lee MacKichan ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Sindbis Virus ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
E2 Glycoprotein ◽  
Human Dendritic Cells ◽  
Antigen Presenting

ABSTRACT The ability to target antigen-presenting cells with vectors encoding desired antigens holds the promise of potent prophylactic and therapeutic vaccines for infectious diseases and cancer. Toward this goal, we derived variants of the prototype alphavirus, Sindbis virus (SIN), with differential abilities to infect human dendritic cells. Cloning and sequencing of the SIN variant genomes revealed that the genetic determinant for human dendritic cell (DC) tropism mapped to a single amino acid substitution at residue 160 of the envelope glycoprotein E2. Packaging of SIN replicon vectors with the E2 glycoprotein from a DC-tropic variant conferred a similar ability to efficiently infect immature human DC, whereupon those DC were observed to undergo rapid activation and maturation. The SIN replicon particles infected skin-resident mouse DC in vivo, which subsequently migrated to the draining lymph nodes and upregulated cell surface expression of major histocompatibility complex and costimulatory molecules. Furthermore, SIN replicon particles encoding human immunodeficiency virus type 1 p55Gag elicited robust Gag-specific T-cell responses in vitro and in vivo, demonstrating that infected DC maintained their ability to process and present replicon-encoded antigen. Interestingly, human and mouse DC were differentially infected by selected SIN variants, suggesting differences in receptor expression between human and murine DC. Taken together, these data illustrate the tremendous potential of using a directed approach in generating alphavirus vaccine vectors that target and activate antigen-presenting cells, resulting in robust antigen-specific immune responses.

scholarly journals Evolution of moth sex pheromone composition by a single amino acid substitution in a fatty acid desaturase

2015 ◽  
Vol 112 (41) ◽  
pp. 12586-12591 ◽  
Author(s):  
Aleš Buček ◽  
Petra Matoušková ◽  
Heiko Vogel ◽  
Petr Šebesta ◽  
Ullrich Jahn ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Functional Characterization ◽  
Fatty Acid Desaturase ◽  
Fatty Acyl ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid

For sexual communication, moths primarily use blends of fatty acid derivatives containing one or more double bonds in various positions and configurations, called sex pheromones (SPs). To study the molecular basis of novel SP component (SPC) acquisition, we used the tobacco hornworm (Manduca sexta), which uses a blend of mono-, di-, and uncommon triunsaturated fatty acid (3UFA) derivatives as SP. We identified pheromone-biosynthetic fatty acid desaturases (FADs) MsexD3, MsexD5, and MsexD6 abundantly expressed in the M. sexta female pheromone gland. Their functional characterization and in vivo application of FAD substrates indicated that MsexD3 and MsexD5 biosynthesize 3UFAs via E/Z14 desaturation from diunsaturated fatty acids produced by previously characterized Z11-desaturase/conjugase MsexD2. Site-directed mutagenesis of sequentially highly similar MsexD3 and MsexD2 demonstrated that swapping of a single amino acid in the fatty acyl substrate binding tunnel introduces E/Z14-desaturase specificity to mutated MsexD2. Reconstruction of FAD gene phylogeny indicates that MsexD3 was recruited for biosynthesis of 3UFA SPCs in M. sexta lineage via gene duplication and neofunctionalization, whereas MsexD5 representing an alternative 3UFA-producing FAD has been acquired via activation of a presumably inactive ancestral MsexD5. Our results demonstrate that a change as small as a single amino acid substitution in a FAD enzyme might result in the acquisition of new SP compounds.

A single amino acid substitution improves the in vivo immunogenicity of the HPV16 oncoprotein E7(11–20) cytotoxic T lymphocyte epitope

Vaccine ◽  
2005 ◽  
Vol 23 (31) ◽  
pp. 4005-4010 ◽  
Author(s):  
Marco W.J. Schreurs ◽  
Esther W.M. Kueter ◽  
Kirsten B.J. Scholten ◽  
François A. Lemonnier ◽  
Chris J.L.M. Meijer ◽  
...  
Keyword(s):  
Amino Acid ◽  
T Lymphocyte ◽  
Amino Acid Substitution ◽  
Cytotoxic T Lymphocyte ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid

scholarly journals A Region in the Bacillus subtilisTranscription Factor Spo0A That Is Important for spoIIGPromoter Activation

1998 ◽  
Vol 180 (14) ◽  
pp. 3578-3583 ◽  
Author(s):  
Cindy M. Buckner ◽  
Ghislain Schyns ◽  
Charles P. Moran
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Amino Acid Substitution ◽  
Dna Binding Protein ◽  
Single Amino Acid ◽  
Promoter Activation ◽  
Additional Amino Acid

ABSTRACT Spo0A is a DNA binding protein in Bacillus subtilisrequired for the activation of spoIIG and other promoters at the onset of endospore formation. Activation of some of these promoters may involve interaction of Spo0A and the ςAsubunit of RNA polymerase. Previous studies identified two single-amino-acid substitutions in ςA, K356E and H359R, that specifically impaired Spo0A-dependent transcription in vivo. Here we report the identification of an amino acid substitution in Spo0A (S231F) that suppressed the sporulation deficiency due to the H359R substitution in ςA. We also found that the S231F substitution partially restored use of the spoIIG promoter by the ςA H359R RNA polymerase in vitro. Alanine substitutions in the 231 region of Spo0A revealed an additional amino acid residue important for spoIIG promoter activation, I229. This amino acid substitution in Spo0A did not affect repression of abrB transcription, indicating that the alanine-substituted Spo0A was not defective in DNA binding. Moreover, the alanine-substituted Spo0A protein activated the spoIIApromoter; therefore, this region of Spo0A is probably not required for Spo0A-dependent, ςH-directed transcription. These and other results suggest that the region of Spo0A near position 229 is involved in ςA-dependent promoter activation.

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