Christian Madsen

Biotin starvation causes mitochondrial protein hyperacetylation and partial rescue by the SIRT3-like deacetylase Hst4p

The essential vitamin biotin is a covalent and tenaciously attached prosthetic group in several carboxylases that play important roles in the regulation of energy metabolism. Here we describe increased acetyl-CoA levels and mitochondrial hyperacetylation as downstream metabolic effects of biotin deficiency. Upregulated mitochondrial acetylation sites correlate with the cellular deficiency of the Hst4p deacetylase, and a biotin-starvation-induced accumulation of Hst4p in mitochondria supports a role for Hst4p in lowering mitochondrial acetylation. We show that biotin starvation and knockout of Hst4p cause alterations in cellular respiration and an increase in reactive oxygen species (ROS). These results suggest that Hst4p plays a pivotal role in biotin metabolism and cellular energy homeostasis, and supports that Hst4p is a functional yeast homologue of the sirtuin deacetylase SIRT3. With biotin deficiency being involved in various metabolic disorders, this study provides valuable insight into the metabolic effects biotin exerts on eukaryotic cells.

High-throughput screening of Erwinia chrysanthemi pectin methylesterase variants using carbohydrate microarrays.

Pectin methylesterases (PMEs) catalyse the removal of methyl esters from the homogalacturonan (HG) backbone domain of pectin, a ubiquitous polysaccharide in plant cell walls. The degree of methyl esterification (DE) impacts upon the functional properties of HG within cell walls and plants produce numerous PMEs that act upon HG in muro. Many microbial plant pathogens also produce PMEs, the activity of which renders HG more susceptible to cleavage by pectin lyase and polygalacturonase enzymes and hence aids cell wall degradation. We have developed a novel microarray‐based approach to investigate the activity of a series of variant enzymes based on the PME from the important pathogen Erwinia chrysanthemi. A library of 99 E. chrysanthemi PME mutants was created in which seven amino acids were altered by various different substitutions. Each mutant PME was incubated with a highly methyl esterified lime pectin substrate and, after digestion the enzyme/substrate mixtures were printed as microarrays. The loss of activity that resulted from certain mutations was detected by probing arrays with a mAb (JIM7) that preferentially binds to HG with a relatively high DE. Active PMEs therefore resulted in diminished JIM7 binding to the lime pectin substrate, whereas inactive PMEs did not. Our findings demonstrate the feasibility of our approach for rapidly testing the effects on PME activity of substituting a wide variety of amino acids at different positions.

A two-step protein quality control pathway for a misfolded DJ-1 variant in fission yeast

Background: A mutation, L166P, in DJ-1, is linked to Parkinson disease. Results: The Sdj1-L169P fission yeast orthologue of DJ1-L166P is misfolded, associated with chaperones, and degraded via two ubiquitin-proteasome dependent pathways. Conclusion: Sdj1-L169P is subject to a two-step degradation pathway. Significance: Mapping the degradation pathways for misfolded proteins is important for our basic understanding of protein quality control in health and disease. A mutation, L166P, in the cytosolic protein, PARK7/DJ-1, causes protein misfolding and is linked to Parkinson disease. Here, we identify the fission yeast protein Sdj1 as the orthologue of DJ-1 and calculate by in silico saturation mutagenesis the effects of point mutants on its structural stability. We also map the degradation pathways for Sdj1-L169P, the fission yeast orthologue of the disease-causing DJ-1 L166P protein. Sdj1-L169P forms inclusions, which are enriched for the Hsp104 disaggregase. Hsp104 and Hsp70-type chaperones are required for efficient degradation of Sdj1-L169P. This also depends on the ribosome-associated E3 ligase Ltn1 and its co-factor Rqc1. Although Hsp104 is absolutely required for proteasomal degradation of Sdj1-L169P aggregates, the degradation of already aggregated Sdj1-L169P occurs independently of Ltn1 and Rqc1. Thus, our data point to soluble Sdj1-L169P being targeted early by Ltn1 and Rqc1. The fraction of Sdj1-L169P that escapes this first inspection then forms aggregates that are subsequently cleared via an Hsp104- and proteasome-dependent pathway.

Preparation of N-(alk-1-enyl) nucleobase compounds by Horner and Horner–Wadsworth–Emmons reactions

A series of new N9-(alk-1-enyl)adenines and N1-(alk-1-enyl)thymines has been prepared by Horner reactions of the new phosphine oxides N9-(diphenylphosphorylmethyl)adenine and N1-(diphenylphosphorylmethyl)thymine derivatives 13a–c, or Horner–Wadsworth–Emmons reactions of the corresponding new phosphonates 14a–b, with benzaldehyde and various ketones. Yields were highest for the Horner reactions (10–79%), and were limited by steric hindrance, enolization of the ketones, and decomposition of some of the N1-(alk-1-enyl)thymines in the presence of excess base (NaH). Butanal gave mixtures of products under Horner conditions, probably because aldol reactions intervened.

A Cross-Cultural Comparison of Motivational Factors in Kenyan and Danish Middle and Long Distance Elite Runners

This study examined similarities and differences in motivational factors and reasons for running between Kenyan and Danish elite runners. Participants were elite middle and long distance runners (distances from 800m to marathon) from Kenya (n = 139, males = 114, females = 25) and Denmark (n = 96, males =69, females = 27) aged 14–42 years, (Mean age = 24.3, SD = 5.6 years). They completed the Task and Ego Orientation in Sport Questionnaire (Duda & Nicholls, 1992 in Duda, 1992) and the Achievement Motives Scale-Sport (Elbe & Wenhold, 2005). Participants also answered open-ended questions about their reasons for running. Descriptive statistics indicate that both samples show higher hope for success than fear of failure, and higher task than ego orientation. MANOVA analyses indicate that Kenyan runners scored significantly higher than Danish runners on task orientation and fear of failure. Additionally, Kenyan runners show more extrinsic reasons for running than the Danish runners. In conclusion, the present study documents cultural similarities and differences in motivational orientation among elite Danish and Kenyan runners. These results underscore the importance of cultural sensitivity in sports psychology studies.