Effects on the ubiquitin proteasome system after closed soft-tissue trauma in rat skeletal muscle.

Previous studies have suggested that an increased catabolic stage of skeletal muscle in pathological situations is mainly a reflection of ubiquitin-proteasome system-controlled proteolysis. The proteolytic mechanisms that occur after local muscle trauma are poorly defined. We investigated the effects of closed soft-tissue trauma on ubiquitin-proteasome dependent protein breakdown in rats (n = 25). The enzymatic activities of the ubiquitination and proteasome reactions were both reduced (p < 0.05) immediately after contusion of the hind limb musculus extensor digitorum longus. The same effect was observed in extracts of lung tissue from the injured animals. Cellular levels of free and protein-conjugated ubiquitin were significantly elevated upon decreased proteolytic activity. Our data support an early-state anti-proteolytic role of the ubiquitin-proteasome pathway after local injury. This further implies that there is a yet-to-be elucidated complex regulatory mechanism of muscle regeneration that involves various proteolytic systems.

Cold-sensitive mutants G680V and G691C of Dictyostelium myosin II confer dramatically different biochemical defects.

Cold-sensitive myosin mutants represent powerful tools for dissecting discrete deficiencies in myosin function. Biochemical characterization of two such mutants, G680V and G691C, has allowed us to identify separate facets of myosin motor function perturbed by each alteration. Compared with wild type, the G680V myosin exhibits a substantially enhanced affinity for several nucleotides, decreased ATPase activity, and overoccupancy or creation of a novel strongly actin-binding state. The properties of the novel strong binding state are consistent with a partial arrest or pausing at the onset of the mechanical stroke. The G691C mutant, on the other hand, exhibits an elevated basal ATPase indicative of premature phosphate release. By releasing phosphate without a requirement for actin binding, the G691C can bypass the part of the cycle involving the mechanical stroke. The two mutants, despite having alterations in glycine residues separated by only 11 residues, have dramatically different consequences on the mechanochemical cycle.

Structure-function studies of the myosin motor domain: importance of the 50-kDa cleft.

We used random mutagenesis to create 21 point mutations in a highly conserved region of the motor domain of Dictyostelium myosin and classified them into three distinct groups based on the ability to complement myosin null cell phenotypes: wild type, intermediate, and null. Biochemical analysis of the mutated myosins also revealed three classes of mutants that correlated well with the phenotypic classification. The mutated myosins that were not fully functional showed defects ranging from ATP nonhydrolyzers to myosins whose enzymatic and mechanical properties are uncoupled. Placement of the mutations onto the three-dimensional structure of myosin showed that the mutated region lay along the cleft that separates the active site from the actin-binding domain and that has been shown to move in response to changes at the active site. These results demonstrate that this region of myosin plays a key role in transduction of chemical energy to mechanical displacement.

Structure-function analysis of the motor domain of myosin.

Motor proteins perform a wide variety of functions in all eukaryotic cells. Recent advances in the structural and mutagenic analysis of the myosin motor has led to insights into how these motors transduce chemical energy into mechanical work. This review focuses on the analysis of the effects of myosin mutations from a variety of organisms on the in vivo and in vitro properties of this ubiquitous motor and illustrates the positions of these mutations on the high-resolution three-dimensional structure of the myosin motor domain.

Myosin structure/function: a combined mutagenesis-crystallography approach.

In the past year, the structure of the regulatory domain of scallop myosin has joined that of the chicken skeletal muscle myosin subfragment 1 and provided insights into the regulation of myosin function. Mutagenesis studies in a variety of systems have used the information provided by these structures to create mutant myosins to test models of chemomechanical transduction and its regulation.

Myosin motor function: structural and mutagenic approaches.

Recent advances in three areas of myosin research--structural biology, in vitro motility assays, and mutagenesis--are leading to a new understanding of the molecular mechanism of chemomechanical transduction by this motor protein. Highlights include rational design of mutants using the crystal structure of subfragment 1, combined in vivo and in vitro mutant analyses using Dictyostelium, and the emergence of baculovirus as an in vitro system for expression of mutated mammalian myosins.

Role of highly conserved lysine 130 of myosin motor domain. In vivo and in vitro characterization of site specifically mutated myosin.

We have created a mutant Dictyostelium myosin II heavy chain gene in which a highly conserved lysine residue (Lys-130) is changed to leucine. Lys-130 is a residue that is known to be trimethylated in skeletal muscle myosin and had been thought to play an integral role in the interaction of myosin with ATP during the actomyosin chemomechanical cycle. We report here the first in vivo and in vitro characterization of an engineered missense mutation in the motor domain of myosin. Expression of the K130L myosin in a Dictyostelium strain that lacks the myosin II heavy chain gene is sufficient to restore the ability of that cell line to undergo cytokinesis and multicellular development, processes that require functional myosin. The K130L myosin purified from these cells displays maximal actin-activated ATPase activities and promotes maximal sliding velocities of actin filaments in an in vitro motility assay that are comparable with those of wild-type myosin. These results demonstrate that this lysine residue is not required for the enzymatic or motile activities of myosin. However, the mutant protein exhibits a 4-fold increase in Km for ATP over wild-type myosin, indicating that this residue participates in the interaction of myosin with its nucleotide substrate.

Enzymatic activities correlate with chimaeric substitutions at the actin-binding face of myosin.

Myosins are a functionally divergent group of mechanochemical enzymes involved in various motile activities in cells. Despite a high degree of conservation in the amino-acid sequence of the 130K motor domain (head region) of the molecule, there are large differences in the enzymatic and motile activities (Tables 1 and 2) of myosins from diverse species and cell types. However, the degree of conservation is not uniform throughout the head sequence; therefore, one reasonable hypothesis is that the functional differences between myosins derive from the poorly conserved areas. The most prominent divergent region occurs at the 50K/20K junction, a region of the molecule sensitive to proteolytic digestion and a binding site for actin. We have now constructed chimaeras of this region of myosin by substituting the 9-amino-acid Dictyostelium junction region with those from myosins from other species and find that the actin-activated ATPase correlates well with the activity of the myosin from which the junction region was derived. Our results suggest that this region, likely to be part of the myosin head that interacts directly with actin, is important in determining the enzymatic activity of myosin.

Molecular genetic approaches to the cytoskeleton in Dictyostelium.

Recent advances in molecular genetic techniques are being applied in Dictyostelium to test and expand prevailing views on the functioning of the actin-based cytoskeleton. Current research involves the disruption, by homologous recombination, of genes encoding cytoskeletal elements. We suggest combining classical and molecular genetic approaches to supplement these investigations.

Manipulation and expression of molecular motors in Dictyostelium discoideum.

The eukaryote Dictyostelium discoideum is an attractive model organism for the study of cytoskeletal proteins and cell motility. The appearance and behavior of this cell closely resembles that of mammalian cells, but unlike mammalian cells, Dictyostelium offers the opportunity specifically to alter the cell physiology by molecular genetic approaches.

Expression and characterization of a functional myosin head fragment in Dictyostelium discoideum.

The isolated head fragment of myosin is a motor protein that is able to use energy liberated from the hydrolysis of adenosine triphosphate to cause sliding movement of actin filaments. Expression of a myosin fragment nearly equivalent to the amino-terminal globular head domain, generally referred to as subfragment 1, has been achieved by transforming the eukaryotic organism Dictyostelium discoideum with a plasmid that carries a 2.6-kilobase fragment of the cloned Dictyostelium myosin heavy chain gene under the control of the Dictyostelium actin-15 promoter. The recombinant fragment of the myosin heavy chain was purified 2400-fold from one of the resulting cell lines and was found to be functional by the following criteria: the myosin head fragment copurified with the essential and regulatory myosin light chains, decorated actin filaments, and displayed actin-activated adenosine triphosphatase activity. In addition, motility assays in vitro showed that the recombinant myosin fragment is capable of supporting sliding movement of actin filaments.