scholarly journals Structure and mechanism of the RNA dependent RNase Cas13a from Rhodobacter capsulatus

2021 ◽  
Author(s):  
Leonhard M Kick ◽  
Marie-Kristin von Wrisberg ◽  
Leander S Runtsch ◽  
Sabine Schneider
Keyword(s):  
Single Molecule ◽  
Molecular Mechanisms ◽  
Rhodobacter Capsulatus ◽  
Purple Bacteria ◽  
Functional Characterization ◽  
Regulation Of Gene Expression ◽  
Nuclease Activity ◽  
Site Directed Mutagenesis ◽  
And Function ◽  
Key Residues

Cas13a are single-molecule effectors of the Class II, Type VI family of CRISPR-Cas systems that are part of the bacterial and archaeal defense systems. These RNA-guided and RNA-activated RNA endonucleases are characterized by their ability to cleave target RNAs complementary to the crRNA-spacer sequence, as well as bystander RNAs in a sequence-unspecific manner. Due to cleavage of cellular transcripts they induce dormancy in the host cell and thus protect the bacterial population by aborting the infectious cycle of RNA-phages. Here we report the structural and functional characterization of a Cas13a enzyme from the photo-auxotrophic purple bacteria Rhodobacter capsulatus. The X-ray crystal structure of the RcCas13a-crRNA complex reveals its distinct crRNA recognition mode as well as the enzyme in its contracted, pre-activation conformation. Using site-directed mutagenesis in combination with mass spectrometry, we identified key-residues responsible for pre-crRNA processing by RcCas13a in its distinct catalytic site, and elucidated the acid-base mediated cleavage reaction mechanism. In addition, RcCas13a cleaves target-RNA as well as bystander-RNAs in Escherichia coli which requires its catalytic active HEPN (higher eukaryotes and prokaryotes nucleotide binding) domain nuclease activity. Our data provide further insights into the molecular mechanisms and function of this intriguing family of RNA-dependent RNA endonucleases that are already employed as efficient tools for RNA detection and regulation of gene expression.

scholarly journals How Microbes Use Force To Control Adhesion

2020 ◽  
Vol 202 (12) ◽  
Author(s):  
Albertus Viljoen ◽  
Johann Mignolet ◽  
Felipe Viela ◽  
Marion Mathelié-Guinlet ◽  
Yves F. Dufrêne
Keyword(s):  
Single Molecule ◽  
Molecular Mechanisms ◽  
Flow Chamber ◽  
Microbial Adhesion ◽  
Single Molecule Level ◽  
Force Microscopy ◽  
Atomic Force ◽  
Adhesive Interactions ◽  
And Function ◽  
Mechanisms Of Adhesion

ABSTRACT Microbial adhesion and biofilm formation are usually studied using molecular and cellular biology assays, optical and electron microscopy, or laminar flow chamber experiments. Today, atomic force microscopy (AFM) represents a valuable addition to these approaches, enabling the measurement of forces involved in microbial adhesion at the single-molecule level. In this minireview, we discuss recent discoveries made applying state-of-the-art AFM techniques to microbial specimens in order to understand the strength and dynamics of adhesive interactions. These studies shed new light on the molecular mechanisms of adhesion and demonstrate an intimate relationship between force and function in microbial adhesins.

scholarly journals Cytolethal Distending Toxin Subunit B: A Review of Structure–Function Relationship

Toxins ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 595 ◽  
Author(s):  
Benoît J. Pons ◽  
Julien Vignard ◽  
Gladys Mirey
Keyword(s):  
Pathogenic Bacteria ◽  
Molecular Mechanisms ◽  
3D Structure ◽  
Cytolethal Distending Toxin ◽  
Experimental Systems ◽  
Bacterial Virulence Factor ◽  
Function Relationship ◽  
And Function ◽  
Key Residues ◽  
Cell Cycle Block

The Cytolethal Distending Toxin (CDT) is a bacterial virulence factor produced by several Gram-negative pathogenic bacteria. These bacteria, found in distinct niches, cause diverse infectious diseases and produce CDTs differing in sequence and structure. CDTs have been involved in the pathogenicity of the associated bacteria by promoting persistent infection. At the host-cell level, CDTs cause cell distension, cell cycle block and DNA damage, eventually leading to cell death. All these effects are attributable to the catalytic CdtB subunit, but its exact mode of action is only beginning to be unraveled. Sequence and 3D structure analyses revealed similarities with better characterized proteins, such as nucleases or phosphatases, and it has been hypothesized that CdtB exerts a biochemical activity close to those enzymes. Here, we review the relationships that have been established between CdtB structure and function, particularly by mutation experiments on predicted key residues in different experimental systems. We discuss the relevance of these approaches and underline the importance of further study in the molecular mechanisms of CDT toxicity, particularly in the context of different pathological conditions.

scholarly journals SFPQ Rescues F508del-CFTR Expression and Function in Cystic Fibrosis Bronchial Epithelial Cells

2021 ◽  
Author(s):  
Parameet Kumar ◽  
Dharmendra Kumar Soni ◽  
Chaitali Sen ◽  
Mads B Larsen ◽  
Krystyna Mazan-Mamczarz ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Regulation Of Gene Expression ◽  
Lung Epithelial Cells ◽  
Regulation Of Expression ◽  
Trafficking Defects ◽  
And Function

Abstract Cystic Fibrosis (CF) occurs as a result of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which lead to misfolding, trafficking defects, and impaired function of the CFTR protein. Splicing factor proline/glutamine-rich (SFPQ) is a multifunctional nuclear RNA-binding protein (RBP) implicated in the regulation of gene expression pathways and intracellular trafficking. Here, we investigated the role of SFPQ in the regulation of the expression and function of F508del-CFTR in CF lung epithelial cells. We find that the expression of SFPQ is reduced in F508del-CFTR CF epithelial cells compared to WT-CFTR control cells. Interestingly, the overexpression of SFPQ in CF cells increases the expression as well as rescues the function of F508del-CFTR. Further, comprehensive transcriptome analyses indicate that SFPQ plays a key role in activating the mutant F508del-CFTR by modulating several cellular signaling pathways. This is the first report on the role of SFPQ in the regulation of expression and function of F508del-CFTR in CF lung disease. Our findings provide new insights into SFPQ-mediated molecular mechanisms and point to possible novel epigenetic therapeutic targets for CF and related pulmonary diseases.

The architecture and function of the light-harvesting apparatus of purple bacteria: from single molecules to in vivo membranes

2006 ◽  
Vol 39 (3) ◽  
pp. 227-324 ◽  
Author(s):  
Richard J. Cogdell ◽  
Andrew Gall ◽  
Jürgen Köhler
Keyword(s):  
Quantum Mechanics ◽  
Energy Transfer ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Light Harvesting ◽  
Structural Information ◽  
Purple Bacteria ◽  
Theoretical Background ◽  
Single Molecule Spectroscopy

1. Introduction 2292. Structures 2342.1 The structure of LH2 2342.2 Natural variants of peripheral antenna complexes 2422.3 RC–LH1 complexes 2423. Spectroscopy 2493.1 Steady-state spectroscopy 2493.2 Factors which affect the position of the Qy absorption band of Bchla 2494. Regulation of biosynthesis and assembly 2574.1 Regulation 2574.1.1 Oxygen 2574.1.2 Light 2584.1.2.1 AppA: blue-light-mediated regulation 2594.1.2.2 Bacteriophytochromes 2594.1.3 From the RC to the mature PSU 2614.2 Assembly 2614.2.1 LH1 2624.2.2 LH2 2635. Frenkel excitons 2655.1 General 2655.2 B800 2675.3 B850 2675.4 B850 delocalization 2736. Energy-transfer pathways: experimental results 2746.1 Theoretical background 2746.2 ‘Follow the excitation energy’ 2766.2.1 Bchla→Bchla energy transfer 2776.2.1.1 B800→B800 2776.2.1.2 B800→B850 2786.2.1.3 B850→B850 2796.2.1.4 B850→B875 2806.2.1.5 B875→RC 2806.2.2 Car[harr ]Bchla energy transfer 2817. Single-molecule spectroscopy 2847.1 Introduction to single-molecule spectroscopy 2847.2 Single-molecule spectroscopy on LH2 2857.2.1 Overview 2857.2.2 B800 2867.2.2.1 General 2867.2.2.2 Intra- and intercomplex disorder of site energies 2877.2.2.3 Electron-phonon coupling 2897.2.2.4 B800→B800 energy transfer revisited 2907.2.3 B850 2938. Quantum mechanics and the purple bacteria LH system 2989. Appendix 2999.1 A crash course on quantum mechanics 2999.2 Interacting dimers 30510. Acknowledgements 30611. References 307This review describes the structures of the two major integral membrane pigment complexes, the RC–LH1 ‘core’ and LH2 complexes, which together make up the light-harvesting system present in typical purple photosynthetic bacteria. The antenna complexes serve to absorb incident solar radiation and to transfer it to the reaction centres, where it is used to ‘power’ the photosynthetic redox reaction and ultimately leads to the synthesis of ATP. Our current understanding of the biosynthesis and assembly of the LH and RC complexes is described, with special emphasis on the roles of the newly described bacteriophytochromes. Using both the structural information and that obtained from a wide variety of biophysical techniques, the details of each of the different energy-transfer reactions that occur, between the absorption of a photon and the charge separation in the RC, are described. Special emphasis is given to show how the use of single-molecule spectroscopy has provided a more detailed understanding of the molecular mechanisms involved in the energy-transfer processes. We have tried, with the help of an Appendix, to make the details of the quantum mechanics that are required to appreciate these molecular mechanisms, accessible to mathematically illiterate biologists. The elegance of the purple bacterial light-harvesting system lies in the way in which it has cleverly exploited quantum mechanics.

scholarly journals MOG1 restores the expression and function of SCN5A-p.R104W through sec23a-mediated forward trafficking

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
L Luo ◽  
Y Wang ◽  
Y Du ◽  
C Dong ◽  
A Ma ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Sodium Current ◽  
Site Directed Mutagenesis ◽  
Hek293 Cells ◽  
Funding Source ◽  
Inherited Disease ◽  
Patch Clamp Recording ◽  
Knock Out ◽  
Cardiac Sodium Channel ◽  
And Function

Abstract Background Brugada syndrome (BrS) is an inherited disease which causes fatal arrhythmias and sudden cardiac death. Mutations in SCN5A gene, which encoding cardiac sodium channel (NaV1.5), are the most common genotype of BrS patients. Some SCN5A-related variants were reported to retain NaV1.5 in endoplasmic reticulum (ER) due to trafficking deficiency. MOG1 was previously reported to interact with NaV1.5 and increased sodium current (INa) through enhancing the trafficking. However, its molecular mechanisms are still unclear. Coat protein complex II (COPII) is responsible for the ER to Golgi transport. Sec23 forms the inner coat of COPII and participates in cargo proteins selection. Purpose To demonstrate that MOG1 rescues SCN5A-related variants by enhancing the forward trafficking through Sec23a-NaV1.5 interaction. Methods Site directed mutagenesis, immunofluorescence staining, biotinylation assay, Western blot analysis and whole-cell patch clamp recording were used. CRISPR/Cas9 was used to knock out Sec23a expression in HEK293 cells. Results We found that SCN5A-p.R104W was characterized as reduced NaV1.5 level and lack of INa. The variant SCN5A-p.R104W was mainly distributed in ER. MOG1 could rescue the total and surface expression of SCN5A-p.R104W but could not restore INa (Figure 1a). Considering that most patients are heterozygous, co-transfection of SCN5A-WT and SCN5A-p.R104W were obtained. We found MOG1 could increase both NaV1.5 level and INa of heterozygous expressed SCN5A-p.R104W. We further revealed an interaction between NaV1.5 and Sec23a by co-immunoprecipitation (Co-IP) assay. The interaction between NaV1.5 and Sec23a was increased by MOG1, which indicates that Sec23a participates in MOG1-mediated increase in NaV1.5 level (Figure 1b). Knockout of Sec23a reduced cell surface, but not total, NaV1.5 level (Figure 1c and 1d). Next, the Sec23a knockout HEK293 cells were co-transfected with SCN5A-p.R104W and pcDNA3 or MOG1. MOG1 could not increase SCN5A-p.R104W protein level in Sec23a knockout cells. Conclusion Our data demonstrated a novel mechanism that MOG1 restores the expression and function of SCN5A-p.R104W by enhancing its forward trafficking through Sec23a-NaV1.5 interaction. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Natural Science Foundation of China

scholarly journals Structure and function of a novel endonuclease acting on branched DNA substrates

2011 ◽  
Vol 39 (1) ◽  
pp. 145-149 ◽  
Author(s):  
Christophe Creze ◽  
Roxane Lestini ◽  
Joelle Kühn ◽  
Alessio Ligabue ◽  
Hubert F. Becker ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Nuclease Activity ◽  
Nuclear Antigen ◽  
Double Stranded Dna ◽  
Dna Junctions ◽  
Branched Dna ◽  
And Function ◽  
Proliferating Cell ◽  
Pyrococcus Abyssi

Branched DNA structures that occur during DNA repair and recombination must be efficiently processed by structure-specific endonucleases in order to avoid cell death. In the present paper, we summarize our screen for new interaction partners for the archaeal replication clamp that led to the functional characterization of a novel endonuclease family, dubbed NucS. Structural analyses of Pyrococcus abyssi NucS revealed an unexpected binding site for ssDNA (single-stranded DNA) that directs, together with the replication clamp, the nuclease activity of this protein towards ssDNA–dsDNA (double-stranded DNA) junctions. Our studies suggest that understanding the detailed architecture and dynamic behaviour of the NucS (nuclease specific for ssDNA)–PCNA (proliferating-cell nuclear antigen) complex with DNA will be crucial for identification of its physiologically relevant activities.

scholarly journals SFPQ rescues F508del-CFTR expression and function in cystic fibrosis bronchial epithelial cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Parameet Kumar ◽  
Dharmendra Kumar Soni ◽  
Chaitali Sen ◽  
Mads B. Larsen ◽  
Krystyna Mazan-Mamczarz ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Regulation Of Gene Expression ◽  
Lung Epithelial Cells ◽  
Regulation Of Expression ◽  
Trafficking Defects ◽  
And Function

AbstractCystic fibrosis (CF) occurs as a result of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which lead to misfolding, trafficking defects, and impaired function of the CFTR protein. Splicing factor proline/glutamine-rich (SFPQ) is a multifunctional nuclear RNA-binding protein (RBP) implicated in the regulation of gene expression pathways and intracellular trafficking. Here, we investigated the role of SFPQ in the regulation of the expression and function of F508del-CFTR in CF lung epithelial cells. We find that the expression of SFPQ is reduced in F508del-CFTR CF epithelial cells compared to WT-CFTR control cells. Interestingly, the overexpression of SFPQ in CF cells increases the expression as well as rescues the function of F508del-CFTR. Further, comprehensive transcriptome analyses indicate that SFPQ plays a key role in activating the mutant F508del-CFTR by modulating several cellular signaling pathways. This is the first report on the role of SFPQ in the regulation of expression and function of F508del-CFTR in CF lung disease. Our findings provide new insights into SFPQ-mediated molecular mechanisms and point to possible novel epigenetic therapeutic targets for CF and related pulmonary diseases.

scholarly journals Autoacetylation of the MYST Lysine Acetyltransferase MOF Protein

2012 ◽  
Vol 287 (42) ◽  
pp. 34917-34926 ◽  
Author(s):  
Chao Yang ◽  
Jiang Wu ◽  
Sarmistha H. Sinha ◽  
John M. Neveu ◽  
Yujun George Zheng
Keyword(s):  
Enzymatic Activity ◽  
Histone Acetylation ◽  
Regulation Of Gene Expression ◽  
Point Mutations ◽  
Cellular Processes ◽  
Lysine Deacetylase ◽  
Highly Sensitive ◽  
And Function ◽  
Key Residues ◽  
The Impact

The MYST family of histone acetyltransferases (HATs) plays critical roles in diverse cellular processes, such as the epigenetic regulation of gene expression. Lysine autoacetylation of the MYST HATs has recently received considerable attention. Nonetheless, the mechanism and function of the autoacetylation process are not well defined. To better understand the biochemical mechanism of MYST autoacetylation and the impact of autoacetylation on the cognate histone acetylation, we carried out detailed analyses of males-absent-on-the-first (MOF), a key member of the MYST family. A number of mutant MOF proteins were produced with point mutations at several key residues near the active site of the enzyme. Autoradiography and immunoblotting data showed that mutation of these residues affects the autoacetylation activity and HAT activity of MOF by various degrees demonstrating that MOF activity is highly sensitive to the chemical changes in those residues. We produced MOF protein in the deacetylated form by using a nonspecific lysine deacetylase. Interestingly, both the autoacetylation activity and the histone acetylation activity of the deacetylated MOF were found to be very close to that of wild-type MOF, suggesting that autoacetylation of MOF only marginally modulates the enzymatic activity. Also, we found that the autoacetylation rates of MOF and deacetylated MOF were much slower than the cognate substrate acetylation. Thus, autoacetylation does not seem to contribute to the intrinsic enzymatic activity in a significant manner. These data provide new insights into the mechanism and function of MYST HAT autoacetylation.

scholarly journals Phosphoregulation of Nap1 Plays a Role in Septin Ring Dynamics and Morphogenesis in Candida albicans

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Zhen-Xing Huang ◽  
Pan Zhao ◽  
Gui-Sheng Zeng ◽  
Yan-Ming Wang ◽  
Ian Sudbery ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Mouse Model ◽  
Cellular Localization ◽  
Molecular Mechanisms ◽  
Functional Characterization ◽  
Content Type ◽  
Septin Ring ◽  
Wide Range ◽  
Ring Dynamics ◽  
And Function

ABSTRACT Nap1 has long been identified as a potential septin regulator in yeasts. However, its function and regulation remain poorly defined. Here, we report functional characterization of Nap1 in the human-pathogenic fungus Candida albicans. We find that deletion of NAP1 causes constitutive filamentous growth and changes of septin dynamics. We present evidence that Nap1’s cellular localization and function are regulated by phosphorylation. Phos-tag gel electrophoresis revealed that Nap1 phosphorylation is cell cycle dependent, exhibiting the lowest level around the time of bud emergence. Mass spectrometry identified 10 phosphoserine and phosphothreonine residues in a cluster near the N terminus, and mutation of these residues affected Nap1’s localization to the septin ring and cellular function. Nap1 phosphorylation involves two septin ring-associated kinases, Cla4 and Gin4, and its dephosphorylation occurs at the septin ring in a manner dependent on the phosphatases PP2A and Cdc14. Furthermore, the nap1Δ/Δ mutant and alleles carrying mutations of the phosphorylation sites exhibited greatly reduced virulence in a mouse model of systemic candidiasis. Together, our findings not only provide new mechanistic insights into Nap1’s function and regulation but also suggest the potential to target Nap1 in future therapeutic design. IMPORTANCE Septins are conserved filament-forming GTPases involved in a wide range of cellular events, such as cytokinesis, exocytosis, and morphogenesis. In Candida albicans, the most prevalent human fungal pathogen, septin functions are indispensable for its virulence. However, the molecular mechanisms by which septin structures are regulated are poorly understood. In this study, we deleted NAP1, a gene encoding a putative septin regulator, in C. albicans and found that cells lacking NAP1 showed abnormalities in morphology, invasive growth, and septin ring dynamics. We identified a conserved N-terminal phosphorylation cluster on Nap1 and demonstrated that phosphorylation at these sites regulates Nap1 localization and function. Importantly, deletion of NAP1 or mutation in the N-terminal phosphorylation cluster strongly reduced the virulence of C. albicans in a mouse model of systemic infection. Thus, this study not only provides mechanistic insights into septin regulation but also suggests Nap1 as a potential antifungal target.

Functional characterization of human brown adipose tissue metabolism

2020 ◽  
Vol 477 (7) ◽  
pp. 1261-1286 ◽  
Author(s):  
Marie Anne Richard ◽  
Hannah Pallubinsky ◽  
Denis P. Blondin
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Functional Characterization ◽  
Human Infants ◽  
Positron Emission ◽  
Brown Adipose ◽  
Treatment Of Obesity ◽  
And Function

Brown adipose tissue (BAT) has long been described according to its histological features as a multilocular, lipid-containing tissue, light brown in color, that is also responsive to the cold and found especially in hibernating mammals and human infants. Its presence in both hibernators and human infants, combined with its function as a heat-generating organ, raised many questions about its role in humans. Early characterizations of the tissue in humans focused on its progressive atrophy with age and its apparent importance for cold-exposed workers. However, the use of positron emission tomography (PET) with the glucose tracer [18F]fluorodeoxyglucose ([18F]FDG) made it possible to begin characterizing the possible function of BAT in adult humans, and whether it could play a role in the prevention or treatment of obesity and type 2 diabetes (T2D). This review focuses on the in vivo functional characterization of human BAT, the methodological approaches applied to examine these features and addresses critical gaps that remain in moving the field forward. Specifically, we describe the anatomical and biomolecular features of human BAT, the modalities and applications of non-invasive tools such as PET and magnetic resonance imaging coupled with spectroscopy (MRI/MRS) to study BAT morphology and function in vivo, and finally describe the functional characteristics of human BAT that have only been possible through the development and application of such tools.

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