Alteration in crossbridge kinetics caused by mutations in actin

The generation of force during muscle contraction results from the interaction of myosin and actin. The kinetics of this force generation vary between different muscle types and within the same muscle type in different species. Most attention has focused on the role of myosin isoforms in determining these differences. The role of actin isoforms has received little attention, largely because of the lack of a suitable cell type in which the myosin isoform remains constant yet the actin isoforms vary. An alternative approach would be to examine the effect of actin mutations, however, most of these cause such gross disruption of muscle structure that mechanical measurements are impossible. We have now identified two actin mutations which, despite involving conserved amino acids, can assemble into virtually normal myofibrils. These amino-acid changes in actin significantly affect the kinetics of force generation by muscle fibres. One of the mutations is not in the putative myosin-binding site, demonstrating the importance of long-range effects of amino acids on actin function.     

Requirement of yeast fimbrin for actin organization and morphogenesis in vivo

The SAC6 gene was found by suppression of a yeast actin mutation. Its protein product, Sac6p (previously referred to as ABP67), was independently isolated by actin-filament affinity chromatography and colocalizes with actin in vivo. Thus Sac6p binds to actin in vitro, and functionally associates with actin structures involved in the development and maintenance of cell polarity in vivo. We report here that Sac6p is an actin-filament bundling protein 43% identical in amino-acid sequence to the vertebrate bundling protein fimbrin. This yeast fimbrin homologue contains two putative actin-binding regions homologous to domains of dystrophin, beta-spectrin, filamin, actin-gelation protein and alpha-actinin. Mutants lacking Sac6p do not form normal actin structures and are defective in morphogenesis. These findings demonstrate an in vivo role for the well-documented biochemical interaction between fimbrin and actin.     

Cell-surface receptor for ecotropic murine retroviruses is a basic amino-acid transporter

The complementary DNA sequence encoding the cell-surface receptor for ecotropic host-range murine retroviruses (ecoR) shows that it contains 622 amino acids and 14 hydrophobic potentially membrane-spanning sequences. Because this receptor occurs on many or all murine cells and is probably essential for viability of cultured fibroblasts, its normal function might be to transport an essential metabolite. We expressed ecoR in Xenopus laevis oocytes by injecting RNA transcribed from the cloned cDNA. These oocytes specifically bound the gp70 envelope glycoprotein from an ecotropic murine leukaemia virus. An inward current was recorded electrophysiologically when a mixture of amino-acids was applied: this resulted from a stereoselective, saturable uptake of lysine, arginine and ornithine; it was independent of sodium and not substantially altered by gp70. Cysteine and homoserine were also taken up, but sodium was necessary for their transport. These properties of ecoR correspond to those of the y+ amino-acid transporter. Our results demonstrate the subversion of a ubiquitous cell membrane protein, in this case a basic amino acid transporter, for use as a retroviral receptor.     

Homology of a yeast actin-binding protein to signal transduction proteins and myosin-I

In yeast, the cortical actin cytoskeleton seems to specify sites of growth of the cell surface. Because the actin-binding protein ABP1p is associated with the cortical cytoskeleton of Saccharomyces cerevisiae, it might be involved in the spatial organization of cell surface growth. ABP1p is localized to the cortical cytoskeleton and its overproduction causes assembly of the cortical actin cytoskeleton at inappropriate sites on the cell surface, resulting in delocalized surface growth. We have now cloned and sequenced the gene encoding ABP1p. ABP1p is a novel protein with a 50 amino-acid C-terminal domain that is very similar to the SH3 domain in the non-catalytic region of nonreceptor tyrosine kinases (including those encoded by the proto-oncogenes c-src and c-abl), in phospholipase C gamma and in alpha-spectrin. We also identified an SH3-related motif in the actin-binding tail domain of myosin-I. The identification of SH3 domains in a family of otherwise unrelated proteins that associate with the membrane cytoskeleton indicates that this domain might serve to bring together signal transduction proteins and their targets or regulators, or both, in the membrane cytoskeleton.     

Identification of a secretory granule-binding protein as caldesmon

Stimulation of adrenal chromaffin cells results in a rise in the concentration of intracellular free calcium which initiates catecholamine secretion by exocytosis. An understanding of the molecular basis of exocytosis will require knowledge of the sites of action of calcium. A role for calmodulin has been implicated in secretion from chromaffin cells, and isolated granule membranes bind both calmodulin and a series of cytosolic proteins in a calcium-dependent fashion. Here, we demonstrate that one of the cytosolic granule-binding proteins with a relative molecular mass (Mr) of 70,000 (70K) is a form of the calmodulin-regulated actin-binding protein caldesmon, first isolated from smooth muscle. Cytoplasmic gels assembled from an adrenal medullary extract in the absence of Ca2+ contained actin and the 70K protein. The association of both of these proteins with the cytoplasmic gel was inhibited by a micromolar concentration of Ca2+. In addition, we have demonstrated that the 70K protein is localized at the periphery of chromaffin cells. These results are consistent with the notion that 70K protein (caldesmon) has a role in regulating the organization of actin filaments of the cell periphery during the secretory process.     

Cloning and characterization of a new actin gene from Oryza sativa L.

Using Rho family member osRACD as bait, a new member of actin gene family -Act was isolated from Oryza sativa by yeast two-hybrid system. The full-length cDNA was cloned with 5' RACE technology, which contains an open reading frame of 1134 bp with a predicted protein of 377 amino acids. Sequence alignment revealed 96% to 81.8% identities with some known actin proteins in plants. The method of bioinformatics was used to analyze the protein modification sites, structure and evolution of the gene. Southern blot analysis showed that Act is a single-copy gene in the genome. The result of RT-PCR showed it is ubiquitously expressed in root, shoot, callus and panicle in a temporal fashion. The relationship between Rho family and actin family in evolution and function was also studied.     

A homologue of the bacterial heat-shock gene DnaJ that alters protein sorting in yeast

Heat-shock proteins have been implicated in assembly of protein complexes, correct protein folding and uptake of proteins into organelles. In Escherichia coli, the heat-shock protein DnaJ and the Hsp70 homologue, DnaK, act together to disassemble a protein complex involved in bacteriophage lambda replication. We report the identification of SCJ1, a gene in the yeast Saccharomyces cerevisiae that encodes a homologue of the bacterial DnaJ protein. SCJ1 was identified by a genetic screen in which increased expression of candidate genes results in missorting of a nuclear-targeted test protein. The predicted amino-acid sequence of SCJ1 is 37% identical to the entire E. coli DnaJ protein. Hybridization experiments indicate that there is a family of yeast genes related to SCJ1. These findings suggest that the Hsp70 DnaK-DnaJ interaction is general to eukaryotes.     

New yeast actin-like gene required late in the cell cycle.

Actin, a major cytoskeletal component of all eukaryotic cells, is one of the most highly conserved proteins. It is involved in various cellular processes such as motility, cytoplasmic streaming, chromosome segregation and cytokinesis. The actin from the yeast Saccharomyces cerevisiae, encoded by the essential ACT1 gene, is 89% identical to mouse cytoplasmic actin and is involved in the organization and polarized growth of the cell surface. We report here the characterization of ACT2, a previously undescribed yeast split gene encoding a putative protein (391 amino acids, relative molecular mass (Mr) 44,073) that is 47% identical to yeast actin. The requirement of the ACT2 gene for vegetative growth of yeast cells and the existence of related genes in other eukaryotes indicate an important and conserved role for these actin-like proteins. Superimposition of the Act2 polypeptide onto the three-dimensional structure of known actins reveals that most of the divergence occurred in loops involved in actin polymerization, DNase I and myosin binding, leaving the core domain mainly unaffected. To our knowledge, the Act2 protein from S. cerevisiae is the first highly divergent actin molecule described. Structural and physiological data suggest that the Act2 protein might have an important role in cytoskeletal reorganization during the cell cycle.     

Structure of hisactophilin is similar to interleukin-1 beta and fibroblast growth factor.

The fast reaction of the actin-based cytoskeleton in motile cells after stimulation with a chemoattractant requires a signal-transduction chain that creates a very specific environment at distinct regions beneath the plasma membrane. Dictyostelium hisactophilin, a unique actin-binding protein, is a submembranous pH sensor that signals slight changes of the H+ concentration to actin by inducing actin polymerization and binding to microfilaments only at pH values below seven. It has a relative molecular mass of 13.5K and its most unusual feature is the presence of 31 histidine residues among its total of 118 amino acids. The transduction of an external signal from the plasma membrane to the cytoskeleton is poorly understood. Here we report the protein's structure in solution determined by nuclear magnetic resonance spectroscopy. The nuclear Overhauser effect intensities of the three-dimensional nuclear Overhauser spectra were used directly in the calculations. The overall folding of histactophilin is similar to that of interleukin-1 beta and fibroblast growth factor, but the primary amino-acid sequence of hisactophilin is unrelated to these two proteins.     

Ankyrin binding to (Na+ + K+)ATPase and implications for the organization of membrane domains in polarized cells

The interaction between membrane proteins and cytoplasmic structural proteins is thought to be one mechanism for maintaining the spatial order of proteins within functional domains on the plasma membrane. Such interactions have been characterized extensively in the human erythrocyte, where a dense, cytoplasmic matrix of proteins comprised mainly of spectrin and actin, is attached through a linker protein, ankyrin, to the anion transporter (Band 3). In several nonerythroid cell types, including neurons, exocrine cells and polarized epithelial cells homologues of ankyrin and spectrin (fodrin) are localized in specific membrane domains. Although these results suggest a functional linkage between ankyrin and fodrin and integral membrane proteins in the maintenance of membrane domains in nonerythroid cells, there has been little direct evidence of specific molecular interactions. Using a direct biological and chemical approach, we show here that ankyrin binds to the ubiquitous (Na+ + K+)ATPase, which has an asymmetrical distribution in polarized cells.     

Molecular cloning and expression of the major protein kinase C substrate of platelets

In platelets, agonists that stimulate phosphoinositide turnover cause the rapid phosphorylation of a protein of apparent relative molecular mass (Mr) 40-47,000, called P47, by protein kinase C (PKC). Diverse identities have been ascribed to P47 including lipocortin, inositol 1,4,5-trisphosphate 5-phosphomonoesterase, pyruvate dehydrogenase alpha subunit and an actin regulatory protein. We have isolated human P47 clones by immunological screening of a lambda gt11 complementary DNA library from HL-60 cells, a human promyelocytic leukaemia cell line. P47 recombinants thus identified hybridized to a 3.0 kilobase (kb) messenger RNA in mature white blood cell lines; the same mRNA was induced in HL-60 cells during differentiation. A 1,050 base pair (bp) open reading frame that could encode a protein of Mr40,087 was confirmed by comparison with peptide sequences from platelet P47, and by expression of the putative recombinant P47 in E. coli and in vitro. The P47 sequence appears to have been conserved throughout vertebrate evolution, and is not similar to any other known sequence including human lipocortin and the alpha subunit of pyruvate dehydrogenase. The P47 protein contains a potential Ca2+-binding 'EF-hand' structure and a region that strongly resembles known PKC phosphorylation sites.     

Two forms of transforming growth factor-beta distinguished by multipotential haematopoietic progenitor cells

Type-beta transforming growth factors (TGF-beta s) are polypeptides that act hormonally to control proliferation and differentiation of many cell types. Two distinct homodimeric TGF-beta polypeptides, TGF-beta 1 and TGF-beta 2 have been identified which show approximately 70% amino-acid sequence similarity. Despite their structural differences, TGF-beta 1 and TGF-beta 2 are equally potent at inhibiting epithelial cell proliferation and adipogenic differentiation. The recent immunohistochemical localization of high levels of TGF-beta in the bone marrow and haematopoietic progenitors of the fetal liver has raised the possibility that TGF-beta s might be involved in the regulation of haematopoiesis. Here we show that TGF-beta 1, but not TGF-beta 2, is a potent inhibitor of haematopoietic progenitor cell proliferation. TGF-beta 1 inhibited colony formation by murine factor-dependent haematopoietic progenitor cells in response to interleukin-3 (IL-3) or granulocyte-macrophage colony stimulating factor (GM-CSF), as well as colony formation by marrow progenitor cells responding to CSF-1 (M-CSF). The progenitor cell lines examined were approximately 100-fold more sensitive to TGF-beta 1 than TGF-beta 2, and displayed type-I TGF-beta receptors with affinity approximately 20-fold higher for TGF-beta 1 than TGF-beta 2. These results identify TGF-beta 1 as a novel regulator of haematopoiesis that acts through type-I TGF-beta receptors to modulate proliferation of progenitor cells in response to haematopoietic growth factors.     

The importance of sequence diversity in the aggregation and evolution of proteins

Incorrect folding of proteins, leading to aggregation and amyloid formation, is associated with a group of highly debilitating medical conditions including Alzheimer's disease and late-onset diabetes. The issue of how unwanted protein association is normally avoided in a living system is particularly significant in the context of the evolution of multidomain proteins, which account for over 70% of all eukaryotic proteins, where the effective local protein concentration in the vicinity of each domain is very high. Here we describe the aggregation kinetics of multidomain protein constructs of immunoglobulin domains and the ability of different homologous domains to aggregate together. We show that aggregation of these proteins is a specific process and that the efficiency of coaggregation between different domains decreases markedly with decreasing sequence identity. Thus, whereas immunoglobulin domains with more than about 70% identity are highly prone to coaggregation, those with less than 30-40% sequence identity do not detectably interact. A bioinformatics analysis of consecutive homologous domains in large multidomain proteins shows that such domains almost exclusively have sequence identities of less than 40%, in other words below the level at which coaggregation is likely to be efficient. We propose that such low sequence identities could have a crucial and general role in safeguarding proteins against misfolding and aggregation.     

Different rhinovirus serotypes neutralized by antipeptide antibodies

Recently, Rossman et al. have described the three-dimensional structure of a human rhinovirus. A possible host cell surface receptor binding site was identified with a cleft on each icosahedral face. Two highly conserved amino-acid sequences found in rhino-, polio-, and foot-and-mouth disease (FMD) viruses are located near the base of this site and could be important in maintaining its topology. We have prepared site-specific antibodies to two synthetic peptides which include these sequences. The antibodies bind to the predicted capsid proteins of rhinovirus and neutralize approximately 60% of 48 rhinovirus serotypes tested. These results could provide a route to a rhinovirus vaccine effective against most of the numerous serotypes of this virus.     

人血红蛋白β链氨基酸对的变异模式

根据氨基酸对可预测性分析人血红蛋白β链的244个变异,以确定哪些氨基酸对对变异更敏感。结果发现4种变异模式：（1）85.66%的变异发生在不可预测的氨基酸对,（2）绝大多数被替换掉的氨基酸对中包含一个或两个氨基酸对它们的实际频率大于预测频率,（3）79.51%的变异中被替换出的氨基酸对包含一个或两个在正常人β血红蛋白中不存在的氨基酸对,（4）大多数变异缩小了氨基酸对实际频率与预测频率之差。说明变异通常导致人血红蛋白β链的构成更随机、更稳定。     

Reversible stress softening of actin networks

The mechanical properties of cells play an essential role in numerous physiological processes. Organized networks of semiflexible actin filaments determine cell stiffness and transmit force during mechanotransduction, cytokinesis, cell motility and other cellular shape changes. Although numerous actin-binding proteins have been identified that organize networks, the mechanical properties of actin networks with physiological architectures and concentrations have been difficult to measure quantitatively. Studies of mechanical properties in vitro have found that crosslinked networks of actin filaments formed in solution exhibit stress stiffening arising from the entropic elasticity of individual filaments or crosslinkers resisting extension. Here we report reversible stress-softening behaviour in actin networks reconstituted in vitro that suggests a critical role for filaments resisting compression. Using a modified atomic force microscope to probe dendritic actin networks (like those formed in the lamellipodia of motile cells), we observe stress stiffening followed by a regime of reversible stress softening at higher loads. This softening behaviour can be explained by elastic buckling of individual filaments under compression that avoids catastrophic fracture of the network. The observation of both stress stiffening and softening suggests a complex interplay between entropic and enthalpic elasticity in determining the mechanical properties of actin networks.     

Protein folding in the cell.

In the cell, as in vitro, the final conformation of a protein is determined by its amino-acid sequence. But whereas some isolated proteins can be denatured and refolded in vitro in the absence of other macromolecular cellular components, folding and assembly of polypeptides in vivo involves other proteins, many of which belong to families that have been highly conserved during evolution.     

Defects in mismatch repair promote telomerase-independent proliferation

Mismatch repair has a central role in maintaining genomic stability by repairing DNA replication errors and inhibiting recombination between non-identical (homeologous) sequences. Defects in mismatch repair have been linked to certain human cancers, including hereditary non-polyposis colorectal cancer (HNPCC) and sporadic tumours. A crucial requirement for tumour cell proliferation is the maintenance of telomere length, and most tumours achieve this by reactivating telomerase. In both yeast and human cells, however, telomerase-independent telomere maintenance can occur as a result of recombination-dependent exchanges between often imperfectly matched telomeric sequences. Here we show that loss of mismatch-repair function promotes cellular proliferation in the absence of telomerase. Defects in mismatch repair, including mutations that correspond to the same amino-acid changes recovered from HNPCC tumours, enhance telomerase-independent survival in both Saccharomyces cerevisiae and a related budding yeast with a degree of telomere sequence homology that is similar to human telomeres. These results indicate that enhanced telomeric recombination in human cells with mismatch-repair defects may contribute to cell immortalization and hence tumorigenesis.     

Cloning of the gene and cDNA for mammalian beta-adrenergic receptor and homology with rhodopsin

The adenylate cyclase system, which consists of a catalytic moiety and regulatory guanine nucleotide-binding proteins, provides the effector mechanism for the intracellular actions of many hormones and drugs. The tissue specificity of the system is determined by the particular receptors that a cell expresses. Of the many receptors known to modulate adenylate cyclase activity, the best characterized and one of the most pharmacologically important is the beta-adrenergic receptor (beta AR). The pharmacologically distinguishable subtypes of the beta-adrenergic receptor, beta 1 and beta 2 receptors, stimulate adenylate cyclase on binding specific catecholamines. Recently, the avian erythrocyte beta 1, the amphibian erythrocyte beta 2 and the mammalian lung beta 2 receptors have been purified to homogeneity and demonstrated to retain binding activity in detergent-solubilized form. Moreover, the beta-adrenergic receptor has been reconstituted with the other components of the adenylate cyclase system in vitro, thus making this hormone receptor particularly attractive for studies of the mechanism of receptor action. This situation is in contrast to that for the receptors for growth factors and insulin, where the primary biochemical effectors of receptor action are unknown. Here, we report the cloning of the gene and cDNA for the mammalian beta 2AR. Analysis of the amino-acid sequence predicted for the beta AR indicates significant amino-acid homology with bovine rhodopsin and suggests that, like rhodopsin, beta AR possesses multiple membrane-spanning regions.