Enhanced Fatty Acid Oxidation and FATP4 Protein Expression after Endurance Exercise Training in Human Skeletal Muscle

FATP1 and FATP4 appear to be important for the cellular uptake and handling of long chain fatty acids (LCFA). These findings were obtained from loss- or gain of function models. However, reports on FATP1 and FATP4 in human skeletal muscle are limited. Aerobic training enhances lipid oxidation; however, it is not known whether this involves up-regulation of FATP1 and FATP4 protein. Therefore, the aim of this project was to investigate FATP1 and FATP4 protein expression in the vastus lateralis muscle from healthy human individuals and to what extent FATP1 and FATP4 protein expression were affected by an increased fuel demand induced by exercise training. Eight young healthy males were recruited to the study. All subjects were non smokers and did not participate in regular physical activity (<1 time per week for the past 6 months, VO2peak 3.4±0.1 l O2 min−1). Subjects underwent an 8 week supervised aerobic training program. Training induced an increase in VO2peak from 3.4±0.1 to 3.9±0.1 l min−1 and citrate synthase activity was increased from 53.7±2.5 to 80.8±3.7 µmol g−1 min−1. The protein content of FATP4 was increased by 33%, whereas FATP1 protein content was reduced by 20%. Interestingly, at the end of the training intervention a significant association (r2 = 0.74) between the observed increase in skeletal muscle FATP4 protein expression and lipid oxidation during a 120 min endurance exercise test was observed. In conclusion, based on the present findings it is suggested that FATP1 and FATP4 proteins perform different functional roles in handling LCFA in skeletal muscle with FATP4 apparently more important as a lipid transport protein directing lipids for lipid oxidation

Circadian oscillator proteins across the kingdoms of life : Structural aspects 06 Biological Sciences 0601 Biochemistry and Cell Biology

Circadian oscillators are networks of biochemical feedback loops that generate 24-hour rhythms and control numerous biological processes in a range of organisms. These periodic rhythms are the result of a complex interplay of interactions among clock components. These components are specific to the organism but share molecular mechanisms that are similar across kingdoms. The elucidation of clock mechanisms in different kingdoms has recently started to attain the level of structural interpretation. A full understanding of these molecular processes requires detailed knowledge, not only of the biochemical and biophysical properties of clock proteins and their interactions, but also the three-dimensional structure of clockwork components. Posttranslational modifications (such as phosphorylation) and protein-protein interactions, have become a central focus of recent research, in particular the complex interactions mediated by the phosphorylation of clock proteins and the formation of multimeric protein complexes that regulate clock genes at transcriptional and translational levels. The three-dimensional structures for the cyanobacterial clock components are well understood, and progress is underway to comprehend the mechanistic details. However, structural recognition of the eukaryotic clock has just begun. This review serves as a primer as the clock communities move towards the exciting realm of structural biology

Assessment of Intracellular Auto-Modification Levels of ARTD10 Using Mono-ADP-Ribose-Specific Macrodomains 2 and 3 of Murine Artd8

Mono-ADP-ribosylation is a posttranslational modification, which is catalyzed in cells by certain members of the ADP-ribosyltransferase diphtheria toxin-like family (ARTD) of ADP-ribosyltransferases (aka PARP enzymes). It involves the transfer of a single residue of ADP-ribose (ADPr) from the cofactor NAD+ onto substrate proteins. Although 12 of the 17 members of the ARTD family have been defined as mono-ARTDs in in vitro assays, relatively little is known about their exact cellular functions. A major challenge is the detection of mono-ADP-ribosylated (MARylated) proteins in cells as no antibodies are available that detect exclusively MARylated proteins. As an alternative to classical antibodies, the MAR-specific binding domains macro2 and macro3 of Artd8 can be utilized alone or in combination, to demonstrate intracellular auto-modification levels of ARTD10 in cells in both co-immunoprecipitation and co-localization experiments. Here we demonstrate that different macrodomain constructs of human ARTD8 and murine Artd8, alone or in combination, exert differences with regard to their interaction with ARTD10 in cells. Precisely, while the macrodomains of murine Artd8 interacted with ARTD10 in cells in a MARylation-dependent manner, the macrodomains of human ARTD8 interacted with ARTD10 independent of its catalytic activity. Moreover, we show that a combination of macro2 and macro3 of murine Artd8 was recruited more efficiently to ARTD10 during co-localization experiments compared to the single domains. Therefore, murine Artd8 macrodomain constructs can serve as a tool to evaluate intracellular ARTD10 auto-modification levels using the described methods, while the human ARTD8 macrodomains are less suited because of ADPr-independent binding to ARTD10. Protocols for co-immunoprecipitation and co-localization experiments are described in detail