Hydrology influences carbon flux through metabolic pathways in the hypolimnion of a Mediterranean reservoir

Global change is modifying meteorological and hydrological factors that influence the thermal regime of water bodies. These modifications can lead to longer stratification periods with enlarged hypolimnetic anoxic periods, which can promote heterotrophic anaerobic processes and alter reservoir carbon cycling. Here, we quantified aerobic and anaerobic heterotrophic processes (aerobic respiration, denitrification, iron and manganese reduction, sulfate reduction, and methanogenesis) on dissolved inorganic carbon (DIC) production in the hypolimnion of a Mediterranean reservoir (El Gergal, Spain) under two contrasting hydrological conditions: a wet year with heavy direct rainfall and frequent water inputs from upstream reservoirs, and a dry year with scarce rainfall and negligible water inputs. During the wet year, water inputs and rainfall induced low water column thermal stability and earlier turnover. By contrast, thermal stratification was longer and more stable during the dry year. During wet conditions, we observed lower DIC accumulation in the hypolimnion, mainly due to weaker sulfate reduction and methanogenesis. By contrast, longer stratification during the dry year promoted higher hypolimnetic DIC accumulation, resulting from enhanced methanogenesis and sulfate reduction, thus increasing methane emissions and impairing reservoir water quality. Aerobic respiration, denitrification and metal reduction produced a similar amount of DIC in the hypolimnion during the two studied years. All in all, biological and geochemical (calcite dissolution) processes explained most of hypolimnetic DIC accumulation during stratification regardless of the hydrological conditions, but there is still ~ 30% of hypolimnetic DIC production that cannot be explained by the processes contemplated in this study and the assumptions made.This research was funded by project Alter-C (PID2020-114024GB-C31, PID2020-114024GB-C32, PID2020-114024GB-C33) of Spanish Ministry of Science and Innovation (Spanish Research Agency, AEI). JJM-P was supported by a Spanish FPI grant (RE2018-083596). EMASESA staff provide essential technical support during field surveys. R.M. acknowledges funding from Generalitat de Catalunya through the Consolidated Research Group 2017SGR1124 and the CERCA programme. Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Funding for open access charge: Universidad de Málaga / CBUA

The Two Caenorhabditis elegans UDP-Glucose:Glycoprotein Glucosyltransferase Homologues Have Distinct Biological Functions

The UDP-Glc:glycoprotein glucosyltransferase (UGGT) is the sensor of glycoprotein conformations in the glycoprotein folding quality control as it exclusively glucosylates glycoproteins not displaying their native conformations. Monoglucosylated glycoproteins thus formed may interact with the lectin-chaperones calnexin (CNX) and calreticulin (CRT). This interaction prevents premature exit of folding intermediates to the Golgi and enhances folding efficiency. Bioinformatic analysis showed that in C. elegans there are two open reading frames (F48E3.3 and F26H9.8 to be referred as uggt-1 and uggt-2, respectively) coding for UGGT homologues. Expression of both genes in Schizosaccharomyces pombe mutants devoid of UGGT activity showed that uggt-1 codes for an active UGGT protein (CeUGGT-1). On the other hand, uggt-2 coded for a protein (CeUGGT-2) apparently not displaying a canonical UGGT activity. This protein was essential for viability, although cnx/crt null worms were viable. We constructed transgenic worms carrying the uggt-1 promoter linked to the green fluorescent protein (GFP) coding sequence and found that CeUGGT-1 is expressed in cells of the nervous system. uggt-1 is upregulated under ER stress through the ire-1 arm of the unfolded protein response (UPR). Real-time PCR analysis showed that both uggt-1 and uggt-2 genes are expressed during the entire C. elegans life cycle. RNAi-mediated depletion of CeUGGT-1 but not of CeUGGT-2 resulted in a reduced lifespan and that of CeUGGT-1 and CeUGGT-2 in a developmental delay. We found that both CeUGGT1 and CeUGGT2 play a protective role under ER stress conditions, since 10 µg/ml tunicamycin arrested development at the L2/L3 stage of both uggt-1(RNAi) and uggt-2(RNAi) but not of control worms. Furthermore, we found that the role of CeUGGT-2 but not CeUGGT-1 is significant in relieving low ER stress levels in the absence of the ire-1 unfolding protein response signaling pathway. Our results indicate that both C. elegans UGGT homologues have distinct biological functions

C-Terminus Glycans with Critical Functional Role in the Maturation of Secretory Glycoproteins

The N-glycans of membrane glycoproteins are mainly exposed to the extracellular space. Human tyrosinase is a transmembrane glycoprotein with six or seven bulky N-glycans exposed towards the lumen of subcellular organelles. The central active site region of human tyrosinase is modeled here within less than 2.5 Å accuracy starting from Streptomyces castaneoglobisporus tyrosinase. The model accounts for the last five C-terminus glycosylation sites of which four are occupied and indicates that these cluster in two pairs - one in close vicinity to the active site and the other on the opposite side. We have analyzed and compared the roles of all tyrosinase N-glycans during tyrosinase processing with a special focus on the proximal to the active site N-glycans, s6:N337 and s7:N371, versus s3:N161 and s4:N230 which decorate the opposite side of the domain. To this end, we have constructed mutants of human tyrosinase in which its seven N-glycosylation sites were deleted. Ablation of the s6:N337 and s7:N371 sites arrests the post-translational productive folding process resulting in terminally misfolded mutants subjected to degradation through the mannosidase driven ERAD pathway. In contrast, single mutants of the other five N-glycans located either opposite to the active site or into the N-terminus Cys1 extension of tyrosinase are temperature-sensitive mutants and recover enzymatic activity at the permissive temperature of 31°C. Sites s3 and s4 display selective calreticulin binding properties. The C-terminus sites s7 and s6 are critical for the endoplasmic reticulum retention and intracellular disposal. Results herein suggest that individual N-glycan location is critical for the stability, regional folding control and secretion of human tyrosinase and explains some tyrosinase gene missense mutations associated with oculocutaneous albinism type I

Minimal in vivo efficacy of iminosugars in a lethal Ebola virus guinea pig model

The antiviral properties of iminosugars have been reported previously in vitro and in small animal models against Ebola virus (EBOV); however, their effects have not been tested in larger animal models such as guinea pigs. We tested the iminosugars N-butyl-deoxynojirimycin (NB-DNJ) and N-(9-methoxynonyl)-1deoxynojirimycin (MON-DNJ) for safety in uninfected animals, and for antiviral efficacy in animals infected with a lethal dose of guinea pig adapted EBOV. 1850 mg/kg/day NB-DNJ and 120 mg/kg/day MON-DNJ administered intravenously, three times daily, caused no adverse effects and were well tolerated. A pilot study treating infected animals three times within an 8 hour period was promising with 1 of 4 infected NB-DNJ treated animals surviving and the remaining three showing improved clinical signs. MON-DNJ showed no protective effects when EBOV-infected guinea pigs were treated. On histopathological examination, animals treated with NB-DNJ had reduced lesion severity in liver and spleen. However, a second study, in which NB-DNJ was administered at equally-spaced 8 hour intervals, could not confirm drug-associated benefits. Neither was any antiviral effect of iminosugars detected in an EBOV glycoprotein pseudotyped virus assay. Overall, this study provides evidence that NB-DNJ and MON-DNJ do not protect guinea pigs from a lethal EBOV-infection at the dose levels and regimens tested. However, the one surviving animal and signs of improvements in three animals of the NB-DNJ treated cohort could indicate that NB-DNJ at these levels may have a marginal beneficial effect. Future work could be focused on the development of more potent iminosugars

UDP-Glucose: Glycoprotein Glucosyltransferase 1,2 (UGGT1,2)

Almost one-third of proteins synthesized by eukaryotic cells belong to the secretory pathway, entering the endoplasmic reticulum (ER) either co- or posttranslationally. In the ER, proteins acquire their native tertiary fold, disulfide bonds are formed, and in some cases, they assemble into oligomeric structures. Numerous folding-assisting enzymes and chaperones are in place to ensure the efficiency of these processes. Additionally, almost 70 % of the secretory pathway proteins are N-glycosylated by the translocon-associated oligosaccharyltransferase complex in the consensus sequence Asn-X-Ser/Thr, in which X can be any amino acid except for Pro (Apweiler et al. 1999). The consensus sequences are N-glycosylated as they emerge into the ER lumen when there are about 12–13 amino acids between the Asn residue and the inner ER membrane surface. In some cases, the same modification may occur posttranslationally (Ruiz-Canada et al. 2009). N-glycosylation is one of the most abundant and relevant protein modifications as N-glycans are central players in molecular recognition events, a function particularly suitable for them given their diverse composition. Additionally, N-glycans may modulate the biophysical behavior of their protein moieties. N-glycans may inhibit protein aggregation, may increase resistance to proteolytic degradation, and may promote acquisition of elements of secondary structure such as turns (Chen et al. 2010). Of particular relevance is the involvement of N-glycans in glycoprotein folding in the ER (Caramelo and Parodi 2007; D’Alessio et al. 2010). In this case, N-glycans act as an epigenetic information platform indicating the folding status of glycoproteins. This information is generated by glycosyltransferases and glycosidases that translate the conformational status of glycoproteins into particular N-glycan structures. Upon their recognition, ER-resident lectins retain the immature species in the ER, thus promoting their proper folding and hindering their Golgi exit. N-glycosylation starts in most eukaryotic cells with the transfer of the entire glycan Glc3Man9GlcNAc2 from a dolichol-P-P derivative.Fil: Parodi, Armando José A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Caramelo, Julio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: D'alessio, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Data-independent acquisition for yeast glycoproteomics

Glycosylation is a complex posttranslational modification that is critical for regulating the functions of diverse proteins. Analysis of protein glycosylation is made challenging by the high degree of heterogeneity in both glycan occupancy and structure. Here, we describe methods for data-independent acquisition (SWATH) mass spectrometry analysis of structure and occupancy of N-glycans from yeast cell wall glycoproteins