Further Investigation into the Biochemical Effects of Phosphorylation of Tropomyosin Tpm1.1(α). Serine-283 Is in Communication with the Midregion.

The phosphorylated and unphosphorylated forms of tropomyosin Tpm1.1(α) are prepared from adult rabbit heart and compared biochemically. Electrophoresis confirms the high level of enrichment of the chromatography fractions and is consistent with a single site of phosphorylation. Covalently bound phosphate groups at position 283 of Tpm1.1(α) increase the rate of digestion at Leu-169, suggestive of a conformational rearrangement that extends to the midregion. Such a rearrangement, which is supported by ellipticity measurements between 25 and 42 °C, is consistent with a phosphorylation-mediated tightening of the interaction between various myofilament components. In a nonradioactive, co-sedimentation assay [30 mM KCl, 1 mM Mg(II), and 4 °C], phosphorylated Tpm1.1(α) displays a higher affinity for F-actin compared to that of the unphosphorylated control (Kd, 0.16 µM vs 0.26 µM). Phosphorylation decreases the concentration of thin filaments (pCa 4 plus ATP) required to attain a half-maximal rate of release of product from a pre-power stroke complex [myosin-S1-2-deoxy-3-O-(N-methylanthraniloyl)ADP-Pi], as investigated by double-mixing stopped-flow fluorescence, suggestive of a change in the proportion of active (turned on) and inactive (turned off) conformers, but similar maximum rates of product release are observed with either type of reconstituted thin filament. Phosphorylated thin filaments (pCa 4 and 8) display a higher affinity for myosin-S1(ADP) versus the control scenario without affecting isotherm steepness. Specific activities of ATP and Tpm1.1(α) are determined during an in vitro incubation of rat cardiac tissue [12 day-old, 50% phosphorylated Tpm1.1(α)] with [32P]orthophosphate. The incorporation of an isotope into tropomyosin lags behind that of ATP by a factor of approximately 10, indicating that transfer is a comparatively slow process.

Private rare deletions in SEC16A and MAMDC4 may represent novel pathogenic variants in familial axial spondyloarthritis.

OBJECTIVE: Axial spondyloarthritis (AxSpA) represents a group of inflammatory axial diseases that share common clinical and histopathological manifestations. Ankylosing spondylitis (AS) is the best characterised subset of AxSpA, and its genetic basis has been extensively investigated. Given that genome-wide association studies account for only 25% of AS heritability, the objective of this study was to discover rare, highly penetrant genetic variants in AxSpA pathogenesis using a well-characterised, multigenerational family. METHODS: HLA-B*27 genotyping and exome sequencing was performed on DNA collected from available family members. Variant frequency was assessed by mining publically available datasets and using fragment analysis of unrelated AxSpA cases and unaffected controls. Gene expression was performed by qPCR, and protein expression was assessed by western blot analysis and immunofluorescence microscopy using patient-derived B-cell lines. Circular dichroism spectroscopy was performed to assess the impact of discovered variants on secondary structure. RESULTS: This is the first report identifying two rare private familial variants in a multigenerational AxSpA family, an in-frame SEC16A deletion and an out-of-frame MAMDC4 deletion. Evidence suggests the causative mechanism for SEC16A appears to be a conformational change induced by deletion of three highly conserved amino acids from the intrinsically disordered Sec16A N-terminus and RNA-mediated decay for MAMDC4. CONCLUSIONS: The results suggest that it is the presence of rare syntenic SEC16A and MAMDC4 deletions that increases susceptibility to AxSpA in family members who carry the HLA-B*27 allele.

Self-association of an activating natural killer cell receptor, KIR2DS1.

As a major component of the innate immune system, natural killer cells are responsible for activating the cytolytic killing of certain pathogen-infected or tumor cells. The self-recognition of natural killer cells is achieved in part by the killer cell immunoglobulin-like receptors (KIRs) protein family. In the current study, using a suite of biophysical methods, we investigate the self-association of an activating KIR, KIR2DS1. This KIR is of particular interest because when in the presence of the HLA-Cw6 protein, KIR2DS1 becomes a major risk factor for psoriasis, an autoimmune chronic skin disease. Using circular dichroism spectroscopy, dynamic light scattering, and atomic force microscopy, we reveal that KIR2DS1 self-associates in a well-defined fashion. Our novel results on an activating KIR allow us to suggest a working model for the KIR2DS1- HLA class I molecular mechanism.

Cold adaptation of tropomyosin.

The conformational stability of unphosphorylated and phosphorylated α,α-striated tropomyosins from rabbit and shark (95% identical sequences) has been investigated. Three additional core positions are occupied by atypical amino acids in the protein from shark: Thr179(d), Ser190(a), and Ser211(a). These changes are thought to have further destabilized most, if not all, of the carboxyl-terminal half of the molecule. Heat-induced unfolding of shark tropomyosin (2 mg/mL, 0.1 M salt, pH 7) as monitored by far-UV circular dichroism is biphasic [T(m1) ∼ 33 °C (main), and T(m2) ∼ 54 °C] and takes place over a wider temperature span than that of the mammalian protein. The relationship between ellipticity (and excess heat) and temperature is insensitive to the presence in either tropomyosin of covalently bound phosphate. At ∼10 mg/mL, the minor endotherm of shark tropomyosin is shifted to ∼60 °C and T(m2) - T(m1) is increased to 25 °C; otherwise, the results of calorimetry are in agreement with those of circular dichroism. Analyses of cyanogen bromide fragments by far-UV circular dichroism and intact protein by near-UV circular dichroism (T(m) ∼ 32 °C) show that the most stable sizable portion of shark tropomyosin is located within the amino-terminal half of the molecule. These findings illuminate those regions in tropomyosin where flexibility is critical and show that substitutions predicted to be unfavorable in one temperature regime are desirable in another.

Calcium enhances the proteolytic activity of BACE1: An in vitro biophysical and biochemical characterization of the BACE1-calcium interaction.

BACE1 is a novel type I transmembrane aspartyl protease that has been implicated in the pathogenesis of Alzheimer's disease. Cleavage of the amyloid precursor protein by the beta-secretase, BACE1, is the first step in the production of the Abeta peptide and is a prime target for therapeutic intervention. Using circular dichroism, we reveal that the secondary structure of BACE1 in a membrane environment is significantly different from what was determined from the previously resolved crystal structure, and, we provide the first evidence that show differences in stability between the active (pH 4.8) and inactive (pH 7.4) forms of BACE1. In this study we have also examined Ca(2+) binding to BACE1, the effect of this binding on the secondary and tertiary structural characteristics of BACE1, and the influence of this binding on the specific activity of the purified protein. Circular dichroism and endogenous tryptophan fluorescence measurements demonstrated that the secondary and tertiary structures, respectively, are sensitive to increasing concentrations of Ca(2+). Isothermal titration calorimetry was then used to characterize the Ca(2+)-BACE1 interaction in more detail. Our results suggest that there is a high affinity of binding (k(d) = 2.0 x microM) between Ca(2+) and BACE1 and that the binding process was exothermic (DeltaH= -3.5 kcal/mol). We also could demonstrate that low concentrations of Ca(2+) (microM range) significantly increased the proteolytic activity of BACE1. Collectively, these results identify a direct interaction between BACE1 and Ca(2+) and suggest that under physiological conditions, the function(s) of BACE1 must also be influenced by Ca(2+).

Shark skeletal muscle tropomyosin is a phosphoprotein.

Shark skeletal muscle tropomyosin is classified as an alpha-type isoform. The chemical structure is characterised by the absence of cysteine and the presence of a sub-stoichiometric amount of covalently bound phosphate. The protein migrates as a single component on a SDS polyacrylamide gel but is resolved into two components by chromatography and electrophoresis both in the presence of urea at mild alkaline pH. The only detectable difference between these components is the presence of phosphoserine in the tropomyosin form of greater net negative charge. Low ionic strength (pH 7) solutions of phosphorylated shark tropomyosin display significantly higher specific viscosity than unphosphorylated, consistent with the presence of a phosphorylation site within the overlap region, serine 283, as well as conservation of the positively charged amino terminal region. Similar observations were made with tropomyosin prepared from the trunk muscle of Atlantic cod. In a steady-state MgATPase assay, thin filaments (Ca2+) reconstituted with shark phosphorylated tropomyosin activate myosin to a greater extent than those composed of unphosphorylated. The difference is attributable chiefly to a change in Vmax. Skeletal muscle tropomyosin is concluded to be phosphorylated in cartilaginous fishes as well as some teleosts.

Biochemical analysis of the interaction of calcium with toposome: a major protein component of the sea urchin egg and embryo.

We have investigated the biochemical and functional properties of toposome, a major protein component of sea urchin eggs and embryos. Atomic force microscopy was utilized to demonstrate that a Ca(2+)-driven change in secondary structure facilitated toposome binding to a lipid bilayer. Thermal denaturation studies showed that toposome was dependent upon calcium in a manner paralleling the effect of this cation on secondary and tertiary structure. The calcium-induced, secondary, and tertiary structural changes had no effect on the chymotryptic cleavage pattern. However, the digestion pattern of toposome bound to phosphatidyl serine liposomes did vary as a function of calcium concentration. We also investigated the interaction of this protein with various metal ions. Calcium, Mg(2+), Ba(2+), Cd(2+), Mn(2+), and Fe(3+) all bound to toposome. In addition, Cd(2+) and Mn(2+) displaced Ca(2+), prebound to toposome, while Mg(2+), Ba(2+), and Fe(3+) had no effect. Collectively, these results further enhance our understanding of the role of Ca(2+) in modulating the biological activity of toposome.

Interaction of toposome from sea-urchin yolk granules with dimyristoyl phosphatidylserine model membranes: a 2H-NMR study.

The yolk granule is the most abundant membrane-bound organelle present in sea urchin eggs and embryos. The major protein component of this organelle, toposome, accounts for approximately 50% of the total yolk protein and has been shown to be localized to the embryonic cell surface. Extensive characterization in several laboratories has defined a role for toposome in mediating membrane-membrane interactions. The current study expands the analysis of toposome-membrane interaction by defining toposome-induced changes to the lipid bilayer. The effect of toposome on the biophysical properties of phosphatidyl serine (PS) multibilayers was investigated using deuterium nuclear magnetic resonance and perdeuterated dimyristoyl PS (DMPS-d(54)). Toposome was found to have little effect on DMPS-d(54) chain orientational order in both the gel and liquid-crystalline phases. Timescales for DMPS-d(54) reorientation were investigated using quadropole echo decay. Echo decay times were sensitive to toposome in the liquid-crystalline phase but not in the gel phase. Additional information about the perturbation of bilayer motions by toposome was obtained by analyzing its effect on the decay of Carr-Purcell-Meiboom-Gill echo trains. Collectively, these results suggest that toposome interacts peripherally with DMPS bilayers and that it increases the amplitude of lipid reorientation, possibly through local enhancement of bilayer curvature.

Biochemical analysis of a Ca2+-dependent membrane-membrane interaction mediated by the sea urchin yolk granule protein, toposome.

Toposome, a high molecular mass protein, is an abundant component of the yolk granule in the sea urchin egg and embryo. Toposome is composed of a 160 kDa polypeptide that is proteolytically processed into smaller species of 120 and 90 kDa during embryonic development. The exact biological function of toposome during early development is unknown. In this study we have examined calcium binding to toposome and the effect of this binding on the secondary and tertiary structural characteristics of the purified protein. Initially, we used equilibrium dialysis to quantify calcium binding to toposome. Monophasic binding of up to 600 M of calcium per mole of protein was detected with an intrinsic dissociation constant (calcium) of 240 microm. Increasing concentrations of calcium resulted in an increase in alpha helical content from 3.0 to 22.0%, which occurred with an apparent dissociation constant (calcium) of 25 microm. In parallel experiments, toposome binding to liposomes required similar concentrations of calcium; an apparent dissociation constant (calcium) of 25 microm was recorded. Endogenous tryptophan fluorescence measurements, both in the presence and absence of liposomes, demonstrated that the tertiary structure is sensitive to increasing concentrations of calcium with an apparent dissociation constant (calcium) of 240 microm. Toposome-driven, liposome aggregation assays revealed a similar calcium requirement. Collectively, these results define a two-step model for calcium modulation of toposome structure and function.