The lipoatrophic caveolin-1 deficient mouse model reveals autophagy in mature adipocytes

Adipose tissue lipoatrophy caused by caveolin gene deletion in mice is not linked to defective adipocyte differentiation. We show that adipose tissue development cannot be rescued by endothelial specific caveolin-1 re-expression, indicating primordial role of caveolin in mature adipocytes. Partial or total caveolin deficiency in adipocytes induced broad protein expression defects, including but not limited to previously described downregulation of insulin receptor. Global alterations in protein turnover, and accelerated degradation of long-lived proteins were found in caveolin-deficient adipocytes. Lipidation of endogenous LC3 autophagy marker and distribution of GFP-LC3 into aggregates demonstrated activated autophagy in the absence of caveolin-1 in adipocytes. Furthermore, electron microscopy revealed autophagic vacuoles in caveolin-1 deficient but not control adipocytes. Surprisingly, significant levels of lipidated LC3-II were found around lipid droplets of normal adipocytes, maintained in nutrient-rich conditions or isolated from fed mice, which do not display autophagy. Altogether, these data indicate that caveolin deficiency induce autophagy in adipocytes, a feature that is not a physiological response to fasting in normal fat cells. This likely resulted from defective insulin and lipolytic responses that converge in chronic nutrient shortage in adipocytes lacking caveolin-1. This is the first report of a pathological situation with autophagy as an adaptative response to adipocyte failure

Improving image analysis in 2DGE-based redox proteomics by labeling protein carbonyl with fluorescent hydroxylamine

Recent advances in redox proteomics have provided significant insight into the role of oxidative modifications in cellular signalling and metabolism. At present, these techniques rely heavily on Western blots to visualize the oxidative modification and corresponding two dimensional (2D) gels for detection of total protein levels, resulting in the duplication of efforts. A major limitation associated with this methodology includes problematic matching up of gels and blots due to the differences in processing and/or image acquisition. In this study, we present a new method which allows detection of protein oxidation and total protein on the same gel to improve matching in image analysis. Furthermore, the digested protein spots are compatible with standard MALDI mass spectrometry protein identification. The methodology highlighted here may be useful in facilitating the development of biomarkers, assessing potential therapeutic targets and elucidating new mechanisms of redox signalling in redox-related conditions

SUN proteins belong to a novel family of β-(1,3)-glucan-modifying enzymes involved in fungal morphogenesis

BACKGROUND: SUN proteins are involved in yeast morphogenesis, but their function is unknown. RESULTS: SUN protein plays a role in the Aspergillus fumigatus morphogenesis. Biochemical properties of recombinant SUN proteins were elucidated. CONCLUSION: Both Candida albicans and Aspergillus fumigatus sun proteins show a β-(1,3)-glucanase activity. SIGNIFICANCE: The mode of action of SUN proteins on β-(1,3)-glucan is unique, new, and original. In yeasts, the family of SUN proteins has been involved in cell wall biogenesis. Here, we report the characterization of SUN proteins in a filamentous fungus, Aspergillus fumigatus. The function of the two A. fumigatus SUN genes was investigated by combining reverse genetics and biochemistry. During conidial swelling and mycelial growth, the expression of AfSUN1 was strongly induced, whereas the expression of AfSUN2 was not detectable. Deletion of AfSUN1 negatively affected hyphal growth and conidiation. A closer examination of the morphological defects revealed swollen hyphae, leaky tips, intrahyphal growth, and double cell wall, suggesting that, like in yeast, AfSun1p is associated with cell wall biogenesis. In contrast to AfSUN1, deletion of AfSUN2 either in the parental strain or in the AfSUN1 single mutant strain did not affect colony and hyphal morphology. Biochemical characterization of the recombinant AfSun1p and Candida albicans Sun41p showed that both proteins had a unique hydrolysis pattern: acting on β-(1,3)-oligomers from dimer up to insoluble β-(1,3)-glucan. Referring to the CAZy database, it is clear that fungal SUN proteins represent a new family of glucan hydrolases (GH132) and play an important morphogenetic role in fungal cell wall biogenesis and septation

Protein Ontology: Enhancing and scaling up the representation of protein entities

The Protein Ontology (PRO; http://purl.obolibrary.org/obo/pr) formally defines and describes taxon-specific and taxon-neutral protein-related entities in three major areas: proteins related by evolution; proteins produced from a given gene; and protein-containing complexes. PRO thus serves as a tool for referencing protein entities at any level of specificity. To enhance this ability, and to facilitate the comparison of such entities described in different resources, we developed a standardized representation of proteoforms using UniProtKB as a sequence reference and PSI-MOD as a post-translational modification reference. We illustrate its use in facilitating an alignment between PRO and Reactome protein entities. We also address issues of scalability, describing our first steps into the use of text mining to identify protein-related entities, the large-scale import of proteoform information from expert curated resources, and our ability to dynamically generate PRO terms. Web views for individual terms are now more informative about closely-related terms, including for example an interactive multiple sequence alignment. Finally, we describe recent improvement in semantic utility, with PRO now represented in OWL and as a SPARQL endpoint. These developments will further support the anticipated growth of PRO and facilitate discoverability of and allow aggregation of data relating to protein entities

Advances in “Omics” Approaches for Improving Toxic Metals/Metalloids Tolerance in Plants

Food safety has emerged as a high-urgency matter for sustainable agricultural production. Toxic metal contamination of soil and water significantly affects agricultural productivity, which is further aggravated by extreme anthropogenic activities and modern agricultural practices, leaving food safety and human health at risk. In addition to reducing crop production, increased metals/metalloids toxicity also disturbs plants’ demand and supply equilibrium. Counterbalancing toxic metals/metalloids toxicity demands a better understanding of the complex mechanisms at physiological, biochemical, molecular, cellular, and plant level that may result in increased crop productivity. Consequently, plants have established different internal defense mechanisms to cope with the adverse effects of toxic metals/metalloids. Nevertheless, these internal defense mechanisms are not adequate to overwhelm the metals/metalloids toxicity. Plants produce several secondary messengers to trigger cell signaling, activating the numerous transcriptional responses correlated with plant defense. Therefore, the recent advances in omics approaches such as genomics, transcriptomics, proteomics, metabolomics, ionomics, miRNAomics, and phenomics have enabled the characterization of molecular regulators associated with toxic metal tolerance, which can be deployed for developing toxic metal tolerant plants. This review highlights various response strategies adopted by plants to tolerate toxic metals/metalloids toxicity, including physiological, biochemical, and molecular responses. A seven-(omics)-based design is summarized with scientific clues to reveal the stress-responsive genes, proteins, metabolites, miRNAs, trace elements, stress-inducible phenotypes, and metabolic pathways that could potentially help plants to cope up with metals/metalloids toxicity in the face of fluctuating environmental conditions. Finally, some bottlenecks and future directions have also been highlighted, which could enable sustainable agricultural production

Diurnal differences in human muscle isometric force and rate of force development in vivo are associated with differential phosphorylation of sarcomeric M-band proteins.

The maximum force of skeletal muscle exhibits circadian variation that is associated with time-of-day differences in athletic performance. We investigated whether the diurnal difference in force is associated with the post-translational state of muscle proteins. Twenty physically active men (mean ± SD; age 26.0 ± 4.4 y, height 177.3 ± 6.8 cm, body mass 75.1 ± 8.2.8 kg) completed 5 familiarisation sessions where-in they practiced all maximal efforts. Thereafter they performed experimental sessions, in the morning (08:00 h) and evening (17:00 h), counterbalanced in order of administration and separated by at least 72 h. Rectal, skin, muscle temperatures and ratings of perceived effort measurements where made after the subjects had reclined for 30 min (rest) and after the 5-min cycle ergometry warm-ups and prior to the measurement of knee extensor maximal voluntary isometric contraction (MVIC; including twitch-interpolation) and peak rate of force development (RFD). Data handling: 10 subjects from the cohort of 20 volunteered for muscle biopsy procedures, hence only their data is reported for temperature, MVIC and RFD to align with proteomic analyses. Samples of vastus lateralis were collected immediately after exercise and were analysed by ‘top-down’ and ‘bottom-up’ proteomic methods. Rectal and muscle temperatures were higher at rest in the evening (mean difference of 0.51°C and 0.69°C; p<0.05) than in the morning. MVIC force in the evening was significantly greater than in the morning (mean difference of 67 N, 9.3%; p<0.05), similarly peak RFD (mean difference of 1080 N/s, 15.3%; p<0.05) was improved in the evening. 2D gel analysis encompassed 122 proteoforms and discovered 6 statistically significant (p<0.05; false discovery rate [FDR] = 10%) diurnal differences. Phosphopeptide analysis identified 1,693 phosphopeptides and detected 140 phosphopeptides from 104 proteins that were more phosphorylated (p<0.05, FDR=22%) in the morning vs. evening. Myomesin 2, muscle creatine kinase and the C-terminus of titin, exhibited the most robust (FDR<10%) diurnal differences. In summary, the effects of time of day where seen in measures of rectal and muscle temperature and muscle performance. Exercise in the morning, compared to the evening, coincided with greater phosphorylation of M-band-associated proteins in human muscle. These protein modifications may alter M-band structure and disrupt force transmission, thus potentially explaining the lower force output in the morning

Exploring the Knowledge Landscape of <em>Escherichia coli</em> Research: A Scientometric Overview

Escherichia coli (E. coli) has the hallmark of being the most extensively studied organism. This is shown by the thousands of articles published since its discovery by T. Escherich in 1885. On the other hand, very little is known about the intellectual landscape in E. coli research. For example, how the trend of publications on E. coli has evolved over time and which scientific topics have been the focus of interest for researchers. In this chapter, we present the results of a large-scale scientometric analysis of about 100,000 bibliographic records from PubMed over the period 1981–2021. To examine the evolution of research topics over time, we divided the dataset into four intervals of equal width. We created co-occurrence networks from keywords indexed in the Medical Subject Headings vocabulary and systematically examined the structure and evolution of scientific knowledge about E. coli. The extracted research topics were visualized in strategic diagrams and qualitatively characterized in terms of their maturity and cohesion

PrP catabolites as determinants of TSE susceptibility

Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases that are characterised by long incubation periods, protein aggregation and vacuolation. During TSE pathogenesis the normal, cellular prion protein, (PrPC), which is encoded by the gene PRNP, misfolds and accumulates as abnormal disease associated prion protein, (PrPSc) within the central nervous system. Variants of the Prion protein gene are associated with susceptibility to TSE disease. For example sheep scrapie disease is modulated by several PRNP alleles, with certain alleles carried by susceptible animals being different from those carried by resistant animals. The mechanisms linking PRNP genetics and disease is poorly understood but may involve protein sequence, PrPC expression levels, and possibly differences in protein processing. Post-translational modification of PrPC leads to specific cleavage (alpha cleavage) between amino acids 115/116 of ovine PrP, producing two fragments C1 and N1. Cleavage of PrP may occur as a protective mechanism, as a response to changes in the cellular environment or as a feature of an as yet unknown biological function. In the context of TSEs, alpha cleavage may inadvertently provide a protective role by reducing available PrPC protein for conversion into PrPSc, assuming that the C1 fragment would be an inefficient substrate for conversion, the opposite theory was also proposed. The former hypothesis became the focus of this present study, with the idea that total full-length PrPC, total C1 or the ratio between full-length PrPC and C1 may be linked to differences in scrapie susceptibility. To investigate these aims the C1 fragment was measured as a percentage of total PrPC in different PRNP genotypes with varying degrees of susceptibility to scrapie and in different brain regions. This study found that PrPC alpha cleavage increased during development from the new born lamb to the adult sheep, which may have consequences for the susceptibility differences related to age. There are also variations in the amount of alpha cleavage between brain regions such as cortex and medulla that may influence scrapie strain targeting. Overall the amount of the C1 fragment in the different brain areas varied as much as 10x (range 5% to 60%). There was a significant difference in the ratio of C1 to the other PrPC forms between two PRNP genotype groups carrying the VRQ and ARQ allele but there was no correlation between C1 level and scrapie susceptibility or scrapie incubation period in our scrapie models. Alpha cleavage of PrPC also occurs in various transgenic mouse models expressing different ruminant PrP sequences. In PrPC over-expressing transgenic mouse models a higher ratio of C1 was observed, this may suggest a link between PrPC expression levels and alpha cleavage. Transgenic mice are therefore important models to further investigate the link between PrPC biology and scrapie disease phenotype. In conclusion, this thesis has shown for the first time that certain ovine PRNP alleles can influence alpha cleavage of the PrPC protein; however it appears not to be a significant indicator of TSE disease susceptibility in sheep