scholarly journals Cas3 Protein—A Review of a Multi-Tasking Machine

Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 208 ◽  
Author(s):  
Liu He ◽  
Michael St. John James ◽  
Marin Radovcic ◽  
Ivana Ivancic-Bace ◽  
Edward L. Bolt
Keyword(s):  
Cell Biology ◽  
Protein A ◽  
Mobile Genetic Elements ◽  
Genetic Elements ◽  
Cellular Processes ◽  
Concise Review ◽  
In Cells

Cas3 has essential functions in CRISPR immunity but its other activities and roles, in vitro and in cells, are less widely known. We offer a concise review of the latest understanding and questions arising from studies of Cas3 mechanism during CRISPR immunity, and highlight recent attempts at using Cas3 for genetic editing. We then spotlight involvement of Cas3 in other aspects of cell biology, for which understanding is lacking—these focus on CRISPR systems as regulators of cellular processes in addition to defense against mobile genetic elements.

scholarly journals Mobile genetic elements: in silico,in vitro,in vivo

2016 ◽  
Vol 25 (5) ◽  
pp. 1027-1031 ◽  
Author(s):  
Irina R. Arkhipova ◽  
Phoebe A. Rice
Keyword(s):  
In Silico ◽  
Mobile Genetic Elements ◽  
Genetic Elements

scholarly journals Antioxidant and food additive BHA prevents TNF cytotoxicity by acting as a direct RIPK1 inhibitor

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Tom Delanghe ◽  
Jon Huyghe ◽  
Seungheon Lee ◽  
Dario Priem ◽  
Samya Van Coillie ◽  
...  
Keyword(s):  
Antioxidant Properties ◽  
In Silico Analysis ◽  
Oxygen Species ◽  
Ros Scavenging ◽  
Cellular Processes ◽  
Silico Analysis ◽  
Surprising Finding ◽  
Necrosis Factor ◽  
In Cells

AbstractButylate hydroxyanisole (BHA) is a synthetic phenol that is widely utilized as a preservative by the food and cosmetic industries. The antioxidant properties of BHA are also frequently used by scientists to claim the implication of reactive oxygen species (ROS) in various cellular processes, including cell death. We report on the surprising finding that BHA functions as a direct inhibitor of RIPK1, a major signaling hub downstream of several immune receptors. Our in silico analysis predicts binding of 3-BHA, but not 2-BHA, to RIPK1 in an inactive DLG-out/Glu-out conformation, similar to the binding of the type III inhibitor Nec-1s to RIPK1. This predicted superior inhibitory capacity of 3-BHA over 2-BHA was confirmed in cells and using in vitro kinase assays. We demonstrate that the reported protective effect of BHA against tumor necrosis factor (TNF)-induced necroptotic death does not originate from ROS scavenging but instead from direct RIPK1 enzymatic inhibition, a finding that most probably extends to other reported effects of BHA. Accordingly, we show that BHA not only protects cells against RIPK1-mediated necroptosis but also against RIPK1 kinase-dependent apoptosis. We found that BHA treatment completely inhibits basal and induced RIPK1 enzymatic activity in cells, monitored at the level of TNFR1 complex I under apoptotic conditions or in the cytosol under necroptosis. Finally, we show that oral administration of BHA protects mice from RIPK1 kinase-dependent lethality caused by TNF injection, a model of systemic inflammatory response syndrome. In conclusion, our results demonstrate that BHA can no longer be used as a strict antioxidant and that new functions of RIPK1 may emerge from previously reported effects of BHA.

scholarly journals The bacterial chromatin protein HupA can remodel DNA and associates with the nucleoid in Clostridium difficile

2018 ◽  
Author(s):  
Ana M. Oliveira Paiva ◽  
Annemieke H. Friggen ◽  
Liang Qin ◽  
Roxanne Douwes ◽  
Remus T. Dame ◽  
...  
Keyword(s):  
Protein A ◽  
Chromatin Protein ◽  
Chromosomal Organization ◽  
Cellular Processes ◽  
Hu Proteins ◽  
Architectural Proteins ◽  
Chromatin Proteins

AbstractThe maintenance and organization of the chromosome plays an important role in the development and survival of bacteria. Bacterial chromatin proteins are architectural proteins that bind DNA, modulate its conformation and by doing so affect a variety of cellular processes. No bacterial chromatin proteins of C. difficile have been characterized to date.Here, we investigate aspects of the C. difficile HupA protein, a homologue of the histone-like HU proteins of Escherichia coli. HupA is a 10 kDa protein that is present as a homodimer in vitro and self-interacts in vivo. HupA co-localizes with the nucleoid of C. difficile. It binds to the DNA without a preference for the DNA G+C content. Upon DNA binding, HupA induces a conformational change in the substrate DNA in vitro and leads to compaction of the chromosome in vivo.The present study is the first to characterize a bacterial chromatin protein in C. difficile and opens the way to study the role of chromosomal organization in DNA metabolism and on other cellular processes in this organism.

scholarly journals Mesangial cell proteoglycans: synthesis and metabolism.

1992 ◽  
Vol 2 (10) ◽  
pp. S88
Author(s):  
M Davies ◽  
G J Thomas ◽  
L D Shewring ◽  
R M Mason
Keyword(s):  
Culture Medium ◽  
Mesangial Cell ◽  
Dermatan Sulfate ◽  
Mesangial Cells ◽  
Core Protein ◽  
Glomerular Mesangial Cells ◽  
Cellular Processes ◽  
Specialized Function ◽  
In Cells

In cultures of human adult glomerular mesangial cells, large chondroitin sulfate proteoglycans (CSPG) and small dermatan sulfate proteoglycans (DSPG) are synthesized. The large CSPG has a core protein, M(r) of 400,000 (major) and M(r) of 500,000 (minor), and binds to hyaluronic acid to form large aggregates. The two small DSPGs (Mr of approximately 350,000 and M(r) of approximately 200,000) were related to biglycan and decorin, respectively. The majority of these proteoglycans were located in the culture medium, but a hydrophobic form of the CSPG was extracted from the cell layer. Mesangial cells in the growing phase synthesized and secreted all three types of proteoglycans, but in cells arrested in G0 by serum deprivation the incorporation of (35S)sulfate in CSPG was drastically reduced. In the same cells stimulated to proliferate by replacing the medium with one containing serum, the synthesis of CSPG dramatically enhanced. The synthesis of CSPG and DSPG was also elevated in cells cocultured with cytokines but in contrast was significantly reduced when cultured in medium containing hyperglycemic levels of glucose. Finally, preliminary experiments are reported that indicate that CSPG and DSPG bind to low-density lipoproteins in vitro. These observations suggest a possible specialized function for proteoglycans in cellular processes characteristic of glomerular disease.

scholarly journals How important is CRISPR-Cas for protecting natural populations of bacteria against infections by mobile genetic elements?

2020 ◽  
Author(s):  
Edze Westra ◽  
Bruce Levin
Keyword(s):  
Computer Simulation ◽  
Immune System ◽  
Natural Populations ◽  
Adaptive Immune System ◽  
Simulation Models ◽  
Mobile Genetic Elements ◽  
Genetic Elements ◽  
Adaptive Immune ◽  
Computer Simulation Models

AbstractArticles on CRISPR commonly open with some variant of the phrase ‘these short-palindromic repeats and their associated endonucleases (Cas) are an adaptive immune system that exists to protect bacteria and archaea from viruses and infections with other mobile genetic elements’. There is an abundance of genomic data consistent with the hypothesis that CRISPR plays this role in natural populations of bacteria and archaea, and experimental demonstrations with a few species of bacteria and their phage and plasmids show that CRISPR-Cas systems can play this role in vitro. Not at all clear are the ubiquity, magnitude and nature of the contribution of CRISPR-Cas systems to the ecology and evolution of natural populations of microbes, and the strength of selection mediated by different types of phage and plasmids to the evolution and maintenance of CRISPR-Cas systems. In this perspective, with the aid of heuristic mathematical-computer simulation models, we explore the a priori conditions under which exposure to lytic and temperate phage and conjugative plasmids will select for and maintain CRISPR-Cas systems in populations of bacteria and archaea. We review the existing literature addressing these ecological and evolutionary questions and highlight the experimental and other evidence needed to fully understand the conditions responsible for the evolution and maintenance of CRISPR-Cas systems and the contribution of these systems to the ecology and evolution of bacteria, archaea and the mobile genetic elements that infect them.SignificanceThere is no question about the importance and utility of CRISPR-Cas for editing and modifying genomes. On the other hand, the mechanisms responsible for the evolution and maintenance of these systems and the magnitude of their importance to the ecology and evolution of bacteria, archaea and their infectious DNAs, are not at all clear. With the aid of heuristic mathematical – computer simulation models and reviews of the existing literature, we raise questions that have to be answered to elucidate the contribution of selection – mediated by phage and plasmids – to the evolution and maintenance of this adaptive immune system and its consequences for the ecology and evolution of prokaryotes and their viruses and plasmids.

scholarly journals It is unclear how important CRISPR-Cas systems are for protecting natural populations of bacteria against infections by mobile genetic elements

2020 ◽  
Vol 117 (45) ◽  
pp. 27777-27785
Author(s):  
Edze R. Westra ◽  
Bruce R. Levin
Keyword(s):  
A Priori ◽  
Natural Populations ◽  
Adaptive Immune System ◽  
Simulation Models ◽  
Mobile Genetic Elements ◽  
Genetic Elements ◽  
Adaptive Immune ◽  
Different Types ◽  
Computer Simulation Models

Articles on CRISPR commonly open with some variant of the phrase “these short palindromic repeats and their associated endonucleases (Cas) are an adaptive immune system that exists to protect bacteria and archaea from viruses and infections with other mobile genetic elements.” There is an abundance of genomic data consistent with the hypothesis that CRISPR plays this role in natural populations of bacteria and archaea, and experimental demonstrations with a few species of bacteria and their phage and plasmids show that CRISPR-Cas systems can play this role in vitro. Not at all clear are the ubiquity, magnitude, and nature of the contribution of CRISPR-Cas systems to the ecology and evolution of natural populations of microbes and the strength of selection mediated by different types of phage and plasmids to the evolution and maintenance of CRISPR-Cas systems. In this perspective, with the aid of heuristic mathematical–computer simulation models, we explore the a priori conditions under which exposure to lytic and temperate phage and conjugative plasmids will select for and maintain CRISPR-Cas systems in populations of bacteria and archaea. We review the existing literature addressing these ecological and evolutionary questions and highlight the experimental and other evidence needed to fully understand the conditions responsible for the evolution and maintenance of CRISPR-Cas systems and the contribution of these systems to the ecology and evolution of bacteria, archaea, and the mobile genetic elements that infect them.

scholarly journals Dynamics between horizontal gene transfer and acquired antibiotic resistance in S. Heidelberg following in vitro incubation in broiler ceca

2019 ◽  
Author(s):  
Adelumola Oladeinde ◽  
Kimberly Cook ◽  
Steven M. Lakin ◽  
Zaid Abdo ◽  
Torey Looft ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Gastrointestinal Tract ◽  
Gut Microbiome ◽  
Genetic Material ◽  
Mobile Genetic Elements ◽  
Important Food ◽  
Genetic Elements ◽  
Food Borne ◽  
Acquired Antibiotic Resistance

AbstractThe chicken gastrointestinal tract harbors taxa of microorganisms that play a role in the health and disease status of the host. The cecum is the part of the gut that carries the highest microbial densities, has the longest residence time of digesta and is a vital site for urea recycling and water regulation. Therefore, the cecum provides a rich environment for bacteria to horizontally transfer genes between one another via mobile genetic elements such as plasmids and bacteriophages. In this study, we used broiler chicken cecum as a model to investigate antibiotic resistance genes that can be transferred in vitro from ceca flora to Salmonella enterica serovar Heidelberg (S. Heidelberg). We used whole genome sequencing and resistome enrichment to decipher the interactions between S. Heidelberg, gut microbiome and acquired antibiotic resistance. After 48 h incubation of ceca under microaerophilic conditions, one S. Heidelberg isolate was recovered with an acquired Inck2 plasmid (88 kb) encoding extended β-lactamase producing gene (blaCMY-2). In vitro, this plasmid was transferrable between E. coli and S. Heidelberg strains, but transfer was unsuccessful between S. Heidelberg strains. An in-depth genetic characterization of transferred plasmids suggests that they share significant homology with P1-like phages. This study contributes to our understanding of the dynamics between an important food-borne pathogen and the chicken gut microbiome.ImportanceS. Heidelberg is a clinically important serovar, linked to food-borne illness and among the top 5 serovars isolated from poultry in USA and Canada. Acquisition of new genetic material from microbial flora in the gastrointestinal tract of food animals, including broilers, may contribute to increased fitness of pathogens like S. Heidelberg and may increase their level of antibiotic tolerance. Therefore, it is critical to gain a better understanding on the dynamic interactions that occur between important pathogens and the commensals present in the animal gut and other agroecosystems. In this study, we show that the native flora in the broiler ceca were capable of transferring mobile genetic elements carrying AmpC β-lactamase (blaCMY-2) gene to an important food-borne pathogen S. Heidelberg. The potential role for P1-like bacteriophage transduction was also discussed.

scholarly journals SCFFbxw5 targets MCAK in G2/M to facilitate ciliogenesis in the following cell cycle

2021 ◽  
Author(s):  
Jörg Schweiggert ◽  
Gregor Habeck ◽  
Sandra Hess ◽  
Felix Mikus ◽  
Klaus Meese ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Primary Cilia ◽  
Protein Microarrays ◽  
Mitotic Progression ◽  
Cellular Processes ◽  
Ubiquitin E3 Ligase ◽  
M Phase ◽  
Cancer Types ◽  
In Cells

AbstractThe microtubule depolymerase Kif2C/MCAK plays important roles in various cellular processes and is frequently overexpressed in different cancer types. Despite the importance of its correct abundance, remarkably little is known about how MCAK levels are regulated in cells.Using comprehensive screening on protein microarrays, we identified 161 candidate substrates of the multi-subunit ubiquitin E3 ligase SCFFbxw5, including MCAK. In vitro reconstitution assays demonstrate that MCAK and its closely related orthologs Kif2A and Kif2B become efficiently polyubiquitylated by neddylated SCFFbxw5 and Cdc34, without requiring preceding modifications. In cells, SCFFbxw5 targets MCAK for proteasomal degradation specifically during G2/M. While this seems largely dispensable for mitotic progression, loss of Fbxw5 leads to increased MCAK levels at basal bodies, which impair formation of primary cilia in the following G1. We have thus identified a novel regulatory event of ciliogenesis that occurs already within the G2/M phase of the preceding cell cycle.

ROCKII Ser1366 phosphorylation reflects the activation status

2012 ◽  
Vol 443 (1) ◽  
pp. 145-151 ◽  
Author(s):  
Hsiang-Hao Chuang ◽  
Chih-Hsuan Yang ◽  
Yeou-Guang Tsay ◽  
Chih-Yi Hsu ◽  
Ling-Ming Tseng ◽  
...  
Keyword(s):  
Phosphorylation Site ◽  
Human Breast ◽  
New Approach ◽  
Cellular Processes ◽  
Downstream Effector ◽  
Conserved Residue ◽  
Major Phosphorylation Site ◽  
Activation Status ◽  
In Cells

ROCK (Rho-associated protein kinase), a downstream effector of RhoA, plays an important role in many cellular processes. Accumulating evidence has shown the involvement of ROCK activation in the pathogenesis of many diseases. However, a reagent capable of detecting ROCK activation directly is lacking. In the present study, we show autophosphorylation of ROCKII in an in vitro kinase reaction. The phosphorylation sites were identified by MS, and the major phosphorylation site was found to be at the highly conserved residue Ser1366. A phospho-specific antibody was generated that can specifically recognize ROCKII Ser1366 phosphorylation. We found that the extent of Ser1366 phosphorylation of endogenous ROCKII is correlated with that of myosin light chain phosphorylation in cells in response to RhoA stimulation, showing that Ser1366 phosphorylation reflects its kinase activity. In addition, ROCKII Ser1366 phosphorylation could be detected in human breast tumours by immunohistochemical staining. The present study provides a new approach for revealing the ROCKII activation status by probing ROCKII Ser1366 phosphorylation directly in cells or tissues.

scholarly journals Microtubule architecture in vitro and in cells revealed by cryo-electron tomography

2018 ◽  
Vol 74 (6) ◽  
pp. 572-584 ◽  
Author(s):  
Joseph Atherton ◽  
Melissa Stouffer ◽  
Fiona Francis ◽  
Carolyn A. Moores
Keyword(s):  
Molecular Motors ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Structural Heterogeneity ◽  
Dimensional Structure ◽  
Cellular Functions ◽  
Cellular Processes ◽  
Cryo Electron Tomography ◽  
In Cells

The microtubule cytoskeleton is involved in many vital cellular processes. Microtubules act as tracks for molecular motors, and their polymerization and depolymerization can be harnessed to generate force. The structures of microtubules provide key information about the mechanisms by which their cellular roles are accomplished and the physiological context in which these roles are performed. Cryo-electron microscopy allows the visualization of in vitro-polymerized microtubules and has provided important insights into their overall morphology and the influence of a range of factors on their structure and dynamics. Cryo-electron tomography can be used to determine the unique three-dimensional structure of individual microtubules and their ends. Here, a previous cryo-electron tomography study of in vitro-polymerized GMPCPP-stabilized microtubules is revisited, the findings are compared with new tomograms of dynamic in vitro and cellular microtubules, and the information that can be extracted from such data is highlighted. The analysis shows the surprising structural heterogeneity of in vitro-polymerized microtubules. Lattice defects can be observed both in vitro and in cells. The shared ultrastructural properties in these different populations emphasize the relevance of three-dimensional structures of in vitro microtubules for understanding microtubule cellular functions.

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