Comparative functional analysis reveals differential nucleotide sensitivity between human and mouse UCP1.

AIM: Mitochondrial uncoupling protein 1 (UCP1) is a unique protein of brown adipose tissue. Upon activation by free fatty acids, UCP1 facilitates a thermogenic net proton flux across the mitochondrial inner membrane. Non-complexed purine nucleotides inhibit this fatty acid-induced activity of UCP1. The most available data have been generated from rodent model systems. In light of its role as a putative pharmacological target for treating metabolic disease, in-depth analyses of human UCP1 activity, regulation, and structural features are essential. METHODS: In the present study, we established a doxycycline-regulated cell model with inducible human or murine UCP1 expression and conducted functional studies using respirometry comparing wild-type and mutant variants of human UCP1. RESULTS: We demonstrate that human and mouse UCP1 exhibit similar specific fatty acid-induced activity but a different inhibitory potential of purine nucleotides. Mutagenesis of non-conserved residues in human UCP1 revealed structural components in α-helix 56 and α-helix 6 crucial for uncoupling function. CONCLUSION: Comparative studies of human UCP1 with other orthologs can provide new insights into the structure-function relationship for this mitochondrial carrier and will be instrumental in searching for new activators.

Comparative assessment of purified saponins as permeabilization agents during respirometry.

Saponins are a diverse group of secondary plant metabolites, some of which display hemolytic toxicity due to plasma membrane permeabilization. This feature is employed in biological applications for transferring hydrophilic molecules through cell membranes. Widely used commercial saponins include digitonin and saponins from soap tree bark, both of which constitute complex mixtures of little definition. We assessed the permeabilization power of pure saponins towards cellular membranes in an effort to detect novel properties and to improve existing applications. In a respirometric assay, we characterized half-maximal permeabilization of the plasma membrane for different metabolites, of the mitochondrial outer membrane for cytochrome C and the full solubilization of mitochondrial inner membrane protein complexes. Beyond the complete list as repository for the field, we highlight several findings with direct applicability. First, we identified and validated α-chaconine as alternative permeabilization agent in respirometric assays of cultured cells and isolated synaptosomes, superior to digitonin in its tolerability for mitochondria. Second, we identified glycyrrhizic acid to form exceptionally small pores impermeable for adenosine diphosphate. Third, in a concentration dependent manner, tomatine proved to be able to selectively permeabilize the mitochondrial outer, but not inner membrane, allowing for novel states in which to determine cytochrome C oxidase activity. In summary, we provide a list of the permeabilization properties of 18 pure saponins. The identification of two saponins, namely tomatine and chaconine, with direct usability in improved or novel cell biological applications within this small subgroup demonstrates the tremendous potential for further functional screening of pure saponins.

Meltome atlas-thermal proteome stability across the tree of life.

We have used a mass spectrometry-based proteomic approach to compile an atlas of the thermal stability of 48,000 proteins across 13 species ranging from archaea to humans and covering melting temperatures of 30-90 °C. Protein sequence, composition and size affect thermal stability in prokaryotes and eukaryotic proteins show a nonlinear relationship between the degree of disordered protein structure and thermal stability. The data indicate that evolutionary conservation of protein complexes is reflected by similar thermal stability of their proteins, and we show examples in which genomic alterations can affect thermal stability. Proteins of the respiratory chain were found to be very stable in many organisms, and human mitochondria showed close to normal respiration at 46 °C. We also noted cell-type-specific effects that can affect protein stability or the efficacy of drugs. This meltome atlas broadly defines the proteome amenable to thermal profiling in biology and drug discovery and can be explored online at http://meltomeatlas.proteomics.wzw.tum.de:5003/ and http://www.proteomicsdb.org.