Movement protein of hordeivirus interacts in vitro and in vivo with coilin, a major structural protein of Cajal bodies

ABSTRACT The cell-to-cell spread of Tobacco mosaic virus infection depends on virus-encoded movement protein (MP), which is believed to form a ribonucleoprotein complex with viral RNA (vRNA) and to participate in the intercellular spread of infectious particles through plasmodesmata. Previous studies in our laboratory have provided evidence that the vRNA movement process is correlated with the ability of the MP to interact with microtubules, although the exact role of this interaction during infection is not known. Here, we have used a variety of in vivo and in vitro assays to determine that the MP functions as a genuine microtubule-associated protein that binds microtubules directly and modulates microtubule stability. We demonstrate that, unlike MP in whole-cell extract, microtubule-associated MP is not ubiquitinated, which strongly argues against the hypothesis that microtubules target the MP for degradation. In addition, we found that MP interferes with kinesin motor activity in vitro, suggesting that microtubule-associated MP may interfere with kinesin-driven transport processes during infection.

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

Journal of Bacteriology ◽

10.1128/jb.183.4.1423-1433.2001 ◽

2001 ◽

Vol 183 (4) ◽

pp. 1423-1433 ◽

Cited By ~ 28

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.

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A Glance at the Nuclear Envelope Spectrin Repeat Protein 3

BioMed Research International ◽

10.1155/2019/1651805 ◽

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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Identification of overlapping DNA-binding and centromere-targeting domains in the human kinetochore protein CENP-C.

Molecular and Cellular Biology ◽

10.1128/mcb.16.7.3576 ◽

1996 ◽

Vol 16 (7) ◽

pp. 3576-3586 ◽

Cited By ~ 77

Author(s):

C H Yang ◽

J Tomkiel ◽

H Saitoh ◽

D H Johnson ◽

W C Earnshaw

Keyword(s):

Dna Binding ◽

Chromosome Segregation ◽

Mitotic Spindle ◽

Mammalian Cells ◽

In Vitro Assays ◽

Centromeric Heterochromatin ◽

Structural Protein ◽

Chromosomal Site

The kinetochore in eukaryotes serves as the chromosomal site of attachment for microtubules of the mitotic spindle and directs the movements necessary for proper chromosome segregation. In mammalian cells, the kinetochore is a highly differentiated trilaminar structure situated at the surface of the centromeric heterochromatin. CENP-C is a basic, DNA-binding protein that localizes to the inner kinetochore plate, the region that abuts the heterochromatin. Microinjection experiments using antibodies specific for CENP-C have demonstrated that this protein is required for the assembly and/or stability of the kinetochore as well as for a timely transition through mitosis. From these observations, it has been suggested that CENP-C is a structural protein that is involved in the organization or the kinetochore. In this report, we wished to identify and map the functional domains of CENP-C. Analysis of CENP-C truncation mutants expressed in vivo demonstrated that CENP-C possesses an autonomous centromere-targeting domain situated at the central region of the CENP-C polypeptide. Similarly, in vitro assays revealed that a region of CENP-C with the ability to bind DNA is also located at the center of the CENP-C molecule, where it overlaps the centromere-targeting domain.

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Combined structural, biochemical and cellular evidence demonstrates that both FGDF motifs in alphavirus nsP3 are required for efficient replication

Open Biology ◽

10.1098/rsob.160078 ◽

2016 ◽

Vol 6 (7) ◽

pp. 160078 ◽

Cited By ~ 20

Author(s):

Tim Schulte ◽

Lifeng Liu ◽

Marc D. Panas ◽

Bastian Thaa ◽

Nicole Dickson ◽

...

Keyword(s):

Viral Replication ◽

Semliki Forest Virus ◽

Protein Structures ◽

Growth Curves ◽

Structural Protein ◽

Binding Studies ◽

Efficient Replication ◽

Hierarchical Interaction

Recent findings have highlighted the role of the Old World alphavirus non-structural protein 3 (nsP3) as a host defence modulator that functions by disrupting stress granules, subcellular phase-dense RNA/protein structures formed upon environmental stress. This disruption mechanism was largely explained through nsP3-mediated recruitment of the host G3BP protein via two tandem FGDF motifs. Here, we present the 1.9 Å resolution crystal structure of the NTF2-like domain of G3BP-1 in complex with a 25-residue peptide derived from Semliki Forest virus nsP3 (nsP3-25). The structure reveals a poly-complex of G3BP-1 dimers interconnected through the FGDF motifs in nsP3-25. Although in vitro and in vivo binding studies revealed a hierarchical interaction of the two FGDF motifs with G3BP-1, viral growth curves clearly demonstrated that two intact FGDF motifs are required for efficient viral replication. Chikungunya virus nsP3 also binds G3BP dimers via a hierarchical interaction, which was found to be critical for viral replication. These results highlight a conserved molecular mechanism in host cell modulation.

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Asthenozoospermia in Mice with Targeted Deletion of the Sperm Mitochondrion-Associated Cysteine-Rich Protein (Smcp) Gene

Molecular and Cellular Biology ◽

10.1128/mcb.22.9.3046-3052.2002 ◽

2002 ◽

Vol 22 (9) ◽

pp. 3046-3052 ◽

Cited By ~ 74

Author(s):

Karim Nayernia ◽

Ibrahim M. Adham ◽

Elke Burkhardt-Göttges ◽

Jürgen Neesen ◽

Mandy Rieche ◽

...

Keyword(s):

Semen Analysis ◽

Computer Assisted ◽

Microscopical Examination ◽

Structural Protein ◽

Wild Type ◽

Vitro Fertilization ◽

In Vivo Experiments ◽

Cysteine Rich Protein

ABSTRACT The sperm mitochondria-associated cysteine-rich protein (SMCP) is a cysteine- and proline-rich structural protein that is closely associated with the keratinous capsules of sperm mitochondria in the mitochondrial sheath surrounding the outer dense fibers and axoneme. To investigate the function of SMCP, we generated mice with a targeted disruption of the gene Smcp by homologous recombination. Homozygous mutant males on a mixed genetic background (C57BL/6J × 129/Sv) are fully fertile, while they are infertile on the 129/Sv background, although spermatogenesis and mating are normal. Homozygous Smcp−/− female mice are fertile on both genetic backgrounds. Electron microscopical examination demonstrated normal structures of sperm head, mitochondria, and tail. In vivo experiments with sperm of Smcp−/− 129/Sv mice revealed that the migration of spermatozoa from the uterus into the oviduct is reduced. This result is supported by the observation that sperm motility as determined by the computer-assisted semen analysis system (CASA) is significantly affected as compared to wild-type spermatozoa. In vitro fertilization assays showed that Smcp-deficient spermatozoa are able to bind to the oocyte but that the number of fertilized eggs is reduced by more than threefold relative to the wild-type control. However, removal of the zona pellucida resulted in an unaffected sperm-egg fusion which was monitored by the presence of pronuclei and generation of blastocyts. These results indicate that the infertility of the male Smcp−/− mice on the 129/Sv background is due to reduced motility of the spermatozoa and decreased capability of the spermatozoa to penetrate oocytes.

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Keratin-Based Nanoparticles as Drug Delivery Carriers

Applied Sciences ◽

10.3390/app11209417 ◽

2021 ◽

Vol 11 (20) ◽

pp. 9417

Author(s):

Claudia Ferroni ◽

Greta Varchi

Keyword(s):

Chemical Properties ◽

Biological Data ◽

The Body ◽

Structural Description ◽

Structural Protein ◽

Antimicrobial Drugs ◽

Biological Environment ◽

Comprehensive Framework

Keratin is a structural protein of mammalian tissues and birds, representing the principal constituent of hair, nails, skin, wool, hooves, horns, beaks, and feathers, and playing an essential role in protecting the body from external harassment. Due to its intrinsic features such as biocompatibility, biodegradability, responsiveness to specific biological environment, and physical–chemical properties, keratin has been extensively explored in the production of nanocarriers of active principles for different biomedical applications. In the present review paper, we aimed to give a literature overview of keratin-based nanoparticles produced starting from human hair, wool, and chicken feathers. Along with the chemical and structural description of keratin nanoparticles, selected in vitro and in vivo biological data are also discussed to provide a more comprehensive framework of possible fields of application of this protein. Despite the considerable number of papers describing the production and use of keratin nanoparticles as carries of anticancer and antimicrobial drugs or as hemostatic and wound healing materials, still, efforts are needed to implement keratin nanoparticles towards their clinical application.

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Duck enteritis virus UL21 is a late gene and encodes a protein that interacts with pUL16

10.21203/rs.2.17186/v2 ◽

2019 ◽

Author(s):

Linjiang Yang ◽

Mingshu Wang ◽

Chunhui Zeng ◽

Yong Shi ◽

Anchun Cheng ◽

...

Keyword(s):

Pseudorabies Virus ◽

Rna Binding ◽

Gene Interaction ◽

Duck Enteritis Virus ◽

Late Gene ◽

Structural Protein ◽

Virus Transport ◽

Enteritis Virus

Abstract Background pUL21 is a conserved protein of Alphaherpesvirinae that performs multiple important functions. The C-terminus of pUL21 in other members of this subfamily has RNA-binding ability; this domain contributes to pseudorabies virus (PRV) retrograde axonal transport in vitro and in vivo and participates in newly replicated viral DNA packaging and intracellular virus transport. However, knowledge regarding duck enteritis virus (DEV) pUL21 is limited. Methods In our study, recombinant pUL21 was expressed using an pET-32c (+) vector in Escherichia coli BL21 cells induced with 0.4 mM isopropyl β-D-thiogalactoside for 8 h at 30°C. The antibody used for the indirect immunofluorescence (IFA) and western blotting (WB) analysis were prepared. Pharmacological inhibition, WB and quantitative reverse transcription PCR (RT-qPCR) were performed. A coimmunoprecipitation (CO-IP) assay was conducted to test the interaction between pUL21 and pUL16. Results We verified that DEV UL21 is a γ2 gene that encodes a structural protein. Moreover, we observed that pUL21 localized to the nucleus and cytoplasm. DEV pUL21 interacted with pUL16 and formed a complex in transfected human embryonic kidney (HEK) 293T cells and DEV-infected duck embryo fibroblasts (DEFs). These results were further confirmed by CO-IP assays. Conclusions The DEV UL21 gene is a late gene, and pUL21 localizes to the nucleus and cytoplasm. DEV UL21 is a virion component. In addition, pUL21 can interact with pUL16. These ﬁndings provide insight into the characteristics of UL21 and the interaction between pUL21 and its binding partner pUL16. Our study enhances the understanding of DEV pUL21. Keywords: Duck enteritis virus, UL21, UL16, late gene, interaction

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Phase separation of both a plant virus movement protein and cellular factors support virus-host interactions

10.1101/2021.05.11.443547 ◽

2021 ◽

Author(s):

Shelby L Brown ◽

Jared P. May

Keyword(s):

Phase Separation ◽

Mosaic Virus ◽

Self Assembly ◽

Electrostatic Interactions ◽

Movement Protein ◽

Virus Movement ◽

In Planta ◽

Ribonucleoprotein Complexes

Phase separation concentrates biomolecules, which should benefit RNA viruses that must sequester viral and host factors during an infection. Here, the p26 movement protein from Pea enation mosaic virus 2 (PEMV2) was found to phase separate and partition in nucleoli and G3BP stress granules (SGs) in vivo . Electrostatic interactions drive p26 phase separation as mutation of basic (R/K-G) or acidic (D/E-G) residues either blocked or reduced phase separation, respectively. During infection, p26 must partition inside the nucleolus and interact with fibrillarin (Fib2) as a pre-requisite for systemic trafficking of viral RNAs. Partitioning of p26 in pre-formed Fib2 droplets was dependent on p26 phase separation suggesting that phase separation of viral movement proteins supports nucleolar partitioning and virus movement. Furthermore, viral ribonucleoprotein complexes containing p26, Fib2, and PEMV2 RNA were formed via phase separation in vitro and could provide the basis for self-assembly in planta . Interestingly, both R/K-G and D/E-G p26 mutants failed to support systemic trafficking of a Tobacco mosaic virus (TMV) vector in Nicotiana benthamiana suggesting that p26 phase separation, proper nucleolar partitioning, and systemic movement are intertwined. p26 also partitioned in SGs and G3BP over-expression restricted PEMV2 accumulation >20-fold. Expression of phase separation-deficient G3BP only restricted PEMV2 5-fold, demonstrating that G3BP phase separation is critical for maximum antiviral activity.

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