Protein Expression in the Gastrocnemius Muscle of a Rodent Shrapnel-Injury Model

With shrapnel injuries, the metal fragment is usually left in place to reduce the risk of morbidity extensive surgery might bring. This means the individual may retain those metals for the remainder of their life. Often the long-term health effects of the embedded metal are not known, especially with respect to protein damage and perturbations of muscle repair pathways. In this study, using homogenates of rat gastrocnemius muscle implanted with pellets of military-relevant metals, we investigated expression of iNOS and eNOS, enzymes involved in nitric oxide production, as well as MMP-2 and MMP-9, matrix metalloproteinases associated with muscle repair. In addition, hydroxynonenal-modified proteins were investigated to assess metal-induced oxidative damage and metal levels in the gastrocnemius determined. Metals were implanted for up to 12 months in order to determine the long-term effects on the expression of muscle-associated proteins. With the exception of iron and cobalt at 1-month post-implantation, there were no significant differences in metal levels in the gastrocnemius in any of the cohorts. Protein expression analysis showed significant decreases in iNOS and eNOS in the 6-month and 12-month lead and depleted uranium groups. Hydroxynonenal-modified proteins were also significantly increased in the iron, copper, lead, and depleted uranium groups. These results suggest that some embedded metals can induce long-term oxidative damage, as well as affect enzyme systems involved in signal transduction.

Malondialdehyde-Acetaldehyde Modified (MAA) Proteins Differentially Effect the Inflammatory Response in Macrophage, Endothelial Cells and Animal Models of Cardiovascular Disease

Chronic inflammation plays a critical role in the pathogenesis of atherosclerosis. Currently, the mechanism(s) by which inflammation contributes to this disease are not entirely understood. Inflammation is known to induce oxidative stress, which can lead to lipid peroxidation. Lipid peroxidation can result in the production of reactive by-products that can oxidatively modify macromolecules including DNA, proteins, and lipoproteins. A major reactive by-product of lipid peroxidation is malondialdehyde (MDA). MDA can subsequently break down to form acetaldehyde (AA). These two aldehydes can covalently interact with the epsilon (ε)-amino group of lysines within proteins and lipoproteins leading to the formation of extremely stable, highly immunogenic malondialdehyde/acetaldehyde adducts (MAA-adducts). The aim of this study was to investigate the inflammatory response to MAA-modified human serum albumin (HSA-MAA) and low-density lipoprotein (LDL-MAA). We found that animals injected with LDL-MAA generate antibodies specific to MAA-adducts. The level of anti-MAA antibodies were further increased in an animal model of atherosclerosis fed a Western diet. An animal model that combined both high fat diet and immunization of MAA-modified protein resulted in a dramatic increase in antibodies to MAA-adducts and vascular fat accumulation compared with controls. In vitro exposure of endothelial cells and macrophages to MAA-modified proteins resulted in increased fat accumulation as well as increased expression of adhesion molecules and pro-inflammatory cytokines. The expression of cytokines varied between the different cell lines and was unique to the individual modified proteins. The results of these studies demonstrate that different MAA-modified proteins elicit unique responses in different cell types. Additionally, the presence of MAA-modified proteins appears to modulate cellular metabolism leading to increased accumulation of triglycerides and further progression of the inflammatory response.

The Landscape and Potential Regulatory Mechanism of Lysine 2-Hydroxyisobutyrylation of Protein in End-Stage Renal Disease

<b><i>Background:</i></b> Acetylation has a vital role in the pathogenesis of end-stage renal disease (ESRD). Lysine 2-hydroxyisobutyrylation (<i>K</i>_hib) is a novel type of acetylation. In this study, we aimed to reveal the key features of <i>K</i>_hib in peripheral blood monocytes (PBMCs) of patients with ESRD. <b><i>Method:</i></b> We combined TMT labeling with LC-MS/MS analysis to compare <i>K</i>_hib modification of PBMCs between 20 ESRD patients and 20 healthy controls. The pan 2-hydroxyisobutyrylation antibody-based affinity enrichment method was used to reveal the features of <i>K</i>_hib, and the bioinformatics analysis was conducted to analyze the pathology of these <i>K</i>_hib-modified proteins. <b><i>Result:</i></b> Compared to healthy controls, we identified 440 upregulated proteins and 552 downregulated proteins in PBMCs of ESRD, among which 579 <i>K</i>_hib sites on 324 upregulated proteins and 287 <i>K</i>_hib sites on 188 downregulated proteins were identified. The site abundance, distribution, and function of the <i>K</i>_hib protein were further analyzed. The bioinformatics analysis revealed that the Rho/ROCK signaling pathway was highly enriched in ESRD, suggesting that it might contribute to renal fibrosis in ESRD patients. <b><i>Conclusion:</i></b> In this study, we found that <i>K</i>_hib-modified proteins correlated with the occurrence and progression of ESRD.

Global Identification and Systematic Analysis of Lysine Malonylation in Maize (Zea mays L.)

Lysine malonylation is a kind of post-translational modifications (PTMs) discovered in recent years, which plays an important regulatory role in plants. Maize (Zea mays L.) is a major global cereal crop. Immunoblotting revealed that maize was rich in malonylated proteins. We therefore performed a qualitative malonylome analysis to globally identify malonylated proteins in maize. In total, 1,722 uniquely malonylated lysine residues were obtained in 810 proteins. The modified proteins were involved in various biological processes such as photosynthesis, ribosome and oxidative phosphorylation. Notably, a large proportion of the modified proteins (45%) were located in chloroplast. Further functional analysis revealed that 30 proteins in photosynthesis and 15 key enzymes in the Calvin cycle were malonylated, suggesting an indispensable regulatory role of malonylation in photosynthesis and carbon fixation. This work represents the first comprehensive survey of malonylome in maize and provides an important resource for exploring the function of lysine malonylation in physiological regulation of maize.

Structure and function of the ROR2 cysteine-rich domain in vertebrate noncanonical WNT5A signaling

The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, Brachydactyly B and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine rich (CRD) and Kringle (Kr) domains of ROR2 and found that, unlike other CRDs, the ROR2 CRD lacks the signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism of receptor action. Functionally, we showed that the ROR2 CRD, but not other domains, is required and minimally sufficient to promote WNT5A signaling, and Robinow mutations in the CRD and the adjacent Kr alter ROR2 function. Moreover, we demonstrated that the activity of the ROR2 CRD requires Frizzled receptors. Thus, ROR2 acts via its CRD to potentiate the function of a receptor supercomplex that includes Frizzleds to transduce WNT5A signals.

Global analysis of protein lysine 2-hydroxyisobutyrylation (Khib) profiles in Chinese herb rhubarb (Dahuang)

Background Lysine 2-hydroxyisobutyrylation (Khib) is a newly discovered protein posttranslational modification (PTM) and is involved in the broad-spectrum regulation of cellular processes that are found in both prokaryotic and eukaryotic cells, including in plants. The Chinese herb rhubarb (Dahuang) is one of the most widely used traditional Chinese medicines in clinical applications. To better understand the physiological activities and mechanism of treating diseases with the herb, it is necessary to conduct intensive research on rhubarb. However, Khib modification has not been reported thus far in rhubarb. Results In this study, we performed the first global analysis of Khib-modified proteins in rhubarb by using sensitive affinity enrichment combined with high-accuracy HPLC-MS/MS tandem spectrometry. A total of 4333 overlapping Khib modification peptides matched on 1525 Khib-containing proteins were identified in three independent tests. Bioinformatics analysis showed that these Khib-containing proteins are involved in a wide range of cellular processes, particularly in protein biosynthesis and central carbon metabolism and are distributed mainly in chloroplasts, cytoplasm, nucleus and mitochondria. In addition, the amino acid sequence motif analysis showed that a negatively charged side chain residue (E), a positively charged residue (K), and an uncharged residue with the smallest side chain (G) were strongly preferred around the Khib site, and a total of 13 Khib modification motifs were identified. These identified motifs can be classified into three motif patterns, and some motif patterns are unique to rhubarb and have not been identified in other plants to date. Conclusions A total of 4333 Khib-modified peptides on 1525 proteins were identified. The Khib-modified proteins are mainly distributed in the chloroplast, cytoplasm, nucleus and mitochondria, and involved in a wide range of cellular processes. Moreover, three types of amino acid sequence motif patterns, including EKhib/KhibE, GKhib and k.kkk….Khib….kkkkk, were extracted from a total of 13 Khib-modified peptides. This study provides comprehensive Khib-proteome resource of rhubarb. The findings from the study contribute to a better understanding of the physiological roles of Khib modification, and the Khib proteome data will facilitate further investigations of the roles and mechanisms of Khib modification in rhubarb.