David B. Hayes

Leiomodin is an Actin Filament Nucleator in Muscle Cells

Initiation of actin polymerization in cells requires nucleation factors. Here we describe an actin-binding protein, leiomodin, that acted as a strong filament nucleator in muscle cells. Leiomodin shared two actin-binding sites with the filament pointed end–capping protein tropomodulin: a flexible N-terminal region and a leucine-rich repeat domain. Leiomodin also contained a C-terminal extension of 150 residues. The smallest fragment with strong nucleation activity included the leucine-rich repeat and C-terminal extension. The N-terminal region enhanced the nucleation activity threefold and recruited tropomyosin, which weakly stimulated nucleation and mediated localization of leiomodin to the middle of muscle sarcomeres. Knocking down leiomodin severely compromised sarcomere assembly in cultured muscle cells, which suggests a role for leiomodin in the nucleation of tropomyosin-decorated filaments in muscles.

Phosphorylation Dependence of Hsp27 Multimeric Size and Molecular Chaperone Function

The molecular chaperone Hsp27 exists as a distribution of large oligomers that are disassembled by phosphorylation at Ser-15, -78, and -82. It is controversial whether the unphosphorylated Hsp27 or the widely used triple Ser-to-Asp phospho-mimic mutant is the more active molecular chaperone in vitro. This question was investigated here by correlating chaperone activity, as measured by the aggregation of reduced insulin or α-lactalbumin, with Hsp27 self-association as monitored by analytical ultracentrifugation. Furthermore, because the phospho-mimic is generally assumed to reproduce the phosphorylated molecule, the size and chaperone activity of phosphorylated Hsp27 were compared with that of the phospho-mimic. Hsp27 was triply phosphorylated by MAPKAP-2 kinase, and phosphorylation was tracked by urea-PAGE. An increasing degree of suppression of insulin or α-lactalbumin aggregation correlated with a decreasing Hsp27 self-association, which was the least for phosphorylated Hsp27 followed by the mimic followed by the unphosphorylated protein. It was also found that Hsp27 added to pre-aggregated insulin did not reverse aggregation but did inhibit these aggregates from assembling into even larger aggregates. This chaperone activity appears to be independent of Hsp27 phosphorylation. In conclusion, the most active chaperone of insulin and α-lactalbumin was the Hsp27 (elongated) dimer, the smallest Hsp27 subunit observed under physiological conditions. Next, the Hsp27 phospho-mimic is only a partial mimic of phosphorylated Hsp27, both in self-association and in chaperone function. Finally, the efficient inhibition of insulin aggregation by Hsp27 dimer led to the proposal of two models for this chaperone activity.

X-Ray Scattering Study of Activated Arp2/3 Complex with Bound Actin-WCA

Previous structures of Arp2/3 complex, determined in the absence of a nucleation-promoting factor and actin, reveal its inactive conformation. The study of the activated structure has been hampered by uncontrollable polymerization. We have engineered a stable activated complex consisting of Arp2/3 complex, the WCA activator region of N-WASP, and one actin monomer, and studied its structure in solution by small angle X-ray scattering (SAXS). The scattering data support a model in which the first actin subunit binds at the barbed end of Arp2, and disqualify an alternative model that places the first actin subunit at the barbed end of Arp3. This location of the first actin and bound W motif constrains the binding site of the C motif to subunits Arp2 and ARPC1, from where the A motif can reach subunits Arp3 and ARPC3. The results support a model of activation that is consistent with most of the biochemical observations.

GDF11 Does Not Rescue Aging-Related Pathological Hypertrophy

RATIONALE Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor-β super family of secreted factors. A recent study showed that reduced GDF11 blood levels with aging was associated with pathological cardiac hypertrophy (PCH) and restoring GDF11 to normal levels in old mice rescued PCH. OBJECTIVE To determine whether and by what mechanism GDF11 rescues aging dependent PCH. METHODS AND RESULTS Twenty-four-month-old C57BL/6 mice were given a daily injection of either recombinant (r) GDF11 at 0.1 mg/kg or vehicle for 28 days. rGDF11 bioactivity was confirmed in vitro. After treatment, rGDF11 levels were significantly increased, but there was no significant effect on either heart weight or body weight. Heart weight/body weight ratios of old mice were not different from 8- or 12-week-old animals, and the PCH marker atrial natriuretic peptide was not different in young versus old mice. Ejection fraction, internal ventricular dimension, and septal wall thickness were not significantly different between rGDF11 and vehicle-treated animals at baseline and remained unchanged at 1, 2, and 4 weeks of treatment. There was no difference in myocyte cross-sectional area rGDF11 versus vehicle-treated old animals. In vitro studies using phenylephrine-treated neonatal rat ventricular myocytes, to explore the putative antihypertrophic effects of GDF11, showed that GDF11 did not reduce neonatal rat ventricular myocytes hypertrophy, but instead induced hypertrophy. CONCLUSIONS Our studies show that there is no age-related PCH in disease-free 24-month-old C57BL/6 mice and that restoring GDF11 in old mice has no effect on cardiac structure or function.

X-ray scattering study of actin polymerization nuclei assembled by tandem W domains

The initiation of actin polymerization in cells requires actin filament nucleators. With the exception of formins, known filament nucleators use the Wiskott–Aldrich syndrome protein (WASP) homology 2 (WH2 or W) domain for interaction with actin. A common architecture, found in Spire, Cobl, VopL, and VopF, consists of tandem W domains that tie together three to four actin monomers to form a polymerization nucleus. Uncontrollable polymerization has prevented the structural investigation of such nuclei. We have engineered stable nuclei consisting of an actin dimer and a trimer stabilized by tandem W domain hybrid constructs and studied their structures in solution by x-ray scattering. We show that Spire-like tandem W domains stabilize a polymerization nucleus by lining up actin subunits along the long-pitch helix of the actin filament. Intersubunit contacts in the polymerization nucleus, thought to involve the DNase I-binding loop of actin, coexist with the binding of the W domain in the cleft between actin subdomains 1 and 3. The successful stabilization of filament-like multiactin assemblies opens the way to the crystallographic investigation of intersubunit contacts in the actin filament.

Crystal structure of the actin-binding domain of α-actinin-4 Lys255Glu mutant implicated in focal segmental glomerulosclerosis

Mutations in alpha-actinin-4 have been linked to familial focal segmental glomerulosclerosis (FSGS), a common renal disorder in humans, and produce an apparent increase in the actin-binding affinity of alpha-actinin-4 in vitro. One of the mutations, in particular, Lys255Glu, falls in the middle of the actin-binding interface of the actin-binding domain (ABD). The ABD consists of tandem calponin homology (CH) domains (CH1 and CH2). The crystal structures of most ABDs display a compact conformation, characterized by extensive inter-CH interactions. However, the conformation of F-actin-bound ABDs is unsettled. Some electron microscopy studies find that the compact conformation is preserved upon binding to F-actin, whereas other studies suggest that the CHs separate and the ABD becomes extended. The Lys255Glu mutation in CH2 is significant in this regard since it removes a crucial inter-CH interaction with Trp147 of CH1, thought to stabilize the compact conformation. Together, the increased actin-binding affinity and the removal of this important inter-CH contact suggested that the Lys255Glu mutation might facilitate the transition toward the open ABD conformation proposed by some of the electron microscopy studies. However, the crystal structure of the ABD of alpha-actinin-4 Lys255Glu mutant described here displays the canonical compact conformation. Furthermore, the sedimentation coefficients by analytical ultracentrifugation of wild-type and FSGS mutant ABDs (Lys255Glu, Ser262Pro, and Thr259Ile) are nearly identical (2.50+/-0.03 S) and are in good agreement with the theoretical value calculated from the crystal structure (2.382 S), implying that the compact conformation is retained in solution. The absence of a structural change suggests that the compact ABD conformation observed in the majority of the structures is highly stable and is preserved in solution, even in FSGS mutant ABDs.

Toxofilin from Toxoplasma gondii forms a ternary complex with an antiparallel actin dimer

Many human pathogens exploit the actin cytoskeleton during infection, including Toxoplasma gondii, an apicomplexan parasite related to Plasmodium, the agent of malaria. One of the most abundantly expressed proteins of T. gondii is toxofilin, a monomeric actin-binding protein (ABP) involved in invasion. Toxofilin is found in rhoptry and presents an N-terminal signal sequence, consistent with its being secreted during invasion. We report the structure of toxofilin amino acids 69–196 in complex with the host mammalian actin. Toxofilin presents an extended conformation and interacts with an antiparallel actin dimer, in which one of the actins is related by crystal symmetry. Consistent with this observation, analytical ultracentrifugation analysis shows that toxofilin binds two actins in solution. Toxofilin folds into five consecutive helices, which form three relatively independent actin-binding sites. Helices 1 and 2 bind the symmetry-related actin molecule and cover its nucleotide-binding cleft. Helices 3–5 bind the other actin and constitute the primary actin-binding region. Helix 3 interacts in the cleft between subdomains 1 and 3, a common binding site for most ABPs. Helices 4 and 5 wrap around actin subdomain 4, and residue Gln-134 of helix 4 makes a hydrogen-bonding contact with the nucleotide in actin, both of which are unique features among ABPs. Toxofilin dramatically inhibits nucleotide exchange on two actin molecules simultaneously. This effect is linked to the formation of the antiparallel actin dimer because a construct lacking helices 1 and 2 binds only one actin and inhibits nucleotide exchange less potently.

On Line Data Acquisition for the Rayleigh Interference Optical System of the Analytical Ultracentrifuge

The fundamental measurement in analytical ultracentrifugation is the concentration as a function of radial position. The Rayleigh interferometer of the analytical ultracentrifuge produces a cell image in which the concentration at each radial position is presented as the vertical displacement of a set of equally-spaced horizontal fringes (Richards and Schachman, 1959). Manual acquisition of data from interferograms is tedious and automated photographic plate readers still require that photographs be taken, processed, aligned and read before data analysis can be performed. Fortunately, a Rayleigh interference image is well suited for television- camera-based data acquisition. Described here are two types of automated Rayleigh interferometers for the Beckman Model E analytical ultracentrifuge. One type of system relays and magnifies the Rayleigh interference image from the usual photographic plane to a television camera located behind this plane. The other system uses a redesigned camera- cylinder lens combination to create a radially demagnified Rayleigh interference image of the cell on the television camera sensor located on the original cylinder lens mount.

Characterization of the self‐association of human interferon‐α2b, albinterferon‐α2b, and pegasys

The self-association of human interferon-α2b (hIFN-α2b), albinterferon-α2b (a recombinant protein with human serum albumin and hIFN-α2b peptides fused together in a single polypeptide chain), and Pegasys (PEGylated hIFN-α2a) was characterized by analytical ultracentrifugation analyses. By examining the apparent sedimentation coefficient distribution profiles of each protein at different concentrations, it was concluded that the above three proteins are self-associating in albinterferon-α2b formulation buffer. By model fitting of sedimentation data using SEDANAL software, the stoichiometry and equilibrium constants of the self-association of these proteins were characterized. The self-association of hIFN-α2b results in the formation of stable dimers, fast-reversible tetramers, octamers, and hexadecamers. In contrast, although both albinterferon-α2b and Pegasys are self-associated, their self-association stoichiometries are significantly different from that of hIFN-α2b. The self-association of albinterferon-α2b results in the formation of reversible dimers and trimers, whereas the self-association of Pegasys gives only reversible dimers. The self-association behaviors of hIFN-α2b and albinterferon-α2b involves attractive electrostatic forces, which can be suppressed to a negligible level in low pH (pH 4.0-4.5) and high salt concentration (400 mM NaCl) buffer, allowing quantification of their size variant contents by sedimentation velocity analysis.

SEDVIEW, Real-time Sedimentation Analysis

The ability to obtain a sedimentation coefficient distribution as the run proceeds, and to get an early idea of the quality of a particular sample, has not been made available in real-time during the run in any of the existing software packages. It is desirable on many occasions to be able to see the number of components present in a sample at an early stage of the run. The ability to ascertain the extent of heterogeneity of sample would help enormously to reduce the amount of time that is necessary to obtain that information. Most software packages currently available require that the run be completed before analysis is carried out or at least some of the early scans analyzed off-line to determine if the run should continue. A software package called SEDVIEW has been developed by us to allow early analysis in real-time.