Mutation in myosin heavy chain 6 causes atrial septal defect.

Atrial septal defect is one of the most common forms of congenital heart malformation. We identified a new locus linked with atrial septal defect on chromosome 14q12 in a large family with dominantly inherited atrial septal defect. The underlying mutation is a missense substitution, I820N, in alpha-myosin heavy chain (MYH6), a structural protein expressed at high levels in the developing atria, which affects the binding of the heavy chain to its regulatory light chain. The cardiac transcription factor TBX5 strongly regulates expression of MYH6, but mutant forms of TBX5, which cause Holt-Oram syndrome, do not. Morpholino knock-down of expression of the chick MYH6 homolog eliminates the formation of the atrial septum without overtly affecting atrial chamber formation. These data provide evidence for a link between a transcription factor, a structural protein and congenital heart disease.

Putting out the fire: coordinated suppression of the innate and adaptive immune systems by SOCS1 and SOCS3 proteins.

The mounting of an effective immune response requires the coordinated function of both the innate and the adaptive arm of the immune system. Cells from both types of immunity respond to antigenic stimuli through a variety of surface and intracellular receptors and produce cytokines that tightly orchestrate the inflammatory response. The operation of feedback control mechanisms that regulate the duration and the amplitude of antigenic and cytokine receptor signaling is therefore required to prevent hyper-activation of the immune system that could lead to tissue destruction or autoimmunity. Suppressor of cytokine signaling (SOCS) proteins have been identified as a negative feedback loop to cytokine signaling. Recently, the generation of genetically engineered mouse models permitted the evaluation of their function in different processes of the immune responses. In this article, we review new insights into the modular structure of SOCS proteins and the function of SOCS1 and SOCS3 to negatively regulate activation and/or differentiation pathways in macrophages, dendritic cells, and T lymphocytes. Thus, SOCS family proteins are components of an emerging immunoregulatory mechanism that maintains the coordinated balance of both innate and adaptive immune responses.

A re-evaluation of the role of type IV antifreeze protein.

A lipoprotein-like antifreeze protein (type IV AFP) has previously been isolated only from the blood plasma of the longhorn sculpin. However, the plasma antifreeze activity in all individuals of this species tested from Newfoundland and New Brunswick waters ranges from low to undetectable. A close relative of the longhorn sculpin, the shorthorn sculpin, does have appreciable antifreeze activity in its blood but this is virtually all accounted for by the alpha-helical, alanine-rich type I AFP, other isoforms of which are also present in the skin of both fishes. We have characterized a putative ortholog of type IV AFP in shorthorn sculpin by cDNA cloning. This 12.2-kDa Gln-rich protein is 87% identical to the longhorn sculpin's type IV AFP. Recombinant versions of both orthologs were produced in bacteria and shown to have antifreeze activity. Immunoblotting with antibodies raised to type IV AFP shows this protein present in longhorn sculpin plasma at levels of less than 100 microg/mL, which are far too low to protect the blood from freezing at the temperature of icy seawater. This confirms the results of direct antifreeze assays on the plasmas. It appears that type IV AFP has the potential to develop as a functional antifreeze in these fishes but may not have been selected for this role because of the presence of type I AFP. Consistent with this hypothesis is the observation that the type IV AFP gene has not been amplified the way functional antifreeze protein genes have in all other species examined.

Metal ion-dependent, reversible, protein filament formation by designed beta-roll polypeptides.

BACKGROUND: A right-handed, calcium-dependent beta-roll structure found in secreted proteases and repeat-in-toxin proteins was used as a template for the design of minimal, soluble, monomeric polypeptides that would fold in the presence of Ca2+. Two polypeptides were synthesised to contain two and four metal-binding sites, respectively, and exploit stacked tryptophan pairs to stabilise the fold and report on the conformational state of the polypeptide. RESULTS: Initial analysis of the two polypeptides in the presence of calcium suggested the polypeptides were disordered. The addition of lanthanum to these peptides caused aggregation. Upon further study by right angle light scattering and electron microscopy, the aggregates were identified as ordered protein filaments that required lanthanum to polymerize. These filaments could be disassembled by the addition of a chelating agent. A simple head-to-tail model is proposed for filament formation that explains the metal ion-dependency. The model is supported by the capping of one of the polypeptides with biotin, which disrupts filament formation and provides the ability to control the average length of the filaments. CONCLUSION: Metal ion-dependent, reversible protein filament formation is demonstrated for two designed polypeptides. The polypeptides form filaments that are approximately 3 nm in diameter and several hundred nm in length. They are not amyloid-like in nature as demonstrated by their behaviour in the presence of congo red and thioflavin T. A capping strategy allows for the control of filament length and for potential applications including the "decoration" of a protein filament with various functional moieties.

Expression and purification of sea raven type II antifreeze protein from Drosophila melanogaster S2 cells.

We present a system for the expression and purification of recombinant sea raven type II antifreeze protein, a cysteine-rich, C-type lectin-like globular protein that has proved to be a difficult target for recombinant expression and purification. The cDNAs encoding the pro- and mature forms of the sea raven protein were cloned into a modified pMT Drosophila expression vector. These constructs produced N-terminally His(6)-tagged pro- and mature forms of the type II antifreeze protein under the control of a metallothionein promoter when transfected into Drosophila melanogaster S2 cells. Upon induction of stable cell lines the two proteins were expressed at high levels and secreted into the medium. The proteins were then purified from the cell medium in a simple and rapid protocol using immobilized metal affinity chromatography and specific protease cleavage by tobacco etch virus protease. The proteins demonstrated antifreeze activity indistinguishable from that of wild-type sea raven antifreeze protein purified from serum as illustrated by ice affinity purification, ice crystal morphology, and their ability to inhibit ice crystal growth. This expression and purification system gave yields of 95 mg/L of fully active mature sea raven type II AFP and 9.6 mg/L of the proprotein. This surpasses all previous attempts to express this protein in Escherichia coli, baculovirus-infected fall armyworm cells and Pichia pastoris and will provide sufficient protein for structural analysis.

The basis for hyperactivity of antifreeze proteins.

Antifreeze proteins (AFPs) bind to the surface of ice crystals and lower the non-equilibrium freezing temperature of the icy solution below its melting point. We have recently reported the discovery of three novel hyperactive AFPs from a bacterium, a primitive insect and a fish, which, like two hyperactive AFPs previously recognized in beetles and moths, are considerably better at depressing the freezing point than most fish AFPs. When cooled below the non-equilibrium freezing temperature, ice crystals formed in the presence of any of five distinct, moderately active fish AFPs grow suddenly along the c-axis. Ice crystals formed in the presence of any of the five evolutionarily and structurally distinct hyperactive AFPs remain stable to lower temperatures, and then grow explosively in a direction normal to the c-axis when cooled below the freezing temperature. We argue that this one consistent distinction in the behaviour of these two classes of AFPs is the key to hyperactivity. Whereas both AFP classes bind irreversibly to ice, the hyperactive AFPs are better at preventing ice growth out of the basal planes.