Altering the stability of the Cdc8 overlap region modulates the ability of this tropomyosin to bind cooperatively to actin and regulate myosin.

Tropomyosin (Tm) is an evolutionarily conserved ?-helical coiled-coil protein, dimers of which form end-to-end polymers capable of associating with and stabilising actin-filaments and regulate myosin function. The fission yeast, Schizosaccharomyces pombe, possesses a single essential Tm, Cdc8, which can be acetylated on its amino terminal methionine to increase its affinity for actin and enhance its ability to regulate myosin function. We have designed and generated a number of novel Cdc8 mutant proteins with amino terminal substitutions to explore how stability of the Cdc8-polymer overlap region affects the regulatory function of this Tm. By correlating the stability of each protein, its propensity to form stable polymers, its ability to associate with actin and to regulate myosin, we have shown the stability of the amino terminal of the Cdc8 ?-helix is crucial for Tm function. In addition we have identified a novel Cdc8 mutant with increased amino-terminal stability, dimers of which are capable of forming Tm-polymers significantly longer than the wild-type protein. This protein had a reduced affinity for actin with respect to wild type, and was unable to regulate actomyosin interactions. The data presented here are consistent with acetylation providing a mechanism for modulating the formation and stability of Cdc8 polymers within the fission yeast cell. The data also provide evidence for a mechanism in which Tm dimers form end-to-end polymers on the actin-filament, consistent with a cooperative model for Tm binding to actin

Formins Determine the Functional Properties of Actin Filaments in Yeast

The actin cytoskeleton executes a broad range of essential functions within a living cell. The dynamic nature of the actin polymer is modulated to facilitate specific cellular processes at discrete locations by actin-binding proteins (ABPs), including the formins and tropomyosins (Tms). Formins nucleate actin polymers, while Tms are conserved dimeric proteins that form polymers along the length of actin filaments. Cells possess different Tm isoforms, each capable of differentially regulating the dynamic and func- tional properties of the actin polymer. However, the mecha- nism by which a particular Tm localizes to a specific actin polymer is unknown. Here we show that specific formin family members dictate which Tm isoform will associate with a particular actin filament to modulate its dynamic and functional properties at specific cellular locations. Exchanging the localization of the fission yeast formins For3 and Cdc12 results in an exchange in localizations of Tm forms on actin polymers. This nucleator-driven switch in filament composition is reflected in a switch in actin dynamics, together with a corresponding change in the filament’s ability to regulate ABPs and myosin motor activity. These data establish a role for formins in dictating which specific Tm variant will associate with a growing actin filament and therefore specify the functional capacity of the actin filaments that they create

Influence of emulsifier type on the spray-drying properties of model infant formula emulsions

The objective of this study was to compare the drying performance and physicochemical properties of model infant formula (IF) emulsions containing 43, 96 and 192 g L−1 protein, oil and maltodextrin (MD), respectively, prepared using different emulsifier systems. Emulsions were stabilised using either whey protein isolate (WPI), whey protein hydrolysate (WPH; DH 8%), WPH + CITREM (9 g L−1), WPH + lecithin (5 g L−1) or WPH conjugated with maltodextrin (DE 12) (WPH-MD). Homogenised emulsions had 32% solids content and oil globules with mean volume diameter WPH + LEC > WPH > WPH- MD > WPI, WPI > WPH > WPH- MD > WPH + LEC > WPH + CIT and WPH- MD > WPI > WPH > WPH + LEC > WPH + CIT, respectively. Additionally, differences in wettability, surface topography and oil globule distribution within the powder matrix and in reconstituted powders were linked to the emulsifier system used. Inclusion of the WPH-MD conjugate in the formulation of IF powder significantly improved drying behaviour and physicochemical properties of the resultant powder, as evidenced by lowest powder build-up during drying and greatest emulsion quality on reconstitution, compared to the other model formula systems

Characterisation of heat-induced protein aggregation in whey protein isolate and the influence of aggregation on the availability of amino groups as measured by the ortho-phthaldialdehyde (OPA) and trinitrobenzenesulfonic acid (TNBS) methods

Whey protein isolate (WPI) solutions, with different levels of aggregated protein, were prepared by heating (5% protein, pH 7, 90 °C for 30 min) WPI solutions with either 20 mM added NaCl (WPI + NaCl), 5 mM N-ethylmaleimide (WPI + NEM) or 20 mM added NaCl and 5 mM NEM (WPI + NaCl + NEM). Gel electrophoresis demonstrated that the heated WPI and WPI + NaCl solutions had higher levels of aggregated protein, due to more covalent interactions between proteins, than the heated WPI + NEM and WPI + NaCl + NEM solutions. There were marked differences in the levels of amino groups between all heated WPI solutions when measured by the OPA and TNBS methods, with lower levels being measured by the TNBS method than by the OPA method. These results demonstrate that the measurement of available amino groups by the OPA method is less impacted than by the TNBS method after heat-induced structural changes, arising from disulfide or sulfhydryl-disulfide bond-mediated aggregation of whey protein molecules

A review of the analytical approaches used for studying the structure, interactions and stability of emulsions in nutritional beverage systems

Nutritional beverage emulsions contain water and oil, stabilised by surfactants, and are both diverse and complex. Their susceptibility to changes induced by manufacturing processes and on storage, results in challenges with their stability, quality and shelf-life. An understanding of the relationship between structure and stability of an emulsion is essential to designing and competently formulating food products with the desired nutritional functionality and sensory properties, while achieving the required shelf-life. This article critically reviews a selection of commonly-used analytical approaches focused on characterisation of emulsion structure in the context of emulsion formation, techno-functional properties and stability to intrinsic and environmental factors

Identification of sequence changes in myosin II that adjust muscle contraction velocity.

The speed of muscle contraction is related to body size; muscles in larger species contract at slower rates. Since contraction speed is a property of the myosin isoform expressed in a muscle, we investigated how sequence changes in a range of muscle myosin II isoforms enable this slower rate of muscle contraction. We considered 798 sequences from 13 mammalian myosin II isoforms to identify any adaptation to increasing body mass. We identified a correlation between body mass and sequence divergence for the motor domain of the 4 major adult myosin II isoforms (β/Type I, IIa, IIb, and IIx), suggesting that these isoforms have adapted to increasing body mass. In contrast, the non-muscle and developmental isoforms show no correlation of sequence divergence with body mass. Analysis of the motor domain sequence of β-myosin (predominant myosin in Type I/slow and cardiac muscle) from 67 mammals from 2 distinct clades identifies 16 sites, out of 800, associated with body mass (padj 0.05). Both clades change the same small set of amino acids, in the same order from small to large mammals, suggesting a limited number of ways in which contraction velocity can be successfully manipulated. To test this relationship, the 9 sites that differ between human and rat were mutated in the human β-myosin to match the rat sequence. Biochemical analysis revealed that the rat-human β-myosin chimera functioned like the native rat myosin with a 2-fold increase in both motility and in the rate of ADP release from the actin-myosin crossbridge (the step that limits contraction velocity). Thus, these sequence changes indicate adaptation of β-myosin as species mass increased to enable a reduced contraction velocity and heart rate

Inositol Phosphate Recycling Regulates Glycolytic and Lipid Metabolism That Drives Cancer Aggressiveness

Cancer cells possess fundamentally altered metabolism that supports their pathogenic features, which includes a heightened reliance on aerobic glycolysis to provide precursors for synthesis of biomass. We show here that inositol polyphosphate phosphatase 1 (INPP1) is highly expressed in aggressive human cancer cells and primary high-grade human tumors. Inactivation of INPP1 leads to a reduction in glycolytic intermediates that feed into the synthesis of the oncogenic signaling lipid lysophosphatidic acid (LPA), which in turn impairs LPA signaling and further attenuates glycolytic metabolism in a feed-forward mechanism to impair cancer cell motility, invasiveness, and tumorigenicity. Taken together these findings reveal a novel mode of glycolytic control in cancer cells that can serve to promote key oncogenic lipid signaling pathways that drive cancer pathogenicity

Temperature sensitive point mutations in fission yeast tropomyosin have long range effects on the stability and function of the actin- tropomyosin copolymer

The actin cytoskeleton is modulated by regulatory actin-binding proteins which fine- tune the dynamic properties of the actin polymer to regulate function. One such actin-binding protein is tropomyosin (Tpm), a highly-conserved alpha-helical dimer which stabilises actin and regulates interactions with other proteins. Temperature sensitive mutants of Tpm are invaluable tools in the study of actin filament dependent processes, critical to the viability of a cell. Here we investigated the molecular basis of the temperature sensitivity of fission yeast Tpm mutants which fail to undergo cytokinesis at the restrictive temperatures. Comparison of Contractile Actomyosin Ring (CAR) constriction as well as cell shape and size revealed the cdc8.110 or cdc8.27 mutant alleles displayed significant differences in their temperature sensitivity and impact upon actin dependent functions during the cell cycle. In vitro analysis revealed the mutant proteins displayed a different reduction in thermostability, and unexpectedly yield two discrete unfolding domains when acetylated on their amino-termini. Our findings demonstrate how subtle changes in structure (point mutations or acetylation) alter the stability not simply of discrete regions of this conserved cytoskeletal protein but of the whole molecule. This differentially impacts the stability and cellular organisation of this essential cytoskeletal protein