Acylation-dependent and-independent membrane targeting and distinct functions of small myristoylated proteins (SMPs) in Leishmania major

Trypanosomatid parasites express a number of mono- and diacylated proteins that are targeted to distinct regions of the plasma membrane including the cell body, the flagellum and the flagellar pocket. The extent to which the acylation status and other protein motifs regulate the targeting and/or retention of these proteins to the distinct membrane domains is poorly defined. We have previously described a family of small myristoylated proteins (SMPs) that are either monoacylated (myristoylated) or diacylated (myristoylated and palmitoylated) and targeted to distinct plasma membrane domains. Diacylated SMP-1 is a major constituent of the flagellar membrane, whereas monoacylated SMP-2 resides in the flagellar pocket in Leishmania major. Here, we show that a third SMP family member, monoacylated SMP-4, localizes predominantly to the pellicular membrane. Density gradient centrifugation of detergent-insoluble membranes indicated that SMP-4 was associated with detergent-insoluble domains but was not tightly associated with the subpellicular cytoskeleton. Based on the localisation of truncated SMP proteins, we conclude that the flagellum targeting of SMP-1 is primarily dependent on the dual-acylation motif. In contrast, the localisation of SMP-4 to the cell body membrane is dependent on N-terminal myristoylation and a C-terminal peptide subdomain with a predicted α-helical structure. Strikingly, a SMP-1 chimera containing the SMP-4 C-terminal extension was selectively trafficked to the distal tip of the flagellum and failed to complement the loss of native SMP-1 in a Δsmp1/2 double knockout strain. Collectively, these results suggest that dual acylation is sufficient to target some SMP proteins to the flagellum, while the unique C-terminal extensions of these proteins may confer additional membrane targeting signals that are important for both localisation and SMP function

Esterification of high amylose starch with short chain fatty acids modulates degradation by Bifidobacterium spp.

Large bowel luminal short chain fatty acids (SCFA) are protective against gut diseases such as colorectal cancer, Crohn’s disease and ulcerative colitis. High amylose maize starches acylated with acetic, propionic or butyric were previously shown to deliver SCFA to the gut and increase gut SCFA. This study examines the ability of five Bifidobacterium spp. (B. longum, B. breve, B. infantis, B. adolescentis and B. bifidum), Ruminococcus bromii and Faecalibacterium prausnitzii to degrade starches acylated with SCFA. Release of SCFA from modified starches was not observed, suggesting no preferential hydrolysis of the SCFA ester bond over the glycosidic bond. Acetylated and propionated starches are more readily degraded than either the base or butyrated starches in tested cultures. These observed differences may depend on modification to the starch structure. These structural differences alter the kinetics of starch digestion, irrespective of bacterial type, which may impact current approaches of prebiotic selection.Ya-Mei Lim, Mary B.Barnes, Sally L.Gras, Chris McSweeney, Trevor Lockett, Mary Ann Augustin, Paul R.Goole

Hydrodynamic radii of solubilized high amylose native and modified starches by pulsed field gradient NMR diffusion measurements

Over the last decade, there has been an increase in the application of pulsed field gradient nuclear magnetic resonance (PFG NMR) to characterize food materials. In this work, PFG NMR was used to examine the impact of chemical modification on properties of solubilized high amylose starches obtained from heated (121 °C for 15 min) starch suspensions (0.5% w/w). The starches examined were high amylose maize starch (HAMS) and high amylose maize starch chemically modified with acetate (HAMSA), propionate (HAMSP) and butyrate (HAMSB) at degree of substitution (DS) of 0.2. The hydrothermal treatment solubilized 3.36–4.96% of the starch in all samples, corresponding to concentrations of 10.36–10.91 μM on glucose equivalents basis. The hydrodynamic radii of the starches were 30 Å (HAMS), 45 Å (HAMSB), 60–70 Å (HAMSA & HAMSP). Contrary to expectations, the radii did not increase with the molecular weight of the fatty acid attached to the starch. Rather, the trend in the observed hydrodynamic radii was related to the physical organization of the starch molecules in higher DS granular dried starch (DS = 0.32–0.40) as observed by others using X-ray diffraction and small angle X-ray scattering, suggesting the preservation of the nano-structure of starch molecules upon solubilization. Upon storage (7 days at 25 °C), there was retrogradation of HAMS but not of modified HAMS. Given the correlation between retrogradation and food spoilage, the inhibition of retrogradation in the soluble fraction of modified HAMS may increase the stability of starch containing foods.Ya-Mei Lim, Shenggen Yao, Sally L.Gras, Chris McSweeney, Trevor Lockett, Mary Ann Augustin, Paul R.Goole

β-subunit myristoylation functions as an energy sensor by modulating the dynamics of AMP-activated Protein Kinase

10.1038/srep39417Scientific Reports63941