Oligomeric state study of prokaryotic rhomboid proteases

AbstractRhomboid peptidases (proteases) play key roles in signaling events at the membrane bilayer. Understanding the regulation of rhomboid function is crucial for insight into its mechanism of action. Here we examine the oligomeric state of three different rhomboid proteases. We subjected Haemophilus influenzae, (hiGlpG), Escherichia coli GlpG (ecGlpG) and Bacillus subtilis (YqgP) to sedimentation equilibrium analysis in detergent-solubilized dodecylmaltoside (DDM) solution. For hiGlpG and ecGlpG, rhomboids consisting of the core 6 transmembrane domains without and with soluble domains respectively, and YqgP, predicted to have 7 transmembrane domains with larger soluble domains at the termini, the predominant species was dimeric with low amounts of monomer and tetramers observed. To examine the effect of the membrane domain alone on oligomeric state of rhomboid, hiGlpG, the simplest form from the rhomboid class of intramembrane proteases representing the canonical rhomboid core of six transmembrane domains, was studied further. Using gel filtration and crosslinking we demonstrate that hiGlpG is dimeric and functional in DDM detergent solution. More importantly co-immunoprecipitation studies demonstrate that the dimer is present in the lipid bilayer suggesting a physiological dimer. Overall these results indicate that rhomboids form oligomers which are facilitated by the membrane domain. For hiGlpG we have shown that these oligomers exist in the lipid bilayer. This is the first detailed oligomeric state characterization of the rhomboid family of peptidases

Acyl-CoA:diacylglycerol acyltransferase: Properties, physiological roles, metabolic engineering and intentional control

Acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the last reaction in the acyl-CoA-dependent biosynthesis of triacylglycerol (TAG). DGAT activity resides mainly in DGAT1 and DGAT2 in eukaryotes and bifunctional wax ester synthase-diacylglycerol acyltransferase (WSD) in bacteria, which are all membrane-bound proteins but exhibit no sequence homology to each other. Recent studies also identified other DGAT enzymes such as the soluble DGAT3 and diacylglycerol acetyltransferase (EaDAcT), as well as enzymes with DGAT activities including defective in cuticular ridges (DCR) and steryl and phytyl ester synthases (PESs). This review comprehensively discusses research advances on DGATs in prokaryotes and eukaryotes with a focus on their biochemical properties, physiological roles, and biotechnological and therapeutic applications. The review begins with a discussion of DGAT assay methods, followed by a systematic discussion of TAG biosynthesis and the properties and physiological role of DGATs. Thereafter, the review discusses the three-dimensional structure and insights into mechanism of action of human DGAT1, and the modeled DGAT1 from Brassica napus. The review then examines metabolic engineering strategies involving manipulation of DGAT, followed by a discussion of its therapeutic applications. DGAT in relation to improvement of traits of farmed animals is also discussed along with DGATs in various other eukaryotic organisms

Understanding Conformational Dynamics of Complex Lipid Mixtures Relevant to Biology

This is a perspective article entitled “Frontiers in computational biophysics: understanding conformational dynamics of complex lipid mixtures relevant to biology” which is following a CECAM meeting with the same name.Fil: Friedman, Ran. Linnæus University; ArgentinaFil: Khalid, Syma. University of Southampton; Reino UnidoFil: Aponte Santamaría, Camilo. Ruprecht-Karls-Universität Heidelberg; Alemania. Universidad de los Andes; ColombiaFil: Arutyunova, Elena. University of Alberta; CanadáFil: Becker, Marlon. Westfälische Wilhelms Universität; AlemaniaFil: Boyd, Kevin J.. University of Connecticut; Estados UnidosFil: Christensen, Mikkel. University Aarhus; DinamarcaFil: Coimbra, João T. S.. Universidad de Porto; PortugalFil: Concilio, Simona. Universita di Salerno; ItaliaFil: Daday, Csaba. Heidelberg Institute for Theoretical Studies; AlemaniaFil: Eerden, Floris J. van. University of Groningen; Países BajosFil: Fernandes, Pedro A.. Universidad de Porto; PortugalFil: Gräter, Frauke. Heidelberg University; Alemania. Heidelberg Institute for Theoretical Studies; AlemaniaFil: Hakobyan, Davit. Westfälische Wilhelms Universität; AlemaniaFil: Heuer, Andreas. Westfälische Wilhelms Universität; AlemaniaFil: Karathanou, Konstantina. Freie Universität Berlin; AlemaniaFil: Keller, Fabian. Westfälische Wilhelms Universität; AlemaniaFil: Lemieux, M. Joanne. University of Alberta; CanadáFil: Marrink, Siewert J.. University of Groningen; Países BajosFil: May, Eric R.. University of Connecticut; Estados UnidosFil: Mazumdar, Antara. University of Groningen; Países BajosFil: Naftalin, Richard. Colegio Universitario de Londres; Reino UnidoFil: Pickholz, Mónica Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Piotto, Stefano. Universita di Salerno; ItaliaFil: Pohl, Peter. Johannes Kepler University; AustriaFil: Quinn, Peter. Colegio Universitario de Londres; Reino UnidoFil: Ramos, Maria J.. Universidad de Porto; PortugalFil: Schiøtt, Birgit. University Aarhus; DinamarcaFil: Sengupta, Durba. National Chemical Laboratory India; IndiaFil: Sessa, Lucia. Universita di Salerno; ItaliaFil: Vanni, Stefano. University Of Fribourg;Fil: Zeppelin, Talia. University Aarhus; DinamarcaFil: Zoni, Valeria. University of Fribourg; SuizaFil: Bondar, Ana-Nicoleta. Freie Universität Berlin; AlemaniaFil: Domene, Carmen. University of Oxford; Reino Unido. University of Bath; Reino Unid

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Multi-site study of additive genetic effects on fractional anisotropy of cerebral white matter:Comparing meta and megaanalytical approaches for data pooling

Combining datasets across independent studies can boost statistical power by increasing the numbers of observations and can achieve more accurate estimates of effect sizes. This is especially important for genetic studies where a large number of observations are required to obtain sufficient power to detect and replicate genetic effects. There is a need to develop and evaluate methods for joint-analytical analyses of rich datasets collected in imaging genetics studies. The ENIGMA-DTI consortium is developing and evaluating approaches for obtaining pooled estimates of heritability through meta-and mega-genetic analytical approaches, to estimate the general additive genetic contributions to the intersubject variance in fractional anisotropy (FA) measured from diffusion tensor imaging (DTI). We used the ENIGMA-DTI data harmonization protocol for uniform processing of DTI data from multiple sites. We evaluated this protocol in five family-based cohorts providing data from a total of 2248 children and adults (ages: 9-85) collected with various imaging protocols. We used the imaging genetics analysis tool, SOLAR-Eclipse, to combine twin and family data from Dutch, Australian and Mexican-American cohorts into one large "mega-family". We showed that heritability estimates may vary from one cohort to another. We used two meta-analytical (the sample-size and standard-error weighted) approaches and a mega-genetic analysis to calculate heritability estimates across-population. We performed leave-one-out analysis of the joint estimates of heritability, removing a different cohort each time to understand the estimate variability. Overall, meta- and mega-genetic analyses of heritability produced robust estimates of heritability

Proline residues in transmembrane segment IV are critical for activity, expression and targeting of the Na+/H+ exchanger isoform 1.

NHE1 (Na+/H+ exchanger isoform 1) is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammalian cells. Proline residues within transmembrane segments have unusual properties, acting as helix breakers and increasing flexibility of membrane segments, since they lack an amide hydrogen. We examined the importance of three conserved proline residues in TM IV (transmembrane segment IV) of NHE1. Pro167 and Pro168 were mutated to Gly, Ala or Cys, and Pro178 was mutated to Ala. Pro168 and Pro178 mutant proteins were expressed at levels similar to wild-type NHE1 and were targeted to the plasma membrane. However, the mutants P167G (Pro167-->Gly), P167A and P167C were expressed at lower levels compared with wild-type NHE1, and a significant portion of P167G and P167C were retained intracellularly, possibly indicating induced changes in the structure of TM IV. P167G, P167C, P168A and P168C mutations abolished NHE activity, and P167A and P168G mutations caused markedly decreased activity. In contrast, the activity of the P178A mutant was not significantly different from that of wild-type NHE1. The results indicate that both Pro167 and Pro168 in TM IV of NHE1 are required for normal NHE activity. In addition, mutation of Pro167 affects the expression and membrane targeting of the exchanger. Thus both Pro167 and Pro168 are strictly required for NHE function and may play critical roles in the structure of TM IV of the NHE

Michael James (1940-2023).

Michael James is remembered