alanine substitution
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Author(s):  
Yue Zhou ◽  
Chelsi D. Cassilly ◽  
Todd B. Reynolds

The fungal phosphatidylserine (PS) synthase, a membrane protein encoded by the CHO1 gene, is a potential drug target for pathogenic fungi, such as Candida albicans. However, both substrate-binding sites of C. albicans Cho1 have not been characterized. Cho1 has two substrates: cytidyldiphosphate-diacylglycerol (CDP-DAG) and serine. Previous studies identified a conserved CDP-alcohol phosphotransferase (CAPT) binding motif, which is present within Cho1. We tested the CAPT motif for its role in PS synthesis by mutating conserved residues using alanine substitution mutagenesis. PS synthase assays revealed that mutations in all but one conserved amino acid within the CAPT motif resulted in decreased Cho1 function. In contrast, there were no clear motifs in Cho1 for binding serine. Therefore, to identify the serine binding site, PS synthase sequences from three fungi were aligned with sequences of a similar enzyme, phosphatidylinositol (PI) synthase, from the same fungi. This revealed a motif that was unique to PS synthases. Using alanine substitution mutagenesis, we found that some of the residues in this motif are required for Cho1 function. Two alanine substitution mutants, L184A and R189A, exhibited contrasting impacts on PS synthase activity, and were characterized for their Michaelis-Menten kinetics. The L184A mutant displayed enhanced PS synthase activity and showed an increased Vmax. In contrast, R189A showed decreased PS synthase activity and increased Km for serine, suggesting that residue R189 is involved in serine binding. These results help to characterize PS synthase substrate binding, and should direct rational approaches for finding Cho1 inhibitors that may lead to better antifungals.


2021 ◽  
Author(s):  
Bryan A Johnson ◽  
Yiyang Zhou ◽  
Kumari G Lokugamage ◽  
Michelle N Vu ◽  
Nathen E Bopp ◽  
...  

While SARS-CoV-2 continues to adapt for human infection and transmission, genetic variation outside of the spike gene remains largely unexplored. This study investigates a highly variable region at residues 203-205 in SARS-CoV-2 nucleocapsid protein. Recreating the alpha variant mutation in an early pandemic (WA-1) background, we found that the R203K-G204R mutation is sufficient to enhance replication, fitness, and pathogenesis of SARS-CoV-2. Importantly, the R203K-G204R mutation increases nucleocapsid phosphorylation, providing a molecular basis for these phenotypes. Notably, an analogous alanine substitution mutant also increases SARS-CoV-2 fitness and phosphorylation, suggesting that infection is enhanced through ablation of the ancestral RG motif. Overall, these results demonstrate that variant mutations outside spike are also key components in SARS-CoV-2 continued adaptation to human infection.


2020 ◽  
Vol 203 (1) ◽  
Author(s):  
Kimberley T. McLean ◽  
Alexandra Tikhomirova ◽  
Erin B. Brazel ◽  
Salomé Legendre ◽  
Gian Haasbroek ◽  
...  

ABSTRACT Streptococcus pneumoniae (the pneumococcus) is a formidable human pathogen that is capable of asymptomatically colonizing the nasopharynx. Progression from colonization to invasive disease involves adaptation to distinct host niches, which vary markedly in the availability of key nutrients such as sugars. We previously reported that cell-cell signaling via the autoinducer 2 (AI-2)/LuxS quorum-sensing system boosts the capacity of S. pneumoniae to utilize galactose as a carbon source by upregulation of the Leloir pathway. This resulted in increased capsular polysaccharide production and a hypervirulent phenotype. We hypothesized that this effect was mediated by phosphorylation of GalR, the transcriptional activator of the Leloir pathway. GalR is known to possess three putative phosphorylation sites, S317, T319, and T323. In the present study, derivatives of S. pneumoniae D39 with putative phosphorylation-blocking alanine substitution mutations at each of these GalR sites (singly or in combination) were constructed. Growth assays and transcriptional analyses revealed complex phenotypes for these GalR mutants, with impacts on the regulation of both the Leloir and tagatose 6-phosphate pathways. The alanine substitution mutations significantly reduced the capacity of pneumococci to colonize the nasopharynx, middle ear, and lungs in a murine intranasal challenge model. IMPORTANCE Pneumococcal survival in the host and capacity to transition from a commensal to a pathogenic lifestyle are closely linked to the organism’s ability to utilize specific nutrients in distinct niches. Galactose is a major carbon source for pneumococci in the upper respiratory tract. We have shown that both the Leloir and tagatose 6-phosphate pathways are necessary for pneumococcal growth in galactose and demonstrated GalR-mediated interplay between the two pathways. Moreover, the three putative phosphorylation sites in the transcriptional regulator GalR play a critical role in galactose metabolism and are important for pneumococcal colonization of the nasopharynx, middle ear, and lungs.


2020 ◽  
Author(s):  
Pierre E. Thibeault ◽  
Rithwik Ramachandran

ABSTRACTThe C-terminal tail of G-protein-coupled receptors contain important regulatory sites that enable interaction with intracellular signalling effectors. Here we examine the relative contribution of the C-tail serine/threonine phosphorylation sites (Ser383-385, Ser387-Thr392) and the helix-8 palmitoylation site (Cys361) in signalling regulation downstream of the proteolytically-activated GPCR, PAR2. We examined Gαq/11-coupled calcium signalling, β-arrestin-1/-2 recruitment, and MAPK activation (p44/42 phosphorylation) by wild-type and mutant receptors expressed in a CRISPR/Cas9 PAR2-knockout HEK-293 cell background. We find that alanine substitution of the membrane proximal serine residues (Ser383-385Ala) had no effect on SLIGRL-NH2- or trypsin-stimulated β-arrestin recruitment. Alanine substitutions in the Ser387-Thr392 cluster resulted in a large (∼50%) decrease in β-arrestin-1/2 recruitment triggered by the activating peptide, SLIGRL-NH2, but was without effect on trypsin-activated β-arrestin-1/-2 recruitment. Additionally, we find that alanine substitution of the helix-8 cysteine residue (Cys361Ala) led to a (∼50%) decrease in β-arrestin-1/-2 recruitment in response to both trypsin and SLIGRL-NH2. We further show that Gαq/11inhibition with YM254890, inhibited ERK phosphorylation by PAR2 agonists, while genetic deletion of β-arrestin-1/-2 by CRISPR/Cas9 enhanced MAPK activation. Knockout of β-arrestins also enhanced Gαq/11-mediated calcium signalling. In line with these findings, C-tail serine/threonine and cysteine residue mutants that have decreased β-arrestin recruitment also showed enhanced ERK activation. Thus, our studies point to multiple mechanisms that regulate β-arrestin interaction with PAR2 to regulate receptor-mediated signalling.


RSC Advances ◽  
2020 ◽  
Vol 10 (46) ◽  
pp. 27331-27335
Author(s):  
Abha Dangi ◽  
Abhishek Ankur Balmik ◽  
Archana Kisan Ghorpade ◽  
Nalini Vijay Gorantla ◽  
Shweta Kishor Sonawane ◽  
...  
Keyword(s):  

In the alanine mutant peptides of AcPHF6* and AcPHF6, only the peptides with glutamine to alanine substitution show aggregation akin to that of the parent peptides.


2019 ◽  
Author(s):  
Alexis Riché ◽  
Linnka Lefebvre-Legendre ◽  
Michel Goldschmidt-Clermont

ABSTRACTPhosphorylation of the core subunits of photosystem II (PSII) is largely governed by a protein kinase and an antagonistic protein phosphatase. In plants the respective mutants show alterations in the architecture of thylakoid membranes and in the repair of PSII after photo-inhibition. However the protein kinase targets several subunits of PSII, as well as other proteins. To specifically investigate the role of phosphorylation of the different PSII subunits, we used site-directed mutagenesis and chloroplast transformation in Chlamydomonas reinhardtii. Major, evolutionarily-conserved sites of phosphorylation in three components of PSII (CP43, D2 and PsbH) were mutated to replace the corresponding serine or threonine residues with alanine. The alanine substitution mutant of D2 had no apparent phenotype, while the mutant of CP43 presented a minor delay in recovery from photo-inhibition. Alanine substitutions of the phosphorylation sites in PsbH had significant effects on the accumulation of PSII or on its recovery from photo-inhibition. When mutations in two of the target subunits were combined through a second cycle of chloroplast transformation, the strongest phenotype was observed in the mutant lacking phosphorylation of both PsbH and CP43, which showed delayed recovery from photo-inhibition. Surprisingly this phenotype was reversed in the mutant defective for phosphorylation of all three subunits. Our analysis indicates a prominent role for the N-terminus of PsbH in the stable accumulation of PSII and of PsbH phosphorylation in its repair cycle.SIGNIFICANCE STATEMENTTo specifically investigate the role of PSII phosphorylation, alanine-substitution mutants of the major phospho-sites in the subunits of PSII were generated individually or in combinations using chloroplast transformation. PSII assembly was defective in some of the PsbH mutants. PSII repair after photo-inhibition was delayed most strongly in the mutant lacking phosphorylation of both PsbC (CP43) and PsbH.


2019 ◽  
Vol 15 (7) ◽  
pp. e1007225 ◽  
Author(s):  
Elizabeth A. Proctor ◽  
David D. Mowrey ◽  
Nikolay V. Dokholyan

2018 ◽  
Vol 13 (6) ◽  
pp. 1631-1639 ◽  
Author(s):  
Charles W. Hespen ◽  
Joel J. Bruegger ◽  
Yirui Guo ◽  
Michael A. Marletta

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