Instability in the Central Region of Tropomyosin Modulates the Function of Its Overlapping Ends

The causal link between disparate tropomyosin (Tm) functions and the structural instability in Tm is unknown. To test the hypothesis that the structural instability in the central region of Tm modulates the function of the overlapping ends of contiguous Tm dimers, we used transgenic mice (Tm_DM) that expressed a mutant α-Tm in the heart; S229E and H276N substitutions induce structural instability in the central region and the overlapping ends of Tm, respectively. In addition, two mouse cardiac troponin T mutants (TnT_1–44Δ and TnT_45–74Δ) that have a divergent effect on the overlapping ends of Tm were employed. The S229E-induced instability in the central region of Tm_DM altered the overlapping ends of Tm_DM, thereby it negated the attenuating effect of H276N on Ca²⁺-activated maximal tension. The rate of cross-bridge detachment (g) decreased in Tm_DM+TnT_WT and Tm_H276N+TnT_WT fibers but increased in Tm_DM+TnT_45–74Δ fibers; however, TnT_45–74Δ did not alter g, demonstrating that S229E in Tm_DM had divergent effects on g. The S229E substitution in Tm_DM ablated the H276N-induced desensitization of myofilament Ca²⁺ sensitivity in Tm_DM+TnT_1–44Δ fibers. To our knowledge, novel findings from this study show that the structural instability in the central region of Tm modifies cardiac contractile function via its effect on the overlapping ends of contiguous Tm.     

Proteolytic Cleavage of the Linker Region of the Human P-glycoprotein Modulates Its ATPase Function

P-glycoprotein (Pgp), an anticancer drug-translocating ATPase, is responsible for multidrug resistance in cancer. We have previously shown (Nuti, S. L., Mehdi, A., and Rao, U. S. (2000) Biochemistry 39, 3424-3432) that tryptic cleavage of Pgp results in the activation of basal and drug-stimulated ATPase functions of Pgp. To understand this phenomenon, we determined the sites cleaved by trypsin and further examined whether the modulation of Pgp function is trypsin-specific or the result of proteolysis in general. The effects of chymotrypsin and proteinase K on Pgp ATPase function were studied. The results show that proteolysis of Pgp irrespective of the protease employed resulted in the activation of basal ATPase activity. However, drug-stimulated ATPase activities were differentially modulated. Immunoblot analysis of proteolytic digests indicated that, irrespective of the protease employed, Pgp was predominantly cleaved in the middle of the molecule. N-terminal amino acid sequencing of Pgp tryptic and chymotryptic peptides indicated Arg(680) and Leu(682) as the sites of cleavage, respectively. These two cleavage sites are part of the predicted linker region that joins the two halves of Pgp. Together, these results suggest that the linker region in Pgp is primarily accessible to protease action and that cleavage of this region modulates Pgp ATPase function.     

Stabilizing the Central Part of Tropomyosin Increases the Bending Stiffness of the Thin Filament

A two-beam optical trap was used to measure the bending stiffness of F-actin and reconstructed thin filaments. A dumbbell was formed by a filament segment attached to two beads that were held in the two optical traps. One trap was static and held a bead used as a force transducer, whereas an acoustooptical deflector moved the beam holding the second bead, causing stretch of the dumbbell. The distance between the beads was measured using image analysis of micrographs. An exact solution to the problem of bending of an elastic filament attached to two beads and subjected to a stretch was used for data analysis. Substitution of noncanonical residues in the central part of tropomyosin with canonical ones, G126R and D137L, and especially their combination, caused an increase in the bending stiffness of the thin filaments. The data confirm that the effect of these mutations on the regulation of actin-myosin interactions may be caused by an increase in tropomyosin stiffness.     

A Code of Mono-phosphorylation Modulates the Function of RB

1. Download high-res image (181KB)
2. Download full-size image

     

The N-Terminal Region of IFITM3 Modulates Its Antiviral Activity by Regulating IFITM3 Cellular Localization

Interferon-inducible transmembrane (IFITM) protein family members IFITM1, -2, and -3 restrict the infection of multiple enveloped viruses. Significant enrichment of a minor IFITM3 allele was recently reported for patients who were hospitalized for seasonal and 2009 H1N1 pandemic flu. This IFITM3 allele lacks the region corresponding to the first amino-terminal 21 amino acids and is unable to inhibit influenza A virus. In this study, we found that deleting this 21-amino-acid region relocates IFITM3 from the endosomal compartments to the cell periphery. This finding likely underlies the lost inhibition of influenza A virus that completes its entry exclusively within endosomes at low pH. Yet, wild-type IFITM3 and the mutant with the 21-amino-acid deletion inhibit HIV-1 replication equally well. Given the pH-independent nature of HIV-1 entry, our results suggest that IFITM3 can inhibit viruses that enter cells via different routes and that its N-terminal region is specifically required for controlling pH-dependent viruses.     

Ang1-Akt途径对大鼠体外血-脊髓屏障功能的调节

检测血管生成素1(angiopoietin1,Ang1)对体外培养脊髓微血管内皮细胞(spinal cord microvascular endothelial cell,SCMEC)屏障功能的调节机制。体外分离培养大鼠SCMEC及胶质细胞,通过双室培养系统建立体外血-脊髓屏障(blood-spinal cord barrier,BSCB)模型;分别用Ang1及Ang1+wortmannin(Akt抑制剂)处理培养细胞,Western-blot检测Akt及连接蛋白质的表达;放射自显影检测Akt激酶活性变化;测定跨内皮电阻(TEER)反映各组细胞屏障功能;细胞免疫荧光检测连接蛋白在细胞接触面的分布变化;免疫共沉淀实验分析连接蛋白之间的相互作用改变。结果显示,Ang1处理组细胞Akt活性及TEER值均有明显升高,Ang1处理后ZO-1及VE-cadherin在细胞连接处的分布增强,occludin/ZO-1、VE-cadherin/β-catenin之间的相互作用均较对照组显著增强,且以上Ang1处理所引起的SCMEC功能改变均可被wortmannin所拮抗。表明Ang1通过Akt活性调节SCMEC连接蛋白的分布及相互作用,从而影响体外BSCB的屏障功能。     

Alternative Splicing Modulates Ubx Protein Function in Drosophila melanogaster

Polyploidy is an important aspect of the evolution of flowering plants. The potential of gene copies to diverge and evolve new functions is influenced by meiotic behavior of chromosomes leading to segregation as a single locus or duplicated loci. Switchgrass (Panicum virgatum) linkage maps were constructed using a full-sib population of 238 plants and SSR and STS markers to access the degree of preferential pairing and the structure of the tetraploid genome and as a step toward identification of loci underlying biomass feedstock quality and yield. The male and female framework map lengths were 1645 and 1376 cM with 97% of the genome estimated to be within 10 cM of a mapped marker in both maps. Each map coalesced into 18 linkage groups arranged into nine homeologous pairs. Comparative analysis of each homology group to the diploid sorghum genome identified clear syntenic relationships and collinear tracts. The number of markers with PCR amplicons that mapped across subgenomes was significantly fewer than expected, suggesting substantial subgenome divergence, while both the ratio of coupling to repulsion phase linkages and pattern of marker segregation indicated complete or near complete disomic inheritance. The proportion of transmission ratio distorted markers was relatively low, but the male map was more extensively affected by distorted transmission ratios and multilocus interactions, associated with spurious linkages.POLYPLOIDY is common among plants (Masterson 1994; Levin 2002) and is an important aspect of plant evolution. Widespread paleopolyploidy in flowering plant lineages suggests that ancient polyploidization events have contributed to the radiation of angiosperms (Soltis et al. 2009; Van de Peer et al. 2009a). Whole genome duplications are thought to be the sources of evolutionary novelty (Osborn et al. 2003; Freeling and Thomas 2006; Chen 2007; Hegarty and Hiscock 2008; Flagel and Wendel 2009; Leitch and Leitch 2008). Other attributes of polyploids considered to promote evolutionary success include increased vigor, masking of recessive alleles, and reproductive barriers arising from loss of one of the duplicate genes (Soltis and Soltis 2000; Comai 2005; Otto 2007; Van de Peer et al. 2009b). Among crop species, polyploidy likely contributed to trait improvement under artificial selection (Paterson 2005; Udall and Wendell 2006; Dubcovsky and Dvorak 2007; Hovav et al. 2008).Disomic inheritance in polyploids, in contrast to polysomic inheritance, presents opportunities for duplicated genes to diverge and evolve new functions. The relative age of whole genome duplications and the extent of homology between subgenomes greatly influence chromosomal pairing at meiosis (Soltis and Soltis 1995; Wolfe 2001; Ramsey and Schemske 2002). Polysomic inheritance resulting from random chromosome pairing is associated with doubling of a single set of chromosomes. Disomic inheritance resulting from preferential pairing is often associated with polyploidy arising from combinations of divergent genomes. The evolutionary process of diploidization leads to a shift from random to preferential pairing that is not well understood but is genetically defined in systems such as Ph1 of wheat (Triticum aestivum) and PrBn of Brassica napus (Riley and Chapman 1958; Vega and Feldman 1998; Jenczewski et al. 2003). The degree of preferential pairing also affects allelic diversity and the ability to detect linkage. Accurate information about chromosome pairing and whole or partial genome duplications is thus important for both evolutionary studies and in linkage analysis.Such information is extremely limited in the C4 panicoid species Panicum virgatum (switchgrass), which is now viewed as a promising energy crop in the United States and Europe (Lewandowski et al. 2003; McLaughlin and Kszos 2005) and is planted extensively for forage and soil conservation (Vogel and Jung 2001). Little is known about either its genome structure or inheritance. Much current bioenergy feedstock development is focused on tetraploid cytotypes (2n = 4x = 36) due to their higher yield potentials, and an initial segregation study indicated a high degree of preferential pairing in a single F1 mapping population (Missaoui et al. 2005). A once-dominant component of the tallgrass prairie in North America, switchgrass is largely self-incompatible (Martinez-Reyna and Vogel 2002) with predominantly tetraploid or octoploid cytotypes (Hultquist et al. 1997; Lu et al. 1998). Limited gene flow appears possible between different cytotypes suggested by DNA content variation within collection sites and seed lots (Nielsen 1944; Hultquist et al. 1997; Narasimhamoorthy et al. 2008). True diploids appear to be rare (Nielsen 1944; Young et al. 2010). Multivalents in meiosis have not been observed in tetraploids or F1 hybrids between upland and lowland tetraploids, although rare univalents occurred (Barnett and Carver 1967; Martinez-Reyna et al. 2001). However, polysomic inheritance may occur with random bivalent pairing (Howard and Swaminathan 1953).Sustainable production of switchgrass for bioenergy to meet the goal of reducing greenhouse gas emissions will require advances in feedstock production that include improvements in yield (Carroll and Somerville 2009). Switchgrass has extensive genetic diversity and potential for genetic improvements, but each cycle of phenotypic selection can take several years (McLaughlin and Kszos 2005; Parrish and Fike 2005; Bouton 2007). Detailed understanding of genome structure to enable efficient marker-assisted selection (MAS) can speed this process considerably. Complete linkage maps are therefore required to both understand chromosome pairing and allow MAS.We report the construction of the first complete linkage maps of two switchgrass genotypes. The linkage maps provide genetic evidence for disomic inheritance in lowland, tetraploid switchgrass. Gene-derived markers enabled a comparative analysis to sorghum, revealing syntenic relationships between the diploid sorghum genome and the tetraploid switchgrass subgenomes. Transmission ratio distortion and multilocus interactions were analyzed in detail to document their potential influence on map accuracy and map-based studies in switchgrass.     

Mitochondrial Calcium Uptake Modulates Synaptic Vesicle Endocytosis in Central Nerve Terminals

Presynaptic calcium influx triggers synaptic vesicle (SV) exocytosis and modulates subsequent SV endocytosis. A number of calcium clearance mechanisms are present in central nerve terminals that regulate intracellular free calcium levels both during and after stimulation. During action potential stimulation, mitochondria rapidly accumulate presynaptic calcium via the mitochondrial calcium uniporter (MCU). The role of mitochondrial calcium uptake in modulating SV recycling has been debated extensively, but a definitive conclusion has not been achieved. To directly address this question, we manipulated the expression of the MCU channel subunit in primary cultures of neurons expressing a genetically encoded reporter of SV turnover. Knockdown of MCU resulted in ablation of activity-dependent mitochondrial calcium uptake but had no effect on the rate or extent of SV exocytosis. In contrast, the rate of SV endocytosis was increased in the absence of mitochondrial calcium uptake and slowed when MCU was overexpressed. MCU knockdown did not perturb activity-dependent increases in presynaptic free calcium, suggesting that SV endocytosis may be controlled by calcium accumulation and efflux from mitochondria in their immediate vicinity.     

Peripheral Nerve Injury Modulates Neurotrophin Signaling in the Peripheral and Central Nervous System

Peripheral nerve injury disrupts the normal functions of sensory and motor neurons by damaging the integrity of axons and Schwann cells. In contrast to the central nervous system, the peripheral nervous system possesses a considerable capacity for regrowth, but regeneration is far from complete and functional recovery rarely returns to pre-injury levels. During development, the peripheral nervous system strongly depends upon trophic stimulation for neuronal differentiation, growth and maturation. The perhaps most important group of trophic substances in this context is the neurotrophins (NGF, BDNF, NT-3 and NT-4/5), which signal in a complex spatial and timely manner via the two structurally unrelated p75^NTR and tropomyosin receptor kinase (TrkA, Trk-B and Trk-C) receptors. Damage to the adult peripheral nerves induces cellular mechanisms resembling those active during development, resulting in a rapid and robust increase in the synthesis of neurotrophins in neurons and Schwann cells, guiding and supporting regeneration. Furthermore, the injury induces neurotrophin-mediated changes in the dorsal root ganglia and in the spinal cord, which affect the modulation of afferent sensory signaling and eventually may contribute to the development of neuropathic pain. The focus of this review is on the expression patterns of neurotrophins and their receptors in neurons and glial cells of the peripheral nervous system and the spinal cord. Furthermore, injury-induced changes of expression patterns and the functional consequences in relation to axonal growth and remyelination as well as to neuropathic pain development will be reviewed.     

Defining the Region of Bacillus subtilis SpoIIIJ That Is Essential for Its Sporulation-Specific Function

Proteins of the YidC/OxaI/Alb3 family play a crucial role in the insertion, folding, and/or assembly of membrane proteins in prokaryotes and eukaryotes. Bacillus subtilis has two YidC-like proteins, denoted SpoIIIJ and YqjG. SpoIIIJ and YqjG are largely exchangeable in function, but SpoIIIJ has a unique role in sporulation, while YqjG stimulates competence development. To obtain more insight into the regions important for the sporulation specificity of SpoIIIJ, a series of SpoIIIJ/YqjG chimeras was constructed. These chimeras were tested for functionality during vegetative growth and for their ability to complement the sporulation defect of a spoIIIJ deletion strain. The data suggest an important role for the domain comprising transmembrane segment 2 (TMS2) and its flanking loops in sporulation specificity, with lesser contributions to specificity by TMS1 and TMS3.     

AKIP1 Expression Modulates Mitochondrial Function in Rat Neonatal Cardiomyocytes

A kinase interacting protein 1 (AKIP1) is a molecular regulator of protein kinase A and nuclear factor kappa B signalling. Recent evidence suggests AKIP1 is increased in response to cardiac stress, modulates acute ischemic stress response, and is localized to mitochondria in cardiomyocytes. The mitochondrial function of AKIP1 is, however, still elusive. Here, we investigated the mitochondrial function of AKIP1 in a neonatal cardiomyocyte model of phenylephrine (PE)-induced hypertrophy. Using a seahorse flux analyzer we show that PE stimulated the mitochondrial oxygen consumption rate (OCR) in cardiomyocytes. This was partially dependent on PE mediated AKIP1 induction, since silencing of AKIP1 attenuated the increase in OCR. Interestingly, AKIP1 overexpression alone was sufficient to stimulate mitochondrial OCR and in particular ATP-linked OCR. This was also true when pyruvate was used as a substrate, indicating that it was independent of glycolytic flux. The increase in OCR was independent of mitochondrial biogenesis, changes in ETC density or altered mitochondrial membrane potential. In fact, the respiratory flux was elevated per amount of ETC, possibly through enhanced ETC coupling. Furthermore, overexpression of AKIP1 reduced and silencing of AKIP1 increased mitochondrial superoxide production, suggesting that AKIP1 modulates the efficiency of electron flux through the ETC. Together, this suggests that AKIP1 overexpression improves mitochondrial function to enhance respiration without excess superoxide generation, thereby implicating a role for AKIP1 in mitochondrial stress adaptation. Upregulation of AKIP1 during different forms of cardiac stress may therefore be an adaptive mechanism to protect the heart.     

An Overlapping Region between the Two Terminal Folding Units of the Outer Surface Protein A (OspA) Controls Its Folding Behavior

Although many naturally occurring proteins consist of multiple domains, most studies on protein folding to date deal with single-domain proteins or isolated domains of multi-domain proteins. Studies of multi-domain protein folding are required for further advancing our understanding of protein folding mechanisms. Borrelia outer surface protein A (OspA) is a β-rich two-domain protein, in which two globular domains are connected by a rigid and stable single-layer β-sheet. Thus, OspA is particularly suited as a model system for studying the interplays of domains in protein folding. Here, we studied the equilibria and kinetics of the urea-induced folding–unfolding reactions of OspA probed with tryptophan fluorescence and ultraviolet circular dichroism. Global analysis of the experimental data revealed compelling lines of evidence for accumulation of an on-pathway intermediate during kinetic refolding and for the identity between the kinetic intermediate and a previously described equilibrium unfolding intermediate. The results suggest that the intermediate has the fully native structure in the N-terminal domain and the single layer β-sheet, with the C-terminal domain still unfolded. The observation of the productive on-pathway folding intermediate clearly indicates substantial interactions between the two domains mediated by the single-layer β-sheet. We propose that a rigid and stable intervening region between two domains creates an overlap between two folding units and can energetically couple their folding reactions.     

A DEK Domain-Containing Protein Modulates Chromatin Structure and Function in Arabidopsis

Chromatin is a major determinant in the regulation of virtually all DNA-dependent processes. Chromatin architectural proteins interact with nucleosomes to modulate chromatin accessibility and higher-order chromatin structure. The evolutionarily conserved DEK domain-containing protein is implicated in important chromatin-related processes in animals, but little is known about its DNA targets and protein interaction partners. In plants, the role of DEK has remained elusive. In this work, we identified DEK3 as a chromatin-associated protein in Arabidopsis thaliana. DEK3 specifically binds histones H3 and H4. Purification of other proteins associated with nuclear DEK3 also established DNA topoisomerase 1α and proteins of the cohesion complex as in vivo interaction partners. Genome-wide mapping of DEK3 binding sites by chromatin immunoprecipitation followed by deep sequencing revealed enrichment of DEK3 at protein-coding genes throughout the genome. Using DEK3 knockout and overexpressor lines, we show that DEK3 affects nucleosome occupancy and chromatin accessibility and modulates the expression of DEK3 target genes. Furthermore, functional levels of DEK3 are crucial for stress tolerance. Overall, data indicate that DEK3 contributes to modulation of Arabidopsis chromatin structure and function.     

A Product of Heme Catabolism Modulates Bacterial Function and Survival

Bilirubin is the terminal metabolite in heme catabolism in mammals. After deposition into bile, bilirubin is released in large quantities into the mammalian gastrointestinal (GI) tract. We hypothesized that intestinal bilirubin may modulate the function of enteric bacteria. To test this hypothesis, we investigated the effect of bilirubin on two enteric pathogens; enterohemorrhagic E. coli (EHEC), a Gram-negative that causes life-threatening intestinal infections, and E. faecalis, a Gram-positive human commensal bacterium known to be an opportunistic pathogen with broad-spectrum antibiotic resistance. We demonstrate that bilirubin can protect EHEC from exogenous and host-generated reactive oxygen species (ROS) through the absorption of free radicals. In contrast, E. faecalis was highly susceptible to bilirubin, which causes significant membrane disruption and uncoupling of respiratory metabolism in this bacterium. Interestingly, similar results were observed for other Gram-positive bacteria, including B. cereus and S. aureus. A model is proposed whereby bilirubin places distinct selective pressure on enteric bacteria, with Gram-negative bacteria being protected from ROS (positive outcome) and Gram-positive bacteria being susceptible to membrane disruption (negative outcome). This work suggests bilirubin has differential but biologically relevant effects on bacteria and justifies additional efforts to determine the role of this neglected waste catabolite in disease processes, including animal models.     

BiP Modulates the Affinity of Its Co-chaperone ERj1 for Ribosomes

Ribosomes synthesizing secretory and membrane proteins are bound to the endoplasmic reticulum (ER) membrane and attach to ribosome-associated membrane proteins such as the Sec61 complex, which forms the protein-conducting channel in the membrane. The ER membrane-resident Hsp40 protein ERj1 was characterized as being able to recruit BiP to ribosomes in solution and to regulate protein synthesis in a BiP-dependent manner. Here, we show that ERj1 and Sec61 are associated with ribosomes at the ER of human cells and that the binding of ERj1 to ribosomes occurs with a binding constant in the picomolar range and is prevented by pretreatment of ribosomes with RNase. However, the affinity of ERj1 for ribosomes dramatically changes upon binding of BiP. This modulation by BiP may be responsible for the dual role of ERj1 at the ribosome, i.e. acting as a recruiting factor for BiP and regulating translation.