scholarly journals Comprehensive analysis of fungal G1 cyclin docking motif sequences that control CDK regulatory potency in vivo

2020 ◽  
Author(s):  
Sushobhana Bandyopadhyay ◽  
Samyabrata Bhaduri ◽  
Mihkel Örd ◽  
Norman E. Davey ◽  
Mart Loog ◽  
...  
Keyword(s):  
Eukaryotic Cell ◽  
Cdk Inhibitor ◽  
Competitive Growth ◽  
Wild Type ◽  
Cyclin Dependent Kinases ◽  
Growth Assay ◽  
Cdk Regulation ◽  
The Impact ◽  
General Method

SUMMARYCyclin-dependent kinases (CDKs) control the ordered series of events during eukaryotic cell division. The stage at which individual CDK substrates are phosphorylated can be dictated by cyclin-specific docking motifs. In budding yeast, substrates with Leu/Pro-rich (LP) docking motifs are recognized by Cln1/2 cyclins in late G1 phase, yet the key sequence features of these motifs and the conservation of this mechanism were unknown. Here we comprehensively analyzed LP motif requirements in vivo by combining a competitive growth assay with mutational scanning and deep sequencing. We quantified the impact of all single-residue replacements in five different LP motifs, using six distinct G1 cyclins from diverse fungi including medical and agricultural pathogens. The results reveal the basis for variations in potency among wild-type motifs, and allow derivation of a quantitative matrix that predicts the potency of other candidate motifs. In one protein, Whi5, we found overlapping LP and phosphorylation motifs with partly redundant effects. In another protein, the CDK inhibitor Sic1, we found that its LP motif is inherently weak due to unfavorable residues at key positions, and this imposes a beneficial delay in its phosphorylation and degradation. The overall results provide a general method for surveying viable docking motif sequences and quantifying their potency in vivo, and they reveal how variations in LP motif potency can tune the strength and timing of CDK regulation.

scholarly journals Activating Phosphorylation of theSaccharomyces cerevisiaeCyclin-dependent Kinase, Cdc28p, Precedes Cyclin Binding

2000 ◽  
Vol 11 (5) ◽  
pp. 1597-1609 ◽  
Author(s):  
Karen E. Ross ◽  
Philipp Kaldis ◽  
Mark J. Solomon
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Eukaryotic Cell ◽  
Regulatory Mechanisms ◽  
Wild Type ◽  
Cycle Progression ◽  
Threonine Residue ◽  
Cyclin Dependent Kinases ◽  
Higher Eukaryotes

Eukaryotic cell cycle progression is controlled by a family of protein kinases known as cyclin-dependent kinases (Cdks). Two steps are essential for Cdk activation: binding of a cyclin and phosphorylation on a conserved threonine residue by the Cdk-activating kinase (CAK). We have studied the interplay between these regulatory mechanisms during the activation of the major Saccharomyces cerevisiaeCdk, Cdc28p. We found that the majority of Cdc28p was phosphorylated on its activating threonine (Thr-169) throughout the cell cycle. The extent of Thr-169 phosphorylation was similar for monomeric Cdc28p and Cdc28p bound to cyclin. By varying the order of the addition of cyclin and Cak1p, we determined that Cdc28p was activated most efficiently when it was phosphorylated before cyclin binding. Furthermore, we found that a Cdc28pT169Amutant, which cannot be phosphorylated, bound cyclin less well than wild-type Cdc28p in vivo. These results suggest that unphosphorylated Cdc28p may be unable to bind tightly to cyclin. We propose that Cdc28p is normally phosphorylated by Cak1p before it binds cyclin. This activation pathway contrasts with that in higher eukaryotes, in which cyclin binding appears to precede activating phosphorylation.

scholarly journals Production of Noncapped Genomic RNAs Is Critical to Sindbis Virus Disease and Pathogenicity

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Autumn T. LaPointe ◽  
V Douglas Landers ◽  
Claire E. Westcott ◽  
Kevin J. Sokoloski
Keyword(s):  
Sindbis Virus ◽  
Virus Disease ◽  
Severe Disease ◽  
Wild Type Virus ◽  
Wild Type ◽  
Genomic Rnas ◽  
Mortality And Morbidity ◽  
The Impact

ABSTRACT Alphaviruses are positive-sense RNA viruses that utilize a 5′ cap structure to facilitate translation of viral proteins and to protect the viral RNA genome. Nonetheless, significant quantities of viral genomic RNAs that lack a canonical 5′ cap structure are produced during alphaviral replication and packaged into viral particles. However, the role/impact of the noncapped genomic RNA (ncgRNA) during alphaviral infection in vivo has yet to be characterized. To determine the importance of the ncgRNA in vivo, the previously described D355A and N376A nsP1 mutations, which increase or decrease nsP1 capping activity, respectively, were incorporated into the neurovirulent AR86 strain of Sindbis virus to enable characterization of the impact of altered capping efficiency in a murine model of infection. Mice infected with the N376A nsP1 mutant exhibited slightly decreased rates of mortality and delayed weight loss and neurological symptoms, although levels of inflammation in the brain were similar to those of wild-type infection. Although the D355A mutation resulted in decreased antiviral gene expression and increased resistance to interferon in vitro, mice infected with the D355A mutant showed significantly reduced mortality and morbidity compared to mice infected with wild-type virus. Interestingly, expression of proinflammatory cytokines was found to be significantly decreased in mice infected with the D355A mutant, suggesting that capping efficiency and the production of ncgRNA are vital to eliciting pathogenic levels of inflammation. Collectively, these data indicate that the ncgRNA have important roles during alphaviral infection and suggest a novel mechanism by which noncapped viral RNAs aid in viral pathogenesis. IMPORTANCE Mosquito-transmitted alphaviruses have been the cause of widespread outbreaks of disease that can range from mild illness to lethal encephalitis or severe polyarthritis. There are currently no safe and effective vaccines or therapeutics with which to prevent or treat alphaviral disease, highlighting the need to better understand alphaviral pathogenesis to develop novel antiviral strategies. This report reveals production of noncapped genomic RNAs (ncgRNAs) to be a novel determinant of alphaviral virulence and offers insight into the importance of inflammation to pathogenesis. Taken together, the findings reported here suggest that the ncgRNAs contribute to alphaviral pathogenesis through the sensing of the ncgRNAs during alphaviral infection and are necessary for the development of severe disease.

scholarly journals Noncapped Genomic RNA Are Critical for Alphaviral Infection and Pathogenicity

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 44
Author(s):  
Autumn T. LaPointe ◽  
Kevin J Sokoloski
Keyword(s):  
Growth Kinetics ◽  
Particle Production ◽  
Immune Cell ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Genomic Rnas ◽  
Viral Rnas ◽  
The Impact

Alphaviruses are positive-sense RNA arthropod-borne viruses that represent a significant threat to public health. During alphaviral replication, significant quantities of viral genomic RNAs that lack a canonical 5’ cap structure are produced and packaged into viral particles, despite the fact that the noncapped genomes cannot be translated and are essentially noninfectious. Previously, we have reported that the capping efficiency of nsP1, the alphaviral capping enzyme, of Sindbis virus (SINV) could be modulated via point mutation. It was found that increasing RNA capping efficiency led to decreased viral growth kinetics via decreased particle production, despite increased innate immune evasion, whereas decreasing capping efficiency led to wild-type growth kinetics and particle production. This led to the conclusion that the noncapped viral RNAs meaningfully contribute to the biology of alphaviral infections at the molecular level. To determine the importance of the noncapped viral RNAs in vivo, we characterized the impact of altered capping efficiency in a murine model of infection utilizing a neurovirulent strain of SINV. Mice infected with the nsP1 mutant with decreased capping exhibited wild-type rates of mortality, weight loss, and neurological symptoms. Conversely, the mice infected with the increased capping nsP1 mutant showed significantly reduced mortality and morbidity compared to mice infected with the wild-type virus. Interestingly, viral titers in the ankle, serum, and brain were equivalent between the wild-type virus and the two mutant viruses. Importantly, examination of the brain tissue revealed that mice infected with the increased capping mutant had significantly reduced immune cell infiltration and expression of proinflammatory cytokines compared to the decreased capping mutant and wild-type virus. Collectively, these data indicate that the noncapped viral RNAs have important roles during the early and late stages of alphaviral infection and suggest a novel mechanism by which noncapped viral RNA aids in viral pathogenesis.

scholarly journals Novel protective role for MAP kinase phosphatase 2 in inflammatory arthritis

RMD Open ◽  
2019 ◽  
Vol 5 (1) ◽  
pp. e000711
Author(s):  
Juliane Schroeder ◽  
Kirsty Ross ◽  
Kathryn McIntosh ◽  
Shilan Jabber ◽  
Stuart Woods ◽  
...  
Keyword(s):  
Inflammatory Arthritis ◽  
Neutrophil Migration ◽  
Protective Role ◽  
Leukotriene B4 ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
Map Kinase Phosphatase ◽  
Tnf Α ◽  
The Impact

ObjectivesWe have previously shown mitogen-activated protein kinase phosphatase 2 (MKP-2) to be a key regulator of proinflammatory cytokines in macrophages. In the study presented here, we investigated the role of MKP-2 in inflammatory arthritis with a particular focus on neutrophils.MethodsTo achieve this, we subjected MKP-2 deficient and wild type mice to collagen antibody induced arthritis, an innate model of arthritis, and determined disease pathology. To further our investigation, we depleted neutrophils in a prophylactic and therapeutic fashion. Last, we used chemotaxis assays to analyse the impact of MKP-2 deletion on neutrophil migration.ResultsMKP-2-/- mice showed a significant increase in disease pathology linked to elevated levels of proarthritic cytokines and chemokines TNF-α, IL-6 and MCP-1 in comparison to wild type controls. This phenotype is prevented or abolished after administration of neutrophil depleting antibody prior or after onset of disease, respectively. While MCP-1 levels were not affected, neutrophil depletion diminished TNF-α and reduced IL-6, thus linking these cytokines to neutrophils. In vivo imaging showed that MKP-2-/- mice had an increased influx of neutrophils into affected joints, which was higher and potentially prolonged than in wild type animals. Furthermore, using chemotaxis assays we revealed that MKP-2 deficient neutrophils migrate faster towards a Leukotriene B4 gradient. This process correlated with a reduced phosphorylation of ERK in MKP-2-/- neutrophils.ConclusionsThis is the first study to show a protective role for MKP-2 in inflammatory arthritis.

Organoid-based ex vivo reconstitution of Kras-driven pancreatic ductal carcinogenesis

2019 ◽  
Vol 41 (4) ◽  
pp. 490-501 ◽  
Author(s):  
Tetsuya Matsuura ◽  
Yoshiaki Maru ◽  
Masashi Izumiya ◽  
Daisuke Hoshi ◽  
Shingo Kato ◽  
...  
Keyword(s):  
Nude Mice ◽  
Tumor Suppressors ◽  
Ex Vivo ◽  
Disease Model ◽  
Intraepithelial Neoplasia ◽  
Culture Technique ◽  
Ductal Adenocarcinoma ◽  
Wild Type ◽  
The Impact

Abstract The organoid culture technique has been recently applied to modeling carcinogenesis in several organs. To further explore its potential and gain novel insights into tumorigenesis, we here investigated whether pancreatic ductal adenocarcinoma (PDA) could be generated as subcutaneous tumors in immunocompromised nude mice, by genetic engineering of normal organoids. As expected, acute induction of KrasG12Din vitro occasionally led to development of tiny nodules compatible with early lesions known as pancreatic intraepithelial neoplasia (PanIN). KrasG12D-expressing cells were enriched after inoculation in the subcutis, yet proved rather declined during culture, suggesting that its advantage might depend on surrounding environments. Depletion of growth factors or concurrent Trp53 deletion resulted in its robust enrichment, invariably leading to development of PanIN or large high-grade adenocarcinoma, respectively, consistent with in vivo mouse studies for the same genotype. Progression from PanIN was also recapitulated by subsequent knockdown of common tumor suppressors, whereas the impact of Tgfbr2 deletion was only partially recapitulated, illustrating genotype-dependent requirement of the pancreatic niche for tumorigenesis. Intriguingly, analysis of tumor-derived organoids revealed that KrasG12D-expressing cells with spontaneous deletion of wild-type Kras were positively selected and exhibited an aging-related mutation signature in nude mice, mirroring the pathogenesis of human PDA, and that the sphere-forming potential and orthotopic tumorigenicity in syngenic mice were significantly augmented. These observations highlighted the relevance of the subcutis of nude mice in promoting PDA development despite its ectopic nature. Taken together, pancreatic carcinogenesis could be considerably recapitulated with organoids, which would probably serve as a novel disease model.

RNA silencing of hydrogenase(-like) genes and investigation of their physiological roles in the green alga Chlamydomonas reinhardtii

2010 ◽  
Vol 431 (3) ◽  
pp. 345-352 ◽  
Author(s):  
James E. Godman ◽  
Attila Molnár ◽  
David C. Baulcombe ◽  
Janneke Balk
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Green Alga ◽  
Rna Silencing ◽  
Aldehyde Oxidase ◽  
Eukaryotic Cell ◽  
Unique Combination ◽  
Wild Type ◽  
Anaerobic Conditions ◽  
Production Cell

The genome of the green alga Chlamydomonas reinhardtii encodes two [FeFe]-hydrogenases, HydA1 and HydA2, and the hydrogenase-like protein HYD3. The unique combination of these proteins in one eukaryotic cell allows for direct comparison of their in vivo functions, which have not been established for HydA2 and HYD3. Using an artificial microRNA silencing method developed recently, the expression of HydA1, HydA2 and HYD3 was specifically down-regulated. Silencing of HydA1 resulted in 4-fold lower hydrogenase protein and activity under anaerobic conditions. In contrast, silencing of HydA2 or HYD3 did not affect hydrogen production. Cell lines with strongly (>90%) decreased HYD3 transcript levels grew more slowly than wild-type. The activity of aldehyde oxidase, a cytosolic Fe-S enzyme, was decreased in HYD3-knockdown lines, whereas Fe-S dependent activities in the chloroplast and mitochondria were unaffected. In addition, the HYD3-knockdown lines grew poorly on hypoxanthine, indicating impaired function of xanthine dehydrogenase, another cytosolic Fe-S enzyme. The expression levels of selected genes in response to hypoxia were unaltered upon HYD3 silencing. Together, our results clearly distinguish the cellular roles of HydA1 and HYD3, and indicate that HYD3, like its yeast and human homologues, has an evolutionary conserved role in the biogenesis or maintenance of cytosolic Fe-S proteins.

scholarly journals Fortilin binds Ca2+ and blocks Ca2+-dependent apoptosis in vivo

2007 ◽  
Vol 408 (2) ◽  
pp. 181-191 ◽  
Author(s):  
Potchanapond Graidist ◽  
Michio Yazawa ◽  
Moltira Tonganunt ◽  
Akiko Nakatomi ◽  
Curtis Chun-Jen Lin ◽  
...  
Keyword(s):  
Biological Significance ◽  
Wild Type ◽  
Apoptotic Activity ◽  
Apoptotic Pathways ◽  
Induced Apoptosis ◽  
The Impact ◽  
Overlay Assay ◽  
Dissociation Constant Kd ◽  
Flow Dialysis

Fortilin, a 172-amino-acid polypeptide present both in the cytosol and nucleus, possesses potent anti-apoptotic activity. Although fortilin is known to bind Ca2+, the biochemistry and biological significance of such an interaction remains unknown. In the present study we report that fortilin must bind Ca2+ in order to protect cells against Ca2+-dependent apoptosis. Using a standard Ca2+-overlay assay, we first validated that full-length fortilin binds Ca2+ and showed that the N-terminus (amino acids 1–72) is required for its Ca2+-binding. We then used flow dialysis and CD spectropolarimetry assays to demonstrate that fortilin binds Ca2+ with a dissociation constant (Kd) of approx. 10 μM and that the binding of fortilin to Ca2+ induces a significant change in the secondary structure of fortilin. In order to evaluate the impact of the binding of fortilin to Ca2+in vivo, we measured intracellular Ca2+ levels upon thapsigargin challenge and found that the lack of fortilin in the cell results in the exaggerated elevation of intracellular Ca2+ in the cell. We then tested various point mutants of fortilin for their Ca2+ binding and identified fortilin(E58A/E60A) to be a double-point mutant of fortilin lacking the ability of Ca2+-binding. We then found that wild-type fortilin, but not fortilin(E58A/E60A), protected cells against thapsigargin-induced apoptosis, suggesting that the binding of fortilin to Ca2+ is required for fortilin to protect cells against Ca2+-dependent apoptosis. Together, these results suggest that fortilin is an intracellular Ca2+ scavenger, protecting cells against Ca2+-dependent apoptosis by binding and sequestering Ca2+ from the downstream Ca2+-dependent apoptotic pathways.

scholarly journals Role of RpoS in the Virulence of Citrobacter rodentium

2008 ◽  
Vol 77 (1) ◽  
pp. 501-507 ◽  
Author(s):  
Tao Dong ◽  
Brian K. Coombes ◽  
Herb E. Schellhorn
Keyword(s):  
Pathogenicity Island ◽  
Competitive Growth ◽  
Citrobacter Rodentium ◽  
Wild Type ◽  
E Coli ◽  
Mouse Colon ◽  
Expression Of Genes

ABSTRACT Citrobacter rodentium is a mouse enteropathogen that is closely related to Escherichia coli and causes severe colonic hyperplasia and bloody diarrhea. C. rodentium infection requires expression of genes of the locus of enterocyte effacement (LEE) pathogenicity island, which simulates infection by enteropathogenic E. coli and enterohemorrhagic E. coli in the human intestine, providing an effective model for studying enteropathogenesis. In this study we investigated the role of RpoS, the stationary phase sigma factor, in virulence in C. rodentium. Sequence analysis showed that the rpoS gene is highly conserved in C. rodentium and E. coli, exhibiting 92% identity. RpoS was critical for survival under heat shock conditions and during exposure to H2O2 and positively regulated the expression of catalase KatE (HPII). The development of the RDAR (red dry and rough) morphotype, an important virulence trait in E. coli, was also mediated by RpoS in C. rodentium. Unlike E. coli, C. rodentium grew well in the mouse colon, and the wild-type strain colonized significantly better than rpoS mutants. However, a mutation in rpoS conferred a competitive growth advantage over the wild type both in vitro in Luria-Bertani medium and in vivo in the mouse colon. Survival analysis showed that the virulence of an rpoS mutant was attenuated. The expression of genes on the LEE pathogenicity island, which are essential for colonization and virulence, was reduced in the rpoS mutant. In conclusion, RpoS is important for the stress response and is required for full virulence in C. rodentium.

scholarly journals Plaque-associated human microglia accumulate lipid droplets in a chimeric model of Alzheimer’s disease

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Christel Claes ◽  
Emma Pascal Danhash ◽  
Jonathan Hasselmann ◽  
Jean Paul Chadarevian ◽  
Sepideh Kiani Shabestari ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Lipid Droplet ◽  
Lipid Droplets ◽  
Foam Cells ◽  
Wild Type ◽  
Human Microglia ◽  
Model Of Alzheimer’S Disease ◽  
The Impact

Abstract Background Disease-associated microglia (DAMs), that surround beta-amyloid plaques, represent a transcriptionally-distinct microglial profile in Alzheimer’s disease (AD). Activation of DAMs is dependent on triggering receptor expressed on myeloid cells 2 (TREM2) in mouse models and the AD TREM2-R47H risk variant reduces microglial activation and plaque association in human carriers. Interestingly, TREM2 has also been identified as a microglial lipid-sensor, and recent data indicates lipid droplet accumulation in aged microglia, that is in turn associated with a dysfunctional proinflammatory phenotype. However, whether lipid droplets (LDs) are present in human microglia in AD and how the R47H mutation affects this remains unknown. Methods To determine the impact of the TREM2 R47H mutation on human microglial function in vivo, we transplanted wild-type and isogenic TREM2-R47H iPSC-derived microglial progenitors into our recently developed chimeric Alzheimer mouse model. At 7 months of age scRNA-seq and histological analyses were performed. Results Here we report that the transcriptome of human wild-type TREM2 and isogenic TREM2-R47H DAM xenografted microglia (xMGs), isolated from chimeric AD mice, closely resembles that of human atherosclerotic foam cells. In addition, much like foam cells, plaque-bound xMGs are highly enriched in lipid droplets. Somewhat surprisingly and in contrast to a recent in vitro study, TREM2-R47H mutant xMGs exhibit an overall reduction in the accumulation of lipid droplets in vivo. Notably, TREM2-R47H xMGs also show overall reduced reactivity to plaques, including diminished plaque-proximity, reduced CD9 expression, and lower secretion of plaque-associated APOE. Conclusions Altogether, these results indicate lipid droplet accumulation occurs in human DAM xMGs in AD, but is reduced in TREM2-R47H DAM xMGs, as it occurs secondary to TREM2-mediated changes in plaque proximity and reactivity.

scholarly journals The experimentally obtained functional impact assessments of GT>GC 5’ splice site variants differ markedly from those predicted

2019 ◽  
Author(s):  
Jian-Min Chen ◽  
Jin-Huan Lin ◽  
Emmanuelle Masson ◽  
Zhuan Liao ◽  
Claude Férec ◽  
...  
Keyword(s):  
Splice Site ◽  
Disease Genes ◽  
Wild Type ◽  
Human Genetic Disease ◽  
Functional Impact ◽  
Prediction Tools ◽  
Human Disease Genes ◽  
The Impact

ABSTRACTGT>GC 5’ splice site (or +2T>C) variants have been frequently reported to cause human genetic disease. However, although we have demonstrated that GT>GC variants in human disease genes may not invariably be pathogenic, none of the currently available splicing prediction tools appear to be capable of reliably distinguishing those GT>GC variants that generate wild-type transcripts from those that do not. Recently, SpliceAI, a novel deep residual neural network tool, has been developed for splicing prediction. Methodologically distinct from previous approaches that either rely on human-engineered features and/or which focus on short nucleotide windows adjoining exon-intron boundaries, SpliceAI assesses splicing determinants by evaluating 10,000 nucleotides of flanking contextual sequence to predict the functional role in splicing of each position in the pre-mRNA transcript. Herein, we evaluated the performance of SpliceAI in the context of three datasets of GT>GC variants, all of which had been characterized functionally in terms of their impact on mRNA splicing. The first two datasets refer to our recently described “in vivo” dataset of 45 disease-causing GT>GC variants and the “in vitro” dataset of 103 GT>GC substitutions. The third dataset comprised 12 BRCA1 GT>GC variants that were recently analyzed by saturation genome editing. We processed all GT>GC variants using the default settings of SpliceAI. Comparison of the SpliceAI-predicted and experimentally obtained functional impact assessments of the analyzed GT>GC variants revealed that although SpliceAI performed rather better than other prediction tools, it was still far from perfect. A key issue is that the impact of GT>GC (as well as GT>GA or +2T>A) variants that generated wild-type transcripts represents a quantitative change that can vary from barely detectable to almost full expression of wild-type transcripts, with wild-type transcripts often co-existing with aberrantly spliced transcripts. Our findings highlight the challenges that we still face in attempting to accurately identify splice-altering variants.

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