O-GlcNAcylation is a cell glucose sensor. The addition of O-GlcNAc moieties to target protein is catalyzed by the O-Linked N-acetylglucosamine transferase (OGT). OGT is encoded by a single gene that yields differentially spliced OGT isoforms. One of them is targeted to mitochondria (mOGT). Although the impact of O-GlcNAcylation on cancer cells biology is well documented, mOGT’s role remains poorly investigated. We performed studies using breast cancer cells with up-regulated mOGT or its catalytic inactive mutant to identify proteins specifically modified by mOGT. Proteomic approaches included isolation of mOGT protein partners and O-GlcNAcylated proteins from mitochondria-enriched fraction followed by their analysis by mass spectrometry. Moreover, we analyzed the impact of mOGT dysregulation on mitochondrial activity and cellular metabolism using a variety of biochemical assays. We found that mitochondrial OGT expression is glucose-dependent. Elevated mOGT expression affected the mitochondrial transmembrane potential and increased intramitochondrial ROS generation. Moreover, mOGT up-regulation caused a decrease in cellular ATP level. We identified many mitochondrial proteins as mOGT substrates. Most of these proteins are localized in the mitochondrial matrix and the inner mitochondrial membrane and participate in mitochondrial respiration, fatty acid metabolism, transport, translation, apoptosis, and mtDNA processes. Our findings suggest that mOGT interacts with and modifies many mitochondrial proteins, and its dysregulation affects cellular bioenergetics and mitochondria function.
Starch granule morphology is highly variable depending on the botanical origin. Moreover, all investigated plant species display intra-tissular variability of granule size. In potato tubers, the size distribution of starch granules follows a unimodal pattern with diameters ranging from 5 to 100 µm. Several evidences indicate that granule morphology in plants is related to the complex starch metabolic pathway. However, the intra-sample variability of starch-binding metabolic proteins remains unknown. Here, we report on the molecular characterization of size-fractionated potato starch granules with average diameters of 14.2 ± 3.7 µm, 24.5 ± 6.5 µm, 47.7 ± 12.8 µm, and 61.8 ± 17.4 µm. In addition to changes in the phosphate contents as well as small differences in the amylopectin structure, we found that the starch-binding protein stoichiometry varies significantly according to granule size. Label-free quantitative proteomics of each granule fraction revealed that individual proteins can be grouped according to four distinct abundance patterns. This study corroborates that the starch proteome may influence starch granule growth and architecture and opens up new perspectives in understanding the dynamics of starch biosynthesis.
Ancient preserved molecules offer the opportunity of gaining a deeper knowledge on their biological past. However, the development of a proteomic workflow remains a challenge. The analysis of fossils must involve a low quantity of material to avoid damaging the samples. In this study an enhanced proteomic protocol was applied to 5‐milligram samples of about 130,000‐year‐old mammalian bones ranging from the end of the Middle Pleistocene up to the earlier Upper Pleistocene, excavated from Scladina Cave (Sclayn, Belgium). Using sequence homology with modern sequences, a biological classification was successfully achieved and the associated taxonomic ranks to each bone were identified consistently with the information gained from osteomorphological studies and palaeoenvironmental and palaeodietary data. Amino acid substitutions on collagens were identified, thus providing new information on extinct species sequences and helping in taxonomy‐based clustering. Considering samples with no osteomorphological information, such as two fragments of bone retouchers, proteomics successfully identified the families providing paleontologists new information on these objects. Combining osteomorphology studies and amino acid variations identified by proteomics, one of the retouchers was potentially identified as belonging to the Ursus spelaeus species.
Saccharomyces cerevisiae yeast is a fungus presenting a peripheral organelle called the cell wall. The cell wall protects the yeast cell from stress and provides means for communication with the surrounding environment. It has a complex molecular structure, composed of an internal part of cross-linked polysaccharides and an external part of mannoproteins. These latter are very interesting owing to their functional properties, dependent on their molecular features with massive mannosylations. Therefore, the molecular characterization of mannoproteins is a must relying on the optimal isolation and preparation of the cell wall fraction. Multiple methods are well reported for yeast cell wall isolation. The most applied one consists of yeast cell lysis by mechanical disruption. However, applying this classical approach to S288C yeast cells showed considerable contamination with noncell wall proteins, mainly comprising mitochondrial proteins. Herein, we tried to further purify the yeast cell wall preparation by two means: ultracentrifugation and Triton X-100 addition. While the first strategy showed limited outcomes in mitochondrial protein removal, the second strategy showed optimal results when Triton X-100 was added at 5%, allowing the identification of more mannoproteins and significantly enriching their amounts. This promising method could be reliably implemented on the lab scale for identification of mannoproteins and molecular characterization and industrial processes for “pure” cell wall isolation.
Peptide mass fingerprinting (PMF) using MALDI-TOF mass spectrometry allows the identification of bone species based on their type I collagen sequence. In the archaeological or paleontological field, PMF is known as zooarchaeology mass spectrometry (ZooMS) and is widely implemented to find markers for most species, including the extinct ones. In addition to the identification of bone species, ZooMS enables dating estimation by measuring the deamidation value of specific peptides. Herein, we report several enhancements to the classical ZooMS technique, which reduces to 10-fold the required bone sample amount (down to the milligram scale) and achieves robust deamidation value calculation in a high-throughput manner. These improvements rely on a 96-well plate samples preparation, a careful optimization of collagen extraction and digestion to avoid spurious post-translational modification production, and PMF at high resolution using matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance (MALDI-FTICR) analysis. This method was applied to the identification of a hundred bones of herbivores from the Middle Paleolithic site of Caours (Somme, France) well dated from the Eemian Last Interglacial climatic optimum. The method gave reliable species identification to bones already identified by their osteomorphology, as well as to more challenging samples consisting of small or burned bone fragments. Deamidation values of bones originating from the same geological layers have a low standard deviation. The method can be applied to archaeological bone remains and offers a robust capacity to identify traditionally unidentifiable bone fragments, thus increasing the number of identified specimens and providing invaluable information in specific contexts.
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