Biochemical and Biological Properties of Actins and Myosins

肌动蛋白和肌球蛋白的生化和生物学特性

• 批准号: 7968962
• 负责人: EDWARD D KORN
• 金额: $ 128.46万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7968962
• 关键词: ATP Hydrolysis; Acanthamoeba; Acanthamoeba castellanii; Accounting; Actins; Actomyosin; Affect; Affinity; Amino Acid Sequence; Amino Acids; Amoeba genus; Basic Amino Acids; Binding; Binding Sites; Biochemical; Biological; Biological Models; Biological Process; Cell division; Cell membrane; Cell surface; Cells; Charge; Chemotaxis; Cleaved cell; Computers; Cyclic AMP; Cyclic AMP Receptors; Cytoplasm; DNA; Databases; Defect; Deoxyribonuclease I; Development; Dictyostelium; Dictyostelium discoideum; Embryonic Development; Exhibits; Filament; Fruit; Genetic Transcription; Glutamine; Goals; Growth and Development function; Hearing; Hydrogen Bonding; Immune response; In Vitro; Inhibitory Concentration 50; Laboratories; Length; Life; MAPK1 gene; Measures; Mediating; Membrane; Membrane Lipids; Membrane Potentials; Membrane Proteins; Microfilaments; Molecular; Molecular Conformation; Muscle; Muscle Cells; Mutate; Mutation; Myosin ATPase; Myosin Type I; N-terminal; Neoplasm Metastasis; Neurons; Nucleotides; Peptide Sequence Determination; Peptides; Phagocytosis; Phospholipids; Phosphorylation; Pinocytosis; Polymers; Positioning Attribute; Property; Protein Isoforms; Protein Region; Proteins; Regulation; Relative (related person); Reproduction spores; Research; Scaffolding Protein; Sequence Homology; Signal Transduction; Stream; Stress; Structure; Subtilisins; Suspension Culture; Tail; Tertiary Protein Structure; Tyrosine; Vesicle; Vision; Work; Wound Healing; actin 2; adenylyl cyclase A; alpha helix; base; beta pleated sheet; cancer cell; cell growth; cell motility; glutamylalanine; human disease; in vivo; inorganic phosphate; interest; monomer; mutant; organelle movement; phenylalanylalanine; polymerization; preference; programs; response; synthetic peptide

Research this year was focused on: (1) the molecular basis of the membrane affinity of class-I myosins and other membrane-associated proteins, (2) the biochemical and biophysical properties of actin with mutations of Tyr-53, (3) the effects of expression of actin Tyr-53 mutants on the growth and development of Dictyostelium. (1) From work from our and other laboratories, it is known that Acanthamoeba myosin IC (AMIC) binds to plasma membranes through a 220-residue basic region in the tail. Last year we showed that full-length AMIC binds non-specifically to acidic phospholipid vesicles in proportion to their negative charge, and, by competitive inhibition by synthetic peptides, that the binding site is most probably a 13-residue sequence of basic-hydrophobic-basic (BHB) amino acids, KVKPFLYVLKRR, within the basic region of the AMIC heavy chain. IC50 values (concentration required for 50% inhibition of binding of AMIC) of synthetic peptides with the sequences of BHB segments in similar positions in the heavy chains of the three Acanthamoeba class-I myosins were proportional to their sequence homologies to the BHB region of AMIC. The relative IC50 values of the synthetic peptides correlated with the localization of the myosins in either membranes (low IC50 values) or cytoplasm (high IC50 values) in the amoebae. This year, we confirmed and extended the previous results by measuring directly the affinities for acidic phospholipid vesicles of the homologous peptides from Acanthamoeba myosins IA, IB and IC and Dictyostelium myosins IB, IC and ID. We found that the basic and hydrophobic amino acid composition of the peptides, but not the sequence of the residues, is the determining factor for binding affinity. We then developed a computer search program (BH-search) for identifying protein regions enriched in basic and hydrophobic amino acids (i.e. potential membrane-binding sites). After demonstrating that BH-search successfully identified all the previously known unstructured membrane-binding sites in 16 membrane-associated proteins, we applied the program to proteins of interest whose membrane-binding sites were not known. BH-searches found previously unknown potential membrane-binding sites in multiple, but not all, class-I myosins, PAKs and CARMIL (a membrane-associated cytoskeletal scaffold protein). Importantly, synthetic peptides and protein domains with these sites bound to acidic phospholipids with high affinities in vitro. Lipid/membrane-binding sites with highly defined tertiary structures containing alpha-helices and beta-sheets, such as PH, FERM, C2, ENTH and other domains, have been identified previously, but there has heretofore been no program for searching protein sequences for potential membrane binding sites in unstructured regions. (2) Actin is one of the most highly conserved proteins. About 95% of all actins have the same amino acid in about 66% of the 375 positions, and in about 74% of the remaining positions the substitution is always the same amino acid, and often a conservative, substitution. For example, Tyr is at position 53 in all but 11 of the 323 actin sequences in the data base, and Phe replaces Tyr in all 11 exceptions. Interestingly, Tyr-53 is dynamically phosphorylated in Dictyostelium during the developmental cycle (50% of the actin in spores is phosphorylated), and when amoebae are subjected to stress. Actin Tyr53 is also phosphorylated in cancer cells grown in culture. Last year we showed that hydrogen bonds are formed between phosphorylated Tyr-53 and Gly-48, Gln-49 and Lys-61, which partially stabilizes the neighboring DNase I-binding loop (D-loop), residues 40-50. As a consequence of the conformational change in the D-loop, Tyr-53 phosphorylation reduces the rate of subtilisin cleavage of the D-loop, the reduces the affinity of monomeric actin for pancreatic DNase I, and, allosterically, the reduces the rate of nucleotide exchange in monomeric actiin. Tyr-53 phosphorylation also affects actin polymerization: the critical concentration is increased, the rates of nucleation and pointed-end elongation are reduced, polymerization and ATP hydrolysis are partially uncoupled, and filaments of Tyr-53-phosphorylated actin (pY53actin) are unstable such that polymerized pY53-actin is predominantly in the form of very short oligomers. These observations raise the question whether the effects of Tyr-53 phosphorylation are due to the loss of tyrosine or the addition of phosphate. This year we found that mutation of Tyr-53 to Phe has no effect on actin's properties but that mutation of Tyr-53 to either Ala or Glu profoundly affects the biochemical and biophysical properties of Dictyostelium actin, in ways similar, but not identical, to phosphorylation of Tyr53. The Ala and Glu mutations modify the conformation of the D-loop so that actin's affinity for DNase I and pointed-end elongation are reduced, the critical concentration is increased, and the polymerized mutant actins form unstable filaments and small oligomers. In addition, the rates of nucleotide exchange by actin monomers and filaments are increased. These results indicate that both Tyr and Phe (but not Ala or Glu) at position 53 maintain the functional conformations of the D-loop and, furthermore, that the conformation of the D-loop allosterically affects the conformation of the nucleotide-binding cleft. The evolutionary preference for Tyr at this position may be explained by the possibility of dynamic regulation of actins properties by phosphorylation. (3) We extended the studies of the effects of mutations of actin Tyr-53 on the properties of purified actin by expessing the Phe, Ala and Glu mutants in live Dictyostelium amoebae. For technical reasons it was necessary to express the proteins with an N-terminal FLAG tag, so we also expressed FLAG-tagged wild-type actin as a control on the possible effect of the FLAG tag. The expressed actins account for about 25% of the total actin in cells. We found that the properties of purified FLAG-tagged wild-type and purified Flag-tagged Phe mutant actins are very similar to the properties of purified endogenous wild-type actin. However, the purified FLAG-tagged Ala and Glu mutant actins and co-polymers of 25% Ala or Glu mutant actin and endogenous actin have modified properties similar to those described in the previous section for the Ala and Glu mutants without the FLAG-tag. All of the expressed FLAG-actins co-localize in the cell with endogenous actin and none of the expressed actins has any effect on cell growth in suspension culture, pinocytosis or phagocytosis. However, cells expressing the FLAG-tagged Ala or Glu mutant actins, but not cells expressing the Flag-tagged wild-type or Phe mutated actins, exhibit serious defects in chemotaxis, streaming and development; the cells chemotax more slowly, do not form streams, and form small mounds which develop into defective fruiting bodies. Cells expressing the Ala and Glu mutated actins have a 50% reduction in cAMP receptor number in both vegetative and developed cells, a 50% reduction in cAMP-induced ERK2 phosphorylation, a 75% reduction in cAMP-induced adenylyl cyclase A activation, and a 30-40% reduction in instant actin polymerization in response to cAMP. These results suggest that the expression of actins with either an Ala and Glu substitution for Tyr-53 (but not the Phe mutation) affects actin filament structure and function in vivo resulting in a substsntial reduction of cAMP receptors on the cell surface, which results in a global inhibition of cAMP-mediated signaling during Dictyostelium development.

今年的研究主要集中在：(1)i类肌球蛋白和其他膜相关蛋白的膜亲和性的分子基础，(2)带tyr1 -53突变的肌动蛋白的生化和生物物理特性，(3)肌动蛋白tyr1 -53突变体的表达对盘基骨桥菌生长发育的影响。