The muscle regulatory and structural protein MLP is a cytoskeletal binding partner of betaI-spectrin

2000 ◽  
Vol 113 (9) ◽  
pp. 1553-1564 ◽  
Author(s):  
M.J. Flick ◽  
S.F. Konieczny
Keyword(s):  
Structural Integrity ◽  
Striated Muscle ◽  
Myogenic Differentiation ◽  
Specific Factor ◽  
Contractile Apparatus ◽  
Structural Protein ◽  
Biochemical Analyses ◽  
Myogenic Program

Muscle LIM protein (MLP) is a striated muscle-specific factor that enhances myogenic differentiation and is critical to maintaining the structural integrity of the contractile apparatus. The ability of MLP to regulate myogenesis is particularly interesting since it exhibits multiple subcellular localizations, being found in both nuclear and cytoplasmic compartments. Despite extensive biochemical analyses on MLP, the mechanism(s) by which it influences the myogenic program remains largely undefined. To further examine the role of MLP as a positive myogenic regulator, a yeast two-hybrid screen was employed to identify cytoplasmic-associated MLP binding partners. From this screen, the cytoskeletal protein betaI-spectrin was isolated. Protein interaction assays demonstrate that MLP and betaI-spectrin associate with one another in vivo as well as when tested under several in vitro binding conditions. betaI-spectrin binds specifically to MLP but not to the MLP related proteins CRP1 and CRP2 or to other LIM domain containing proteins. The MLP:beta-spectrin interaction is mediated by the second LIM motif of MLP and by repeat 7 of beta-spectrin. Confocal microscopy studies also reveal that MLP co-localizes with beta-spectrin at the sarcolemma overlying the Z- and M-lines of myofibrils in both cardiac and skeletal muscle tissue. Given that beta-spectrin is a known costamere protein, we propose that sarcolemma-associated MLP also serves as a key costamere protein, stabilizing the association of the contractile apparatus with the sarcolemma by linking the beta-spectrin network to the alpha-actinin crosslinked actin filaments of the myofibril.

scholarly journals Heterodimer Formation of the Homodimeric ABC Transporter OpuA

2021 ◽  
Vol 22 (11) ◽  
pp. 5912
Author(s):  
Patricia Alvarez-Sieiro ◽  
Hendrik R. Sikkema ◽  
Bert Poolman
Keyword(s):  
Abc Transporter ◽  
Conformational Dynamics ◽  
Host Organism ◽  
Affinity Tag ◽  
Practical Applications ◽  
Fundamental Research ◽  
Protein Multimerization ◽  
Biochemical Analyses

Many proteins have a multimeric structure and are composed of two or more identical subunits. While this can be advantageous for the host organism, it can be a challenge when targeting specific residues in biochemical analyses. In vitro splitting and re-dimerization to circumvent this problem is a tedious process that requires stable proteins. We present an in vivo approach to transform homodimeric proteins into apparent heterodimers, which then can be purified using two-step affinity-tag purification. This opens the door to both practical applications such as smFRET to probe the conformational dynamics of homooligomeric proteins and fundamental research into the mechanism of protein multimerization, which is largely unexplored for membrane proteins. We show that expression conditions are key for the formation of heterodimers and that the order of the differential purification and reconstitution of the protein into nanodiscs is important for a functional ABC-transporter complex.

scholarly journals Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis

2011 ◽  
Vol 80 (1) ◽  
pp. 3-13 ◽  
Author(s):  
Chen Li ◽  
Kurniyati ◽  
Bo Hu ◽  
Jiang Bian ◽  
Jianlan Sun ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Biofilm Formation ◽  
Adult Population ◽  
Transcriptional Analysis ◽  
Wild Type ◽  
Content Type ◽  
Multiple Organs ◽  
Biochemical Analyses

ABSTRACTThe oral bacteriumPorphyromonas gingivalisis a key etiological agent of human periodontitis, a prevalent chronic disease that affects up to 80% of the adult population worldwide.P. gingivalisexhibits neuraminidase activity. However, the enzyme responsible for this activity, its biochemical features, and its role in the physiology and virulence ofP. gingivalisremain elusive. In this report, we found thatP. gingivalisencodes a neuraminidase, PG0352 (SiaPg). Transcriptional analysis showed thatPG0352is monocistronic and is regulated by a sigma70-like promoter. Biochemical analyses demonstrated that SiaPgis an exo-α-neuraminidase that cleaves glycosidic-linked sialic acids. Cryoelectron microscopy and tomography analyses revealed that thePG0352deletion mutant (ΔPG352) failed to produce an intact capsule layer. Compared to the wild type,in vitrostudies showed that ΔPG352 formed less biofilm and was less resistant to killing by the host complement.In vivostudies showed that while the wild type caused a spreading type of infection that affected multiple organs and all infected mice were killed, ΔPG352 only caused localized infection and all animals survived. Taken together, these results demonstrate that SiaPgis an important virulence factor that contributes to the biofilm formation, capsule biosynthesis, and pathogenicity ofP. gingivalis, and it can potentially serve as a new target for developing therapeutic agents againstP. gingivalisinfection.

scholarly journals Interaction of Proteus mirabilis Urease Apoenzyme and Accessory Proteins Identified with Yeast Two-Hybrid Technology

2001 ◽  
Vol 183 (4) ◽  
pp. 1423-1433 ◽  
Author(s):  
Susan R. Heimer ◽  
Harry L. T. Mobley
Keyword(s):  
Proteus Mirabilis ◽  
Accessory Proteins ◽  
Structural Protein ◽  
Yeast Two Hybrid ◽  
Nickel Ions ◽  
Catalytically Active ◽  
Tract Infections ◽  
Two Hybrid

ABSTRACT Proteus mirabilis, a gram-negative bacterium associated with complicated urinary tract infections, produces a metalloenzyme urease which hydrolyzes urea to ammonia and carbon dioxide. The apourease is comprised of three structural subunits, UreA, UreB, and UreC, assembled as a homotrimer of individual UreABC heterotrimers (UreABC)3. To become catalytically active, apourease acquires divalent nickel ions through a poorly understood process involving four accessory proteins, UreD, UreE, UreF, and UreG. While homologues of UreD, UreF, and UreG have been copurified with apourease, it remains unclear specifically how these polypeptides associate with the apourease or each other. To identify interactions among P. mirabilis accessory proteins, in vitro immunoprecipitation and in vivo yeast two-hybrid assays were employed. A complex containing accessory protein UreD and structural protein UreC was isolated by immunoprecipitation and characterized with immunoblots. This association occurs independently of coaccessory proteins UreE, UreF, and UreG and structural protein UreA. In a yeast two-hybrid screen, UreD was found to directly interact in vivo with coaccessory protein UreF. Unique homomultimeric interactions of UreD and UreF were also detected in vivo. To substantiate the study of urease proteins with a yeast two-hybrid assay, previously described UreE dimers and homomultimeric UreA interactions among apourease trimers were confirmed in vivo. Similarly, a known structural interaction involving UreA and UreC was also verified. This report suggests that in vivo, P. mirabilis UreD may be important for recruitment of UreF to the apourease and that crucial homomultimeric associations occur among these accessory proteins.

scholarly journals TRIM72 is required for effective repair of alveolar epithelial cell wounding

2014 ◽  
Vol 307 (6) ◽  
pp. L449-L459 ◽  
Author(s):  
Seong Chul Kim ◽  
Thomas Kellett ◽  
Shaohua Wang ◽  
Miyuki Nishi ◽  
Nagaraja Nagre ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Molecular Mechanisms ◽  
Striated Muscle ◽  
Alveolar Epithelial Cell ◽  
Alveolar Epithelial Cells ◽  
Lung Cells ◽  
Alveolar Epithelial ◽  
High Tidal Volume Ventilation

The molecular mechanisms for lung cell repair are largely unknown. Previous studies identified tripartite motif protein 72 (TRIM72) from striated muscle and linked its function to tissue repair. In this study, we characterized TRIM72 expression in lung tissues and investigated the role of TRIM72 in repair of alveolar epithelial cells. In vivo injury of lung cells was introduced by high tidal volume ventilation, and repair-defective cells were labeled with postinjury administration of propidium iodide. Primary alveolar epithelial cells were isolated and membrane wounding and repair were labeled separately. Our results show that absence of TRIM72 increases susceptibility to deformation-induced lung injury whereas TRIM72 overexpression is protective. In vitro cell wounding assay revealed that TRIM72 protects alveolar epithelial cells through promoting repair rather than increasing resistance to injury. The repair function of TRIM72 in lung cells is further linked to caveolin 1. These data suggest an essential role for TRIM72 in repair of alveolar epithelial cells under plasma membrane stress failure.

scholarly journals Functional Organization of the Yeast SAGA Complex: Distinct Components Involved in Structural Integrity, Nucleosome Acetylation, and TATA-Binding Protein Interaction

1999 ◽  
Vol 19 (1) ◽  
pp. 86-98 ◽  
Author(s):  
David E. Sterner ◽  
Patrick A. Grant ◽  
Shannon M. Roberts ◽  
Laura J. Duggan ◽  
Rimma Belotserkovskaya ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Structural Integrity ◽  
Functional Organization ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Saga Complex ◽  
Specific Domain ◽  
Nucleosomal Histone

ABSTRACT SAGA, a recently described protein complex in Saccharomyces cerevisiae, is important for transcription in vivo and possesses histone acetylation function. Here we report both biochemical and genetic analyses of members of three classes of transcription regulatory factors contained within the SAGA complex. We demonstrate a correlation between the phenotypic severity of SAGA mutants and SAGA structural integrity. Specifically, null mutations in the Gcn5/Ada2/Ada3 or Spt3/Spt8 classes cause moderate phenotypes and subtle structural alterations, while mutations in a third subgroup, Spt7/Spt20, as well as Ada1, disrupt the complex and cause severe phenotypes. Interestingly, double mutants (gcn5Δ spt3Δand gcn5Δ spt8Δ) causing loss of a member of each of the moderate classes have severe phenotypes, similar tospt7Δ, spt20Δ, or ada1Δmutants. In addition, we have investigated biochemical functions suggested by the moderate phenotypic classes and find that first, normal nucleosomal acetylation by SAGA requires a specific domain of Gcn5, termed the bromodomain. Deletion of this domain also causes specific transcriptional defects at the HIS3 promoter in vivo. Second, SAGA interacts with TBP, the TATA-binding protein, and this interaction requires Spt8 in vitro. Overall, our data demonstrate that SAGA harbors multiple, distinct transcription-related functions, including direct TBP interaction and nucleosomal histone acetylation. Loss of either of these causes slight impairment in vivo, but loss of both is highly detrimental to growth and transcription.

Genetic interaction between transcription elongation factor TFIIS and RNA polymerase II

1992 ◽  
Vol 12 (9) ◽  
pp. 4142-4152
Author(s):  
J Archambault ◽  
F Lacroute ◽  
A Ruet ◽  
J D Friesen
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Structural Integrity ◽  
Genetic Interaction ◽  
Elongation Factor ◽  
Chemical Mutagenesis ◽  
Yeast Genome ◽  
Transcript Elongation

Little is known about the regions of RNA polymerase II (RNAPII) that are involved in the process of transcript elongation and interaction with elongation factors. One elongation factor, TFIIS, stimulates transcript elongation by binding to RNAPII and facilitating its passage through intrinsic pausing sites in vitro. In Saccharomyces cerevisiae, TFIIS is encoded by the PPR2 gene. Deletion of PPR2 from the yeast genome is not lethal but renders cells sensitive to the uracil analog 6-azauracil (6AU). Here, we show that mutations conferring 6AU sensitivity can also be isolated in the gene encoding the largest subunit of S. cerevisiae RNAPII (RPO21). A screen for mutations in RPO21 that confer 6AU sensitivity identified seven mutations that had been generated by either linker-insertion or random chemical mutagenesis. All seven mutational alterations are clustered within one region of the largest subunit that is conserved among eukaryotic RNAPII. The finding that six of the seven rpo21 mutants failed to grow at elevated temperature underscores the importance of this region for the functional and/or structural integrity of RNAPII. We found that the 6AU sensitivity of the rpo21 mutants can be suppressed by increasing the dosage of the wild-type PPR2 gene, presumably as a result of overexpression of TFIIS. These results are consistent with the proposal that in the rpo21 mutants, the formation of the RNAPII-TFIIS complex is rate limiting for the passage of the mutant enzyme through pausing sites. In addition to implicating a region of the largest subunit of RNAPII in the process of transcript elongation, our observations provide in vivo evidence that TFIIS is involved in transcription by RNAPII.

New Diagnostic and Monitoring Method for Osteoporosis

2017 ◽  
pp. 1851-1884
Author(s):  
Sofia Panteliou
Keyword(s):  
Bone Quality ◽  
Structural Integrity ◽  
Assessment Tool ◽  
Bone Diseases ◽  
Damping Factor ◽  
In Vivo Measurement ◽  
Modal Damping ◽  
Invasive Method

Osteoporosis is chronic disease affecting most postmenopausal females and 30% of males with biological, behavioral and financial consequences. A non invasive method to assess bone structural integrity is presented, based on in-vitro or in-vivo measurement of bone dynamic characteristics (Modal Damping Factor) by applying vibration excitation in the range of acoustic frequencies, in the form of an acoustic sweep signal. This method has been applied on metallic structures and composites, including bones, and is supported by analytical and arithmetic tool based on model's theory. Experimental MDF results are compared to results acquired with conventional methods for bone quality assessment and show impressive correlations between damping factor and indices of bone quality in an advantageous manner. Evaluation of these research findings strengthens the potential of the proposed method to consist a valuable assessment tool for diagnosis and monitoring of bone integrity, in metabolic bone diseases, especially osteoporosis.

scholarly journals The C-terminal region of the plasmid partitioning protein TubY is a tetramer that can bind membranes and DNA

2020 ◽  
Vol 295 (51) ◽  
pp. 17770-17780
Author(s):  
Ikuko Hayashi
Keyword(s):  
Lipid Membranes ◽  
Binding Protein ◽  
Coiled Coil ◽  
Type Iii ◽  
Daughter Cells ◽  
Lysine Residues ◽  
Stable Maintenance ◽  
Biochemical Analyses

Bacterial low-copy-number plasmids require partition (par) systems to ensure their stable inheritance by daughter cells. In general, these systems consist of three components: a centromeric DNA sequence, a centromere-binding protein and a nucleotide hydrolase that polymerizes and functions as a motor. Type III systems, however, segregate plasmids using three proteins: the FtsZ/tubulin-like GTPase TubZ, the centromere-binding protein TubR and the MerR-like transcriptional regulator TubY. Although the TubZ filament is sufficient to transport the TubR-centromere complex in vitro, TubY is still necessary for the stable maintenance of the plasmid. TubY contains an N-terminal DNA-binding helix-turn-helix motif and a C-terminal coiled-coil followed by a cluster of lysine residues. This study determined the crystal structure of the C-terminal domain of TubY from the Bacillus cereus pXO1-like plasmid and showed that it forms a tetrameric parallel four-helix bundle that differs from the typical MerR family proteins with a dimeric anti-parallel coiled-coil. Biochemical analyses revealed that the C-terminal tail with the conserved lysine cluster helps TubY to stably associate with the TubR-centromere complex as well as to nonspecifically bind DNA. Furthermore, this C-terminal tail forms an amphipathic helix in the presence of lipids but must oligomerize to localize the protein to the membrane in vivo. Taken together, these data suggest that TubY is a component of the nucleoprotein complex within the partitioning machinery, and that lipid membranes act as mediators of type III systems.

scholarly journals Conditional Deletion of Dicer in Adult Mice Impairs Skeletal Muscle Regeneration

2019 ◽  
Vol 20 (22) ◽  
pp. 5686 ◽  
Author(s):  
Satoshi Oikawa ◽  
Minjung Lee ◽  
Takayuki Akimoto
Keyword(s):  
Skeletal Muscle ◽  
Muscle Regeneration ◽  
Myogenic Differentiation ◽  
Tibialis Anterior Muscle ◽  
Critical Factor ◽  
Skeletal Muscle Regeneration ◽  
Small Noncoding Rnas ◽  
Adult Mice

Skeletal muscle has a remarkable regenerative capacity, which is orchestrated by multiple processes, including the proliferation, fusion, and differentiation of the resident stem cells in muscle. MicroRNAs (miRNAs) are small noncoding RNAs that mediate the translational repression or degradation of mRNA to regulate diverse biological functions. Previous studies have suggested that several miRNAs play important roles in myoblast proliferation and differentiation in vitro. However, their potential roles in skeletal muscle regeneration in vivo have not been fully established. In this study, we generated a mouse in which the Dicer gene, which encodes an enzyme essential in miRNA processing, was knocked out in a tamoxifen-inducible way (iDicer KO mouse) and determined its regenerative potential after cardiotoxin-induced acute muscle injury. Dicer mRNA expression was significantly reduced in the tibialis anterior muscle of the iDicer KO mice, whereas the expression of muscle-enriched miRNAs was only slightly reduced in the Dicer-deficient muscles. After cardiotoxin injection, the iDicer KO mice showed impaired muscle regeneration. We also demonstrated that the number of PAX7+ cells, cell proliferation, and the myogenic differentiation capacity of the primary myoblasts did not differ between the wild-type and the iDicer KO mice. Taken together, these data demonstrate that Dicer is a critical factor for muscle regeneration in vivo.

scholarly journals A Glance at the Nuclear Envelope Spectrin Repeat Protein 3

2019 ◽  
Vol 2019 ◽  
pp. 1-8
Author(s):  
Liwei Liao ◽  
Rongmei Qu ◽  
Jun Ouang ◽  
Jingxing Dai
Keyword(s):  
Nuclear Envelope ◽  
Active Role ◽  
Structural Protein ◽  
Linc Complex ◽  
Repeat Protein ◽  
Spectrin Repeat ◽  
Physiological Importance ◽  
Functional Perspective

Nuclear envelope spectrin repeat protein 3 (nesprin-3) is an evolutionarily-conserved structural protein, widely-expressed in vertebrate cells. Along with other nesprin family members, nesprin-3 acts as an essential component of the linker of nucleoskeleton and cytoskeleton (LINC) complex. Naturally, nesprin-3 shares many functions with LINC, including the localization of various cellular structures and bridging of the nucleoskeleton and cytoskeleton, observed in vitro. When nesprin-3 was knocked down in vivo, using zebrafish and mouse models, however, the animals were minimally affected. This paradoxical observation should not limit the physiological importance of nesprin-3, as recently, nesprin-3 has reignited the interest of the research community in studies on cancer cells migration. Moreover, nesprin-3 also plays an active role in certain developmental conditions such as adipogenesis and spermatogenesis, although more studies are needed. Meanwhile, the various protein binding partners of nesprin-3 should also be emphasized, as they are necessary for maintaining the structure of nesprin-3 and enabling it to carry out its various physiological and pathological functions. Nesprin-3 promises to further our understanding of these complex cellular events. Therefore, this review will focus on nesprin-3, examining it from a genetic, structural, and functional perspective. The final part of the review will in turn address the limitations of existing research and the future perspectives for the study of nesprin-3.

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