Loss of lysosomal ion channel transient receptor potential channel mucolipin-1 (TRPML1) leads to cathepsin B-dependent apoptosis

Mucolipidosis type IV (MLIV) is a lysosomal storage disease caused by mutations in the gene MCOLN1, which codes for the transient receptor potential family ion channel TRPML1. MLIV has an early onset and is characterized by developmental delays, motor and cognitive deficiencies, gastric abnormalities, retinal degeneration, and corneal cloudiness. The degenerative aspects of MLIV have been attributed to cell death, whose mechanisms remain to be delineated in MLIV and in most other storage diseases. Here we report that an acute siRNA-mediated loss of TRPML1 specifically causes a leak of lysosomal protease cathepsin B (CatB) into the cytoplasm. CatB leak is associated with apoptosis, which can be prevented by CatB inhibition. Inhibition of the proapoptotic protein Bax prevents TRPML1 KD-mediated apoptosis but does not prevent cytosolic release of CatB. This is the first evidence of a mechanistic link between acute TRPML1 loss and cell death.     

Sialylation of N-linked glycans mediates apical delivery of endolyn in MDCK cells via a galectin-9-dependent mechanism

The sialomucin endolyn is implicated in adhesion, migration, and differentiation of various cell types. Along rat kidney tubules, endolyn is variously localized to the apical surface and endosomal/lysosomal compartments. Apical delivery of newly synthesized rat endolyn predominates over direct lysosomal delivery in polarized Madin-Darby canine kidney cells. Apical sorting depends on terminal processing of a subset of lumenal N-glycans. Here we dissect the requirements of N-glycan processing for apical targeting and investigate the underlying mechanism. Modulation of glycan branching and subsequent polylactosamine elongation by knockdown of N-acetylglucosaminyltransferase III or V had no effect on apical delivery of endolyn. In contrast, combined but not individual knockdown of sialyltransferases ST3Gal-III, ST3Gal-IV, and ST6Gal-I, which together are responsible for addition of α2,3- and α2,6-linked sialic acids on N-glycans, dramatically decreased endolyn surface polarity. Endolyn synthesized in the presence of kifunensine, which blocks terminal N-glycan processing, reduced its interaction with several recombinant canine galectins, and knockdown of galectin-9 (but not galectin-3, -4, or -8) selectively disrupted endolyn polarity. Our data suggest that sialylation enables recognition of endolyn by galectin-9 to mediate efficient apical sorting. They raise the intriguing possibility that changes in glycosyltransferase expression patterns and/or galectin-9 distribution may acutely modulate endolyn trafficking in the kidney.     

Metabolic monitoring and assessment of anaerobic threshold by means of breath biomarkers

Volatile breath constituents such as acetone and ammonia have been linked to dextrose, fat, and protein metabolism. Non-invasive breath analysis, therefore, may be used for metabolic monitoring, identification of fuel sources actually used for energy production and determination of the anaerobic threshold (AT). This study was intended to assess correlations between exhaled volatile organic compound (VOC) concentrations, metabolism, and physiological parameters. In addition, we tried to find out whether AT could be determined by means of non-invasive analysis of VOCs in breath. Exhaled concentrations of acetone, ammonia, and isoprene were determined in 21 healthy volunteers under controlled ergometric exercise by means of continuous real time Proton Transfer Reaction Mass Spectrometry (PTR-MS). In parallel, spiro-ergometric parameters ( $ {\dot{\text{V}}} $ O₂, $ {\dot{\text{V}}} $ CO₂, respiratory rate and minute ventilation) and hemodynamic data such as heart rate were recorded. AT was determined from serum lactate, by means of respiratory exchange rate and by means of exhaled acetone concentrations. Exhaled acetone concentrations mirrored exercise induced changes of dextrose metabolism and lipolysis. Bland?CAltman statistics showed good agreement between lactate threshold, respiratory compensation point (RCP), and determination of AT by means of exhaled acetone. Exhaled ammonia concentration seemed to be linked to protein metabolism and changes of pH under exercise. Isoprene concentrations showed a close correlation to cardiac output and minute ventilation. Breath biomarkers represent a promising alternative for metabolic monitoring under exercise as they can be determined non-invasively and continuously. In addition, these markers may add complementary information on biochemistry, energy production and fuel consumption.     

The negatively charged amino acids in the lumenal loop influence the pigment binding and conformation of the major light-harvesting chlorophyll a/b complex of photosystem II

The major chlorophyll (Chl) a/b complexes of photosystem II (LHCIIb), in addition to their primary light-harvesting function, play key roles in the organization of the granal ultrastructure of the thylakoid membranes and in various regulatory processes. These functions depend on the structural stability and flexibility of the complexes. The lumenal side of LHCIIb is exposed to broadly variable pH environments, due to the build-up and decay of the pH gradient during photosynthesis. Therefore, the negatively charged amino acids in the lumenal loop might be of paramount importance for adjusting the structure and functions of LHCIIb. In order to clarify the structural roles of these residues, we investigated the pigment stoichiometries, absorption, linear and circular dichroism spectra of the reconstituted LHCIIb complexes, in which the negatively charged amino acids in the lumenal loop were exchanged to neutral ones (E94G, E107V and D111V). The mutations influenced the pigment binding and the molecular architecture of the complexes. Exchanging E94 to G destabilized the 3(10) helix in the lumenal loop structure and led to an acquired pH sensitivity of the LHCIIb structure. We conclude that these amino acids are important not only for pigment binding in the complexes, but also in stabilizing the conformation of LHCIIb at different pHs.     

SUMO-conjugating and SUMO-deconjugating enzymes from Arabidopsis

Posttranslational protein modification by the small ubiquitin-like modifier (SUMO) is a highly dynamic and reversible process. To analyze the substrate specificity of SUMO-conjugating and -deconjugating enzymes from Arabidopsis (Arabidopsis thaliana), we reconstituted its SUMOylation cascade in vitro and tested the capacity of this system to conjugate the Arabidopsis SUMO isoforms AtSUMO1, 2, and 3 to the model substrate ScPCNA from yeast (Saccharomyces cerevisiae). This protein contains two in vivo SUMOylated lysine residues, namely K127 and K164. Under in vitro conditions, the Arabidopsis SUMOylation system specifically conjugates all tested SUMO isoforms to lysine-127, but not to lysine-164, of ScPCNA. The SUMO isoforms AtSUMO1 and AtSUMO2, but not AtSUMO3, were found to form polymeric chains on ScPCNA due to a self-SUMOylation process. In a complementary approach, we analyzed both the SUMO isopeptidase activity and the pre-SUMO-processing capacity of the putative Arabidopsis SUMO proteases At1g60220, At1g10570, and At5g60190 using the known SUMO isopeptidases ScULP1, XopD, and ESD4 (At4g15880) as reference enzymes. Interestingly, At5g60190 exhibits no SUMO protease activity but processes the pre-form of Arabidopsis Rub1. The other five enzymes represent SUMO isopeptidases that show different substrate preferences. All these enzymes cleave AtSUMO1 and AtSUMO2 conjugates of ScPCNA, whereas only the putative bacterial virulence factor XopD is able to release AtSUMO3. In addition, all five enzymes cleave pre-AtSUMO1 and pre-AtSUMO2 peptides, but none of the proteins efficiently produce mature AtSUMO3 or AtSUMO5 molecules from their precursors.     

The hypervariable D3 domain of Salmonella flagellin is an autonomous folding unit

The hypervariable D3 domain of Salmonella flagellin, composed of the 190-285 segment, is the major determinant of flagellar antigenicity. D3 was cloned and overexpressed in E. coli. Although previous studies concluded that D3 is stabilized by interactions with the D2 domain, our calorimetric experiments have revealed that isolated D3 has a stable tertiary structure which is highly resistant against proteolytic digestion. Repeated heating experiments demonstrated that unfolding of D3 is reversible. Its small size and stable structure makes D3 a promising protein scaffold for the development of artificial binding proteins by directed evolution.     

Do associated microbial abundances impact marine demosponge pumping rates and tissue densities?

The evolution of marine demosponges has led to two basic life strategies: one involving close associations with large and diverse communities of microorganisms, termed high microbial abundance (HMA) species, and one that is essentially devoid of associated microorganisms, termed low microbial abundance (LMA) species. This dichotomy has previously been suggested to correlate with morphological differences, with HMA species having a denser mesohyl and a more complex aquiferous systems composed of longer and narrower water canals that should necessitate slower seawater filtration rates. We measured mesohyl density for a variety of HMA and LMA sponges in the Florida Keys, and seawater pumping rates for a select group of these sponges using an in situ dye technique. HMA sponges were substantially denser than LMA species, and had per unit volume pumping rates 52–94% slower than the LMA sponges. These density and pumping rate differences suggest that evolutionary differences between HMA and LMA species may have resulted in profound morphological and physiological differences between the two groups. The LMA sponge body plan moves large quantities of water through their porous tissues allowing them to rapidly acquire the small particulate organic matter (POM) that supplies the majority of their nutritional needs. In contrast, the HMA sponge body plan is suited to host large and tightly packed communities of microorganisms and has an aquiferous system that increases contact time between seawater and the sponge/microbial consortium that feeds on POM, dissolved organic matter and the raw inorganic materials for chemolithotrophic sponge symbionts. The two evolutionary patterns represent different, but equally successful patterns and illustrate how associated microorganisms can potentially have substantial effects on host evolution. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Using Auditory Steady State Responses to Outline the Functional Connectivity in the Tinnitus Brain

Background

Tinnitus is an auditory phantom perception that is most likely generated in the central nervous system. Most of the tinnitus research has concentrated on the auditory system. However, it was suggested recently that also non-auditory structures are involved in a global network that encodes subjective tinnitus. We tested this assumption using auditory steady state responses to entrain the tinnitus network and investigated long-range functional connectivity across various non-auditory brain regions.

Methods and Findings

Using whole-head magnetoencephalography we investigated cortical connectivity by means of phase synchronization in tinnitus subjects and healthy controls. We found evidence for a deviating pattern of long-range functional connectivity in tinnitus that was strongly correlated with individual ratings of the tinnitus percept. Phase couplings between the anterior cingulum and the right frontal lobe and phase couplings between the anterior cingulum and the right parietal lobe showed significant condition x group interactions and were correlated with the individual tinnitus distress ratings only in the tinnitus condition and not in the control conditions.

Conclusions

To the best of our knowledge this is the first study that demonstrates existence of a global tinnitus network of long-range cortical connections outside the central auditory system. This result extends the current knowledge of how tinnitus is generated in the brain. We propose that this global extend of the tinnitus network is crucial for the continuos perception of the tinnitus tone and a therapeutical intervention that is able to change this network should result in relief of tinnitus.