Assembly Of Structural Proteins Research Articles

Phosphoproteomic insight into the changes in structural proteins of muscle architecture associated with texture softening of grass carp (Ctenopharyngodon idella) fillets during chilling storage.

Texture is an important sensory attribute of fish affected by modifications of structural proteins in muscle architecture. To investigate the changes in protein phosphorylation during texture softening of fish, the proteins of grass carp muscle after chilling storage of 0day and 6days were compared by phosphoproteomics, and their association with texture was analyzed. Totally 1026 unique phosphopeptides on 656 phosphoproteins were identified as differential. They were mainly classified as intracellular myofibril and cytoskeleton, and extracellular matrix, of which the molecular function and biological process were binding into supramolecular assembly and myofilament contraction. The concomitant dephosphorylation of kinases and assembly regulators indicated dephosphorylation and disassembly tendency of sarcomeric architecture. Correlation analysis defined the relation between texture and dephosphorylation of myosin light chain, actin, collagen and cytoskeleton. This study revealed that protein phosphorylation may affect the texture of fish muscle through regulating sarcomeric assembly of structural proteins in muscle architecture.

Synthesis and evaluation of 2-pyridinylpyrimidines as inhibitors of HIV-1 structural protein assembly

In an effort to identify an HIV-1 capsid assembly inhibitor with improved solubility and potency, we synthesized two series of pyrimidine analogues based on our earlier lead compound N-(4-(ethoxycarbonyl)phenyl)-2-(pyridine-4-yl)quinazoline-4-amine. In vitro binding experiments showed that our series of 2-pyridine-4-ylpyrimidines had IC50 values higher than 28μM. Our series of 2-pyridine-3-ylpyrimidines exhibited IC50 values ranging from 3 to 60μM. The congeners with a fluoro substituent introduced at the 4-N-phenyl moiety, along with a methyl at C-6, represent potent HIV capsid assembly inhibitors binding to the C-terminal domain of the capsid protein.

Strain-enhanced stress relaxation impacts nonlinear elasticity in collagen gels

The extracellular matrix (ECM) is a complex assembly of structural proteins that provides physical support and biochemical signaling to cells in tissues. The mechanical properties of the ECM have been found to play a key role in regulating cell behaviors such as differentiation and malignancy. Gels formed from ECM protein biopolymers such as collagen or fibrin are commonly used for 3D cell culture models of tissue. One of the most striking features of these gels is that they exhibit nonlinear elasticity, undergoing strain stiffening. However, these gels are also viscoelastic and exhibit stress relaxation, with the resistance of the gel to a deformation relaxing over time. Recent studies have suggested that cells sense and respond to both nonlinear elasticity and viscoelasticity of ECM, yet little is known about the connection between nonlinear elasticity and viscoelasticity. Here, we report that, as strain is increased, not only do biopolymer gels stiffen but they also exhibit faster stress relaxation, reducing the timescale over which elastic energy is dissipated. This effect is not universal to all biological gels and is mediated through weak cross-links. Mechanistically, computational modeling and atomic force microscopy (AFM) indicate that strain-enhanced stress relaxation of collagen gels arises from force-dependent unbinding of weak bonds between collagen fibers. The broader effect of strain-enhanced stress relaxation is to rapidly diminish strain stiffening over time. These results reveal the interplay between nonlinear elasticity and viscoelasticity in collagen gels, and highlight the complexity of the ECM mechanics that are likely sensed through cellular mechanotransduction.

Non-Enzymatic Remodeling of Fibrin Biopolymers via Photothermally Triggered Radical-Generating Nanoparticles

Thrombosis is a hallmark of several chronic diseases leading to potentially fatal heart attacks and strokes. Frontline interventions include intravenous delivery of potent, enzymatic fibrinolytics that possess a high risk for inducing systemic bleeding. As a conceptual countermeasure, we have developed a water-soluble PEGylated gold nanoparticle appended with the enediyne diamine (Z)-octa-4-en-2,6-diyne-1,8-diamine that is capable of photothermally generating 1,4-diradical species under visible excitation (λ = 514 nm, 100 mW, 2–6 h). In the absence of biopolymer substrate, photothermal excitation of these particles leads to self-quenching polymer coating formation in water. When these radical-generating nanoparticles are intrinsically applied toward the blood clot structural protein assembly fibrin, as well as its nonpolymerized precursor protein fibrinogen, scanning electron microscopy images reveal significantly modified fibrin clot morphology, as evidenced by larger void spaces and collapsed fiber regi...

Structural Aspects of the Interaction of Dairy Phages with Their Host Bacteria

Knowledge of phage-host interactions at a fundamental level is central to the design of rational strategies for the development of phage-resistant strains that may be applied in industrial settings. Phages infecting lactic acid bacteria, in particular Lactococcus lactis and Streptococcus thermophilus, negatively impact on dairy fermentation processes with serious economic implications. In recent years a wealth of information on structural protein assembly and topology has become available relating to phages infecting Escherichia coli, Bacillus subtilis and Lactococcus lactis, which act as models for structural analyses of dairy phages. In this review, we explore the role of model tailed phages, such as T4 and SPP1, in advancing our knowledge regarding interactions between dairy phages and their hosts. Furthermore, the potential of currently investigated dairy phages to in turn serve as model systems for this particular group of phages is discussed.

Mutations in the Sensitive Giant Titin Result in a Broken Heart

### Truncations of Titin Causing Dilated Cardiomyopathy Herman et al N Engl J Med. 2012;366:619–628. The specialized cytoskeleton of striated muscle cells consists of highly ordered structures, the single contractile units termed sarcomeres. Although once believed to be simply a scaffold for the assembly of structural proteins to generate force and motion, it is now widely recognized that the sarcomeric cytoskeleton is dynamic and is intimately involved in numerous cellular signaling processes that respond to a wide range of extracellular cues. Sarcomeres in vertebrates are built of an intricate array of myosin-containing thick filaments, actin-containing thin filaments, and titin filaments (Figure, A), along with a plethora of other structural and regulatory proteins. Titin (also known as connectin) is the largest protein discovered in humans to date.1,2 The human titin gene is enormous; at 363 exons, it encompasses the largest number of exons in any single gene and is predicted to encode up to 38 138 residues, or a 4.2-MDa protein.3 Remarkably, a single molecule of titin spans half the length of a sarcomere with its NH2 terminus embedded in the Z-disc and its COOH terminus anchored at the center of the sarcomere, the M-line (Figure, A). This corresponds to a molecular length of approximately 0.9 to >1.5 μm, depending on sarcomere stretch! Because titin filaments from opposite half-sarcomeres fully overlap in the Z-discs and at the M-line, titin filaments form a continuous system within myofibrils. Figure. Striated muscle sarcomere (Panel A) with locations of titin mutations linked to human dilated cardiomyopathy (Panel B). Mutations are markedly missing from the Z disc and M line; they are highly enriched in the thick filament associated A band region. While some patients presented with identical mutations, numbers reflect unique mutations. Although many unique functions for titin have been described, it is most known for its function as a molecular spring; it limits …

Effect of deoxynojirimycin derivatives on morphogenesis of hepatitis C virus

Viral hepatitis C is a dangerous, widespread human disease. The choice of drugs for treatment of chronic hepatitis C virus (HCV) infection is limited, and prophylactic vaccines do not exist. Thus, the development of new antiviral strategies and substances is an issue of great importance. The targeting of viral morphogenesis might be used as an alternative approach to existing strategies of HCV blocking. The glycosylation of viral envelope proteins is an important step of viral particle morphogenesis, which determines the correct assembly of HCV virions. Derivatives of a glucose analog deoxynojirimycin (DNJ) act as an α-glucosidase inhibitor and can impair the assembly of structural proteins and HCV particle formation. In the present work, the effects of alkylated DNJ derivatives, N-pentyl-DNJ and N-benzyl-DNJ, on HCV morphogenesis were studied in a model system of insect cells that produce three viral structural proteins with the formation of virus-like particles. It was shown that DNJ derivatives impair the intracellular N-glycosylation of HCV envelope glycoproteins. At the concentration of 1 mM, these substances cause an increase in the levels of gpE1 and gpE2 glycoproteins and a decrease in their electrophoretic mobility, apparently due to the inhibition of α-glucosidase in the endoplasmic reticulum and the accumulation of hyperglycosylated N-glycans in HCV glycoproteins. The interaction of the latter with calnexin results in the formation of unproductive dimers and blocks the productive assembly of virus-like particles.

Enterotoxigenic Escherichia coli CS6 gene products and their roles in CS6 structural protein assembly and cellular adherence

Enterotoxigenic Escherichia coli (ETEC) produces a variety of colonization factors necessary for attachment to the host cell, among which CS6 is one of the most prevalent in ETEC isolates from developing countries. The CS6 operon is composed of 4 genes, cssA, cssB, cssC, and cssD. The molecular mechanism of CS6 assembly and cell surface presentation, and the contribution of each protein to the attachment of the bacterium to intestinal cells remain unclear. In the present study, a series of css gene-deletion mutants of the CS6 operon were constructed in the ETEC genetic background, and their effect on adhesion to host cells and CS6 assembly was studied. Each subunit deletion resulted in a reduction in the adhesion to intestinal cells to the same level of laboratory E. coli strains, and this effect was restored by complementary plasmids, suggesting that the 4 proteins are necessary for CS6 expression. Bacterial cell fractionation and western blotting of the mutant strains suggested that the formation of a CssA–CssB–CssC complex is necessary for recognition by CssD and transport of CssA–CssB to the outer membrane as a colonization factor.

Biophysical properties of normal and diseased renal glomeruli.

The mechanical properties of tissues and cells including renal glomeruli are important determinants of their differentiated state, function, and responses to injury but are not well characterized or understood. Understanding glomerular mechanics is important for understanding renal diseases attributable to abnormal expression or assembly of structural proteins and abnormal hemodynamics. We use atomic force microscopy (AFM) and a new technique, capillary micromechanics, to measure the elastic properties of rat glomeruli. The Young's modulus of glomeruli was 2,500 Pa, and it was reduced to 1,100 Pa by cytochalasin and latunculin, and to 1,400 Pa by blebbistatin. Cytochalasin or latrunculin reduced the F/G actin ratios of glomeruli but did not disrupt their architecture. To assess glomerular biomechanics in disease, we measured the Young's moduli of glomeruli from two mouse models of primary glomerular disease, Col4a3(-/-) mice (Alport model) and Tg26(HIV/nl) mice (HIV-associated nephropathy model), at stages where glomerular injury was minimal by histopathology. Col4a3(-/-) mice express abnormal glomerular basement membrane proteins, and Tg26(HIV/nl) mouse podocytes have multiple abnormalities in morphology, adhesion, and cytoskeletal structure. In both models, the Young's modulus of the glomeruli was reduced by 30%. We find that glomeruli have specific and quantifiable biomechanical properties that are dependent on the state of the actin cytoskeleton and nonmuscle myosins. These properties may be altered early in disease and represent an important early component of disease. This increased deformability of glomeruli could directly contribute to disease by permitting increased distension with hemodynamic force or represent a mechanically inhospitable environment for glomerular cells.

Structural changes in human cytomegalovirus cytoplasmic assembly sites in the absence of UL97 kinase activity

Studies of human cytomegalovirus (HCMV) UL97 kinase deletion mutant (ΔUL97) indicated a multi-step role for this kinase in early and late phases of the viral life cycle, namely, in DNA replication, capsid maturation and nuclear egress. Here, we addressed its possible involvement in cytoplasmic steps of HCMV assembly. Using the ΔUL97 and the UL97 kinase inhibitor NGIC-I, we demonstrate that the absence of UL97 kinase activity results in a modified subcellular distribution of the viral structural protein assembly sites, from compact structures impacting upon the nucleus to diffuse perinuclear structures punctuated by large vacuoles. Infection by either wild type or ΔUL97 viruses induced a profound reorganization of wheat germ agglutinin (WGA)-positive Golgi-related structures. Importantly, the viral-induced Golgi remodeling along with the reorganization of the nuclear architecture was substantially altered in the absence of UL97 kinase activity. These findings suggest that UL97 kinase activity might contribute to organization of the viral cytoplasmic assembly sites.

Construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivative.

The introduction of a label which can be detected in living cells opens new possibilities for the direct analysis of dynamic processes in virus replication, such as the transport and assembly of structural proteins. Our aim was to generate a tool for the analysis of the trafficking of the main structural protein of human immunodeficiency virus type 1 (HIV-1), Gag, as well as for the analysis of virus-host cell interactions in an authentic setting. We describe here the construction and characterization of infectious HIV derivatives carrying a label within the Gag polyprotein. Based on our initial finding that a short epitope tag could be inserted near the C terminus of the matrix domain of Gag without affecting viral replication, we constructed HIV derivatives carrying the egfp gene at the analogous position, resulting in the expression of a Gag-EGFP fusion protein in the authentic viral context. Particles displaying normal viral protein compositions were released from transfected cells, and Gag-EGFP was efficiently processed by the viral protease, yielding the expected products. Furthermore, particles with mature morphology were observed by thin-section electron microscopy. The modified virus was even found to be infectious, albeit with reduced relative infectivity. By preparing mixed particles containing equimolar amounts of Gag-EGFP and Gag, we were able to obtain highly fluorescently labeled virion preparations which displayed normal morphology and full wild-type infectivity, demonstrating that the process of HIV particle assembly displays a remarkable flexibility. The fluorescent virus derivative is a useful tool for investigating the interaction of HIV with live cells.

Novel multi-nonsense suppressor in bacteriophage T4D.

A novel bacteriophage T 4 D nonsense suppressor, psu + SB , has been isolated as a pseudo-wild type revertant of a multiple amber strain of bacteriophage T 4 D. The suppressor suppresses, with varying efficiencies, amber (UAG), ochre (UAA) and opal (UGA) nonsense mutations. Only suppression of mutations in late genes is demonstrated. Suppression by psu + SB is temperature sensitive. From the experimental data we have been able to obtain, the psu + SB locus appears to be located between genes 32 and 31 on the bacteriophage T 4 map, unlike previously reported phage suppressors, which map in the tRNA region. The psu + SB locus codes for a function that is essential for phage viability whether or not amber mutations are present on the same genome. The suppressor strain has a reduced burst size and a delayed time of lysis compared to wild type, and the suppressor function is dominant to wild type. Lysates of the suppressor strain examined under the electron microscope show abnormal assembly of structural proteins. Models explaining these data in terms of suppression are presented.

Characterization of three complementation groups of vesicular stomatitis virus

The complementation properties of 25 temperature-sensitive mutants of VSV have been tested by measuring the yield of virus in mixedly infected cells at the nonpermissive temperature, 38°C. Two of the mutants, ts10W and ts16BW, complement all the mutants except each other and therefore form one distinct complementation group, A. Two other mutants, ts12W and ts29W do not complement each other but complement nearly all the other mutants and have been assigned to a second complementatation group, B. A group of six mutants, all of which show an increased heat lability of the virion, show no complementation within the group and have been assigned to a third group, C. Most of the remaining mutants complement at least one member of each of groups A, B, and C but have not yet been assigned to other groups since many of the crosses of these mutants with group C mutants fail to show complementation. Virus-specific RNA synthesis in L cells infected with the various mutants was investigated by measuring the incorporation of 14C-labeled uridine. All the group C mutants show little or no virus-specific RNA synthesis at 38°C, even at high multiplicities, suggesting that in these mutants the defect is in the structural RNA polymerase. The two mutants of group A show little or no virus-specific RNA synthesis at 38°C at low input multiplicities but considerable RNA synthesis at high multiplicities. Since this synthesis is not reduced by the presence of puromycin it would appear that these mutants have a structural polymerase which is functional at 38°C but are defective in some other type of RNA synthesis. Mutants of group B show RNA synthesis at 38°C comparable to that of the wild type at all multiplicities but fail to produce infectious virus after a shift from 30 to 38°C at any time during the growth cycle. The defect in these mutants therefore appears to be in some function such as the synthesis or assembly of structural proteins.

Synthesis of viral ribonucleic acid during restricted adenovirus infection.

The mechanism by which simian virus 40 converts the abortive adenovirus type 7 infection of monkey cells into an efficient lytic infection has been investigated. Analysis of ribonucleic acid (RNA) synthesis during unenhanced and enhanced infection of monkey cells has shown that adenovirus RNA synthesized in the abortive infection contains both "early" and "late" sequences. In hybridization competition experiments, early adenovirus RNA from human cells prevented the hybridization of only 20% of the adenovirus RNA transcribed in unenhanced infection. Further, the RNA from unenhanced cells was able to completely block the hybridization of RNA synthesized during enhanced infection. Finally, virus-associated RNA, which is a late RNA transcribed in lytic adenovirus infection, is also produced in the unenhanced infection. An accompanying paper describes a marked deficiency in adenoviral capsid protein synthesis in the unenhanced infection. We conclude that RNA sequences, which are sufficient to code for the synthesis and assembly of structural proteins of adenovirus, are transcribed but are not efficiently translated in the unenhanced adenovirus infection of monkey cells.

Biogenesis of 50 s particles in exponentially growing Escherichia coli.

Abstract Synthesis and assembly of structural proteins into 50 s ribosomal subunits were investigated in exponentially growing Escherichia coli with a doubling time of 120 minutes. The study was designed to determine the rate at which pulse-labeled ribosomal proteins appeared on the 50 s subunit. Examination of the total ribosomal fraction for label following a one-minute pulse with [14C]leucine and subsequent chase with unlabeled leucine revealed that all of the labeled ribosomal protein appeared in the ribosomal fraction only after a chase period of approximately 30 minutes. A study of individual 50 s ribosomal proteins fractionated by polyacrylamide gel electrophoresis indicated that the rate of appearance of labeled proteins varied. The average half-life time of labeled ribosomal proteins prior to their appearance on 50 s subunits was found to be approximately five to six minutes. The half-life times of individual 50 s ribosomal proteins varied between two and eleven minutes. From the rate of appearance of labeled protein on the 50 s subunit, the amount of 50 s ribosomal protein not yet associated with mature 50 s subunits was estimated to be about 5% of the total cellular 50 s ribosomal protein. The amount of individual 50 s ribosomal proteins not associated with mature subunits varied from approximately 2 to 9%. Examination of the labeled proteins on the 50 s subunit after a 15-second pulse with [14C]leucine and no further incubation suggests that there exist at least three groups of 50 s ribosomal proteins added at different rates, consistent with the contention that proteins are added during the maturation of ribosomal subunits forming precursor particles and finally the mature 50 s subunit.

Synthesis and assembly of ribosomal protein into 50 s subunits during recovery from chloramphenicol treatment

Synthesis and assembly of structural proteins into 50 s ribosomal subunits were examined in Escherichia coli recovering from chloramphenicol treatment. The study was designed to determine whether all ribosomal proteins were synthesized to the same extent during recovery from chloramphenicol treatment. This investigation provides evidence that four proteins were preferentially synthesized and assembled into 50 s subunits early during recovery from chloramphenicol treatment and that considerable variation occurred in the rate of assembly of the various proteins into 50 s subunits. Those proteins synthesized to the greatest extent and most rapidly incorporated into 50 s subunits were the “split” proteins which were removed by treating ribosomes with cesium chloride. Later in recovery, cells preferentially synthesized the complement to the proteins synthesized early. After one hour of recovery, the cells resumed the normal pattern of ribosomal protein synthesis.