Chemical crosslinking and mass spectrometry to elucidate the topology of integral membrane proteins.

Here we made an attempt to obtain partial structural information on the topology of multispan integral membrane proteins of yeast by isolating organellar membranes, removing peripheral membrane proteins at pH 11.5 and introducing chemical crosslinks between vicinal amino acids either using homo- or hetero-bifunctional crosslinkers. Proteins were digested with specific proteases and the products analysed by mass spectrometry. Dedicated software tools were used together with filtering steps optimized to remove false positive crosslinks. In proteins of known structure, crosslinks were found only between loops residing on the same side of the membrane. As may be expected, crosslinks were mainly found in very abundant proteins. Our approach seems to hold to promise to yield low resolution topological information for naturally very abundant or strongly overexpressed proteins with relatively little effort. Here, we report novel XL-MS-based topology data for 17 integral membrane proteins (Akr1p, Fks1p, Gas1p, Ggc1p, Gpt2p, Ifa38p, Ist2p, Lag1p, Pet9p, Pma1p, Por1p, Sct1p, Sec61p, Slc1p, Spf1p, Vph1p, Ybt1p)

Proteome-Wide Effect of 17-β-Estradiol and Lipoxin A4 in an Endometriotic Epithelial Cell Line.

Endometriosis affects approximately 10% of women of reproductive age. This chronic, gynecological inflammatory disease results in a decreased quality of life for patients, with the main symptoms including chronic pelvic pain and infertility. The steroid hormone 17-β Estradiol (E2) plays a key role in the pathology. Our previous studies showed that the anti-inflammatory lipid Lipoxin A4 (LXA4) acts as an estrogen receptor-alpha agonist in endometrial epithelial cells, inhibiting certain E2-mediated effects. LXA4 also prevents the progression of endometriosis in a mouse model via anti-proliferative mechanisms and by impacting mediators downstream of ER signaling. The aim of the present study was therefore to examine global proteomic changes evoked by E2 and LXA4 in endometriotic epithelial cells. E2 impacted a greater number of proteins in endometriotic epithelial cells than LXA4. Interestingly, the combination of E2 and LXA4 resulted in a reduced number of regulated proteins, with LXA4 mediating a suppressive effect on E2-mediated signaling. These proteins are involved in diverse pathways of relevance to endometriosis pathology and metabolism, including mRNA translation, growth, proliferation, proteolysis, and immune responses. In summary, this study sheds light on novel pathways involved in endometriosis pathology and further understanding of signaling pathways activated by estrogenic molecules in endometriotic epithelial cells

Impacts of streamflow alteration on benthic macroinvertebrates by mini‑hydro diversion in Sri Lanka

Our study focused on quantifying the alterations of streamflow at a weir site due to the construction of a mini-hydropower plant in the Gurugoda Oya (Sri Lanka), and evaluating the spatial responses of benthic macroinvertebrates to altered flow regime. The HEC-HMS 3.5 model was applied to the Gurugoda Oya sub-catchment to generate streamflows for the time period 1991-2013. Pre-weir flows were compared to post-weir flows with 32 Indicators of Hydrologic Alteration using the range of variability approach (RVA). Concurrently, six study sites were established upstream and downstream of the weir, and benthic macroinvertebrates were sampled monthly from May to November 2013 (during the wet season). The key water physico-chemical parameters were also determined. RVA analysis showed that environmental flow was not maintained below the weir. The mean rate of non-attainment was similar to 45% suggesting a moderate level of hydrologic alteration. Benthic macroinvertebrate communities significantly differed between the study sites located above and below the weir, with a richness reduction due to water diversion. The spatial distribution of zoobenthic fauna was governed by water depth, dissolved oxygen content and volume flow rate. Our work provides first evidence on the effects of small hydropower on river ecosystem in a largely understudied region. Studies like this are important to setting-up adequate e-flows

Quantitative proteomics of rat livers shows that unrestricted feeding is stressful for proteostasis with implications on life span.

Studies in young mammals on the molecular effects of food restriction leading to prolong adult life are scares. Here, we used high-throughput quantitative proteomic analysis of whole rat livers to address the molecular basis for growth arrest and the apparent life-prolonging phenotype of the food restriction regimen. Over 1800 common proteins were significantly quantified in livers of ad libitum, restriction- and re-fed rats, which summed up into 92% of the total protein mass of the cells. Compared to restriction, ad libitum cells contained significantly less mitochondrial catabolic enzymes and more cytosolic and ER HSP90 and HSP70 chaperones, which are hallmarks of heat- and chemically-stressed tissues. Following re-feeding, levels of HSPs nearly reached ad libitum levels. The quantitative and qualitative protein values indicated that the restriction regimen was a least stressful condition that used minimal amounts of HSP-chaperones to maintain optimal protein homeostasis and sustain optimal life span. In contrast, the elevated levels of HSP-chaperones in ad libitum tissues were characteristic of a chronic stress, which in the long term could lead to early aging and shorter life span

Fine-Tuning of Optimal TCR Signaling in Tumor-Redirected CD8 T Cells by Distinct TCR Affinity-Mediated Mechanisms

Redirecting CD8 T cell immunity with self/tumor-specific affinity-matured T cell receptors (TCRs) is a promising approach for clinical adoptive T cell therapy, with the aim to improve treatment efficacy. Despite numerous functional-based studies, little is known about the characteristics of TCR signaling (i.e., intensity, duration, and amplification) and the regulatory mechanisms underlying optimal therapeutic T cell responses. Using a panel of human SUP-T1 and primary CD8 T cells engineered with incremental affinity TCRs against the cancer-testis antigen NY-ESO-1, we found that upon activation, T cells with optimal-affinity TCRs generated intense and sustained proximal (CD3 zeta, LCK) signals associated with distal (ERK1/2) amplification-gain and increased function. In contrast, in T cells with very high affinity TCRs, signal initiation was rapid and strong yet only transient, resulting in poor MAPK activation and low proliferation potential even at high antigen stimulation dose. Under resting conditions, the levels of surface TCR/CD3e, CD8 beta, and CD28 expression and of CD3. phosphorylation were significantly reduced in those hypo-responsive cells, suggesting the presence of TCR affinity-related activation thresholds. We also show that SHP phosphatases were involved along the TCR affinity gradient, but displayed spatially distinct regulatory roles. While PTPN6/SHP-1 phosphatase activity controlled TCR signaling initiation and subsequent amplification by counteracting CD3. and ERK1/2 phosphorylation, PTPN11/SHP-2 augmented MAPK activation without affecting proximal TCR signaling. Together, our findings indicate that optimal TCR signaling can be finely tuned by TCR affinity-dependent SHP-1 and SHP-2 activity, and this may readily be determined at the TCR/CD3 complex level. We propose that these TCR affinity-associated regulations represent potential protective mechanisms preventing high affinity TCR-mediated autoimmune diseases

Bacterial Hsp90 Facilitates the Degradation of Aggregation-Prone Hsp70-Hsp40 Substrates.

In eukaryotes, the 90-kDa heat shock proteins (Hsp90s) are profusely studied chaperones that, together with 70-kDa heat shock proteins (Hsp70s), control protein homeostasis. In bacteria, however, the function of Hsp90 (HtpG) and its collaboration with Hsp70 (DnaK) remains poorly characterized. To uncover physiological processes that depend on HtpG and DnaK, we performed comparative quantitative proteomic analyses of insoluble and total protein fractions from unstressed wild-type (WT) Escherichia coli and from knockout mutants ΔdnaKdnaJ (ΔKJ), ΔhtpG (ΔG), and ΔdnaKdnaJΔhtpG (ΔKJG). Whereas the ΔG mutant showed no detectable proteomic differences with wild-type, ΔKJ expressed more chaperones, proteases and ribosomes and expressed dramatically less metabolic and respiratory enzymes. Unexpectedly, we found that the triple mutant ΔKJG showed higher levels of metabolic and respiratory enzymes than ΔKJ, suggesting that bacterial Hsp90 mediates the degradation of aggregation-prone Hsp70-Hsp40 substrates. Further in vivo experiments suggest that such Hsp90-mediated degradation possibly occurs through the HslUV protease

Closely related dermatophyte species produce different patterns of secreted proteins.

Dermatophytes are the most common infectious agents responsible for superficial mycosis in humans and animals. Various species in this group of fungi show overlapping characteristics. We investigated the possibility that closely related dermatophyte species with different behaviours secrete distinct proteins when grown in the same culture medium. Protein patterns from culture filtrates of several strains of the same species were very similar. In contrast, secreted protein profiles from various species were different, and so a specific signature could be associated with each of the six analysed species. In particular, protein patterns were useful to distinguish Trichophyton tonsurans from Trichophyton equinum, which cannot be differentiated by ribosomal DNA sequencing. The secreted proteases Sub2, Sub6 and Sub7 of the subtilisin family, as well as Mep3 and Mep4 of the fungalisin family were identified. SUB6, SUB7, MEP3 and MEP4 genes were cloned and sequenced. Although the protein sequence of each protease was highly conserved across species, their level of secretion by the various species was not equivalent. These results suggest that a switch of habitat could be related to a differential expression of genes encoding homologous secreted proteins

Morphologic and genetic variability in the Barbus fishes (Teleostei, Cyprinidae) of Central Italy

© 2019 Royal Swedish Academy of Sciences Italian freshwaters are highly biodiverse, with species present including the native fishes Barbus plebejus and Barbus tyberinus that are threatened by habitat alteration, fish stocking and invasive fishes, especially European barbel Barbus barbus. In central Italy, native fluvio-lacustrine barbels are mainly allopatric and so provide an excellent natural system to evaluate the permeability of the Apennine Mountains. Here, the morphologic and genetic distinctiveness was determined for 611 Barbus fishes collected along the Padany–Venetian (Adriatic basins; PV) and Tuscany–Latium (Tyrrhenian basins; TL) districts. Analyses of morphological traits and mitochondrial DNA sequence data explored the natural and anthropogenic factors that have shaped their distribution ranges. Over 100 alien B. barbus were recorded in the Tiber basin (TL district) and Metauro basin (PV district). Comparisons of genetic and morphometric data revealed that morphometric data could identify alien B. barbus from native Barbus, but could not differentiate between B. tyberinus and B. plebejus. Genetic analyses revealed ~50 D-loop mtDNA haplotypes and identified a distinct Barbus lineage present only in the Vomano River at the southern boundary of PV district. Demographic expansion and molecular variance analyses revealed a lack of geographic structuring across the sampling regions. While the contemporary B. plebejus distribution has been driven primarily by anthropogenic fish translocations, the dispersal of B. tyberinus has been via natural dispersion, including their crossing of the Apennine Mountains via temporary river connectivity. The results also revealed that the Barbus fishes of the mid-Adriatic region of Europe have a complex pattern of local endemism. To conserve these patterns of genetic uniqueness, especially in the mid-Adriatic basins, Barbus fishes should be managed by treating them as unique evolutionary units and ceasing translocations of all Barbus fishes between river basins