In-depth Profiling and Quantification of the Lysine Acetylome in Hepatocellular Carcinoma with a Trapped Ion Mobility Mass Spectrometer

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide with limited therapeutic options. Comprehensive investigation of protein posttranslational modifications in HCC is still limited. Lysine acetylation is one of the most common types of posttranslational modification involved in many cellular processes and plays crucial roles in the regulation of cancer. In this study, we analyzed the proteome and K-acetylome in eight pairs of HCC tumors and normal adjacent tissues using a timsTOF Pro instrument. As a result, we identified 9219 K-acetylation sites in 2625 proteins, of which 1003 sites exhibited differential acetylation levels between tumors and normal adjacent tissues. Interestingly, many novel tumor-specific K-acetylation sites were characterized, for example, filamin A (K865), filamin B (K697), and cofilin (K19), suggesting altered activities of these cytoskeleton-modulating molecules, which may contribute to tumor metastasis. In addition, we observed an overall suppression of protein K-acetylation in HCC tumors, especially for enzymes from various metabolic pathways, for example, glycolysis, tricarboxylic acid cycle, and fatty acid metabolism. Moreover, the expression of deacetylase sirtuin 2 (SIRT2) was upregulated in HCC tumors, and its role of deacetylation in HCC cells was further explored by examining the impact of SIRT2 overexpression on the proteome and K-acetylome in Huh7 HCC cells. SIRT2 overexpression reduced K-acetylation of proteins involved in a wide range of cellular processes, including energy metabolism. Furthermore, cellular assays showed that overexpression of SIRT2 in HCC cells inhibited both glycolysis and oxidative phosphorylation. Taken together, our findings provide valuable information to better understand the roles of K-acetylation in HCC and to treat this disease by correcting the aberrant acetylation patterns.     

Glycosylflavonoids from Cecropia pachystachya Trécul are quorum sensing inhibitors

The Cecropia genus is widely distributed in Latin America including at least 60 species, and some of them are commonly used in traditional medicine for the treatment of several diseases. We used Cecropia pachystachya Trécul to search for quorum sensing (QS) inhibitors compounds and found that the aqueous extract of C. pachystachya leaves is a promising source of substances with this activity. Using as biosensor Chromobacterium violaceum ATCC 31532 and Escherichia coli pSB403, the compounds chlorogenic acid (2), isoorientin (3), orientin (4), isovitexin (6), vitexin (7), and rutin (9) were identified as QS inhibitors. None of these compounds inhibited the growth of neither the used biosensors nor the microorganisms Staphylococcus aureus ATCC 23591, Escherichia coli ATCC 25922 and Saccharomyces cerevisiae, used here as growth inhibition controls. Along with the rutin, here we presented for the first time the QS-inhibition potential of the C-glycosyl flavonoids. The prospective of this evidence lead to the use of these compounds as antipathogenic drugs or antifoulants.     

Methylation of KvDMR1 involved in regulating the imprinting of CDKN1C gene in cattle

The CDKN1C gene encodes a cyclin‐dependent kinase inhibitor and is one of the key genes involved in the development of Beckwith–Wiedemann syndrome and cancer. In this study, using a direct sequencing approach based on a single nucleotide polymorphism (SNP) at genomic DNA and cDNA levels, we show that CDKN1C exhibits monoallelic expression in all seven studied organs (heart, liver, spleen, lung, kidney, muscle and subcutaneous fat) in cattle. To investigate how methylation regulates imprinting of CDKN1C in cattle, allele‐specific methylation patterns in two putative differential methylation regions (DMRs), the CDKN1C DMR and KvDMR1, were analyzed in three tissues (liver, spleen and lung) using bisulfite sequencing PCR. Our results show that in the CDKN1C DMR both parental alleles were unmethylated in all three analyzed tissues. In contrast, KvDMR1 was differentially methylated between the two parental alleles in the same tissues. Statistical analysis showed that there is a significant difference in the methylation level between the two parental alleles (P < 0.01), confirming that this region is the DMR of KvDMR1 and that it may be correlated with CDKN1C imprinting.     

Anti-fibrillogenic and fibril destabilizing effects of metal ions on cystatin fibrils

Metals such as Cu²⁺, Fe³⁺, and Zn²⁺ are major contributors to the biology of a brain in stages of health, aging, and disease because of their unique effects on both protein structures (misfolding) and oxidative stress. The relationship between metal ions and neurodegenerative diseases is very complicated. Our study highlights how metal ions influence amyloid formation at low pH and on preformed amyloid fibrils. By using thioflavin T assay, ANS fluorescence, Congo red assay, circular dichroism, and microscopy to elucidate the effects of Cu²⁺, Fe³⁺, and Zn²⁺ on goat brain cystatin (GBC) aggregation at low pH. Results showed that Cu²⁺ and Fe³⁺ inhibit fibril formation of GBC by promoting amorphous aggregates. However, Zn²⁺ exclusively promotes fibril formation at low pH, leading to the formation of more ordered aggregates. Furthermore, the combined results of these complementary methods also suggested that Cu²⁺ and Fe³⁺ destabilize the β-sheet secondary structure of preformed amyloid fibrils of GBC.     

Characterization of Bradyrhizobium strains indigenous to Western Australia and South Africa indicates remarkable genetic diversity and reveals putative new species

Bradyrhizobium are N₂-fixing microsymbionts of legumes with relevant applications in agricultural sustainability, and we investigated the phylogenetic relationships of conserved and symbiotic genes of 21 bradyrhizobial strains. The study included strains from Western Australia (WA), isolated from nodules of Glycine spp. the country is one genetic center for the genus and from nodules of other indigenous legumes grown in WA, and strains isolated from forage Glycine sp. grown in South Africa. The 16S rRNA phylogeny divided the strains in two superclades, of B. japonicum and B. elkanii, but with low discrimination among the species. The multilocus sequence analysis (MLSA) with four protein-coding housekeeping genes (dnaK, glnII, gyrB and recA) pointed out seven groups as putative new species, two within the B. japonicum, and five within the B. elkanii superclades. The remaining eleven strains showed higher similarity with six species, B. lupini, B. liaoningense, B. yuanmingense, B. subterraneum, B. brasilense and B. retamae. Phylogenetic analysis of the nodC symbiotic gene clustered 13 strains in three different symbiovars (sv. vignae, sv. genistearum and sv. retamae), while seven others might compose new symbiovars. The genetic profiles of the strains evaluated by BOX-PCR revealed high intra- and interspecific diversity. The results point out the high level of diversity still to be explored within the Bradyrhizobium genus, and further studies might confirm new species and symbiovars.     

UHRF2, a Ubiquitin E3 Ligase,Acts as a Small Ubiquitin-like Modifier E3 Ligase for Zinc Finger Protein 131

Small ubiquitin-like modifier (SUMO), a member of the ubiquitin-related protein family, is covalently conjugated to lysine residues of its substrates in a process referred to as SUMOylation. SUMOylation occurs through a series of enzymatic reactions analogous to that of the ubiquitination pathway, resulting in modification of the biochemical and functional properties of substrates. To date, four mammalian SUMO isoforms, a single heterodimeric SUMO-activating E1 enzyme SAE1/SAE2, a single SUMO-conjugating E2 enzyme ubiquitin-conjugating enzyme E2I (UBC9), and a few subgroups of SUMO E3 ligases have been identified. Several SUMO E3 ligases such as topoisomerase I binding, arginine/serine-rich (TOPORS), TNF receptor-associated factor 7 (TRAF7), and tripartite motif containing 27 (TRIM27) have dual functions as ubiquitin E3 ligases. Here, we demonstrate that the ubiquitin E3 ligase UHRF2 also acts as a SUMO E3 ligase. UHRF2 effectively enhances zinc finger protein 131 (ZNF131) SUMOylation but does not enhance ZNF131 ubiquitination. In addition, the SUMO E3 activity of UHRF2 on ZNF131 depends on the presence of SET and RING finger-associated and nuclear localization signal-containing region domains, whereas the critical ubiquitin E3 activity RING domain is dispensable. Our findings suggest that UHRF2 has independent functional domains and regulatory mechanisms for these two distinct enzymatic activities.     

Trace elements profile in the blood of Huntington’ disease patients

Huntington’ disease (HD) is an autosomal dominant neurodegenerative disease characterized by progressive motor, psychiatric, and cognitive deterioration. HD is, together with spinocerebellar ataxias, spinobulbar muscular atrophy and dentatorubral-pallido- luysian atrophy, one of the nine disorders caused by an expansion of glutamine residues in the causative protein where the polyglutamine expansion cause aberrant protein folding. Since an excessive metal’s accumulation in organs may induce protein misfolding and oxidative stress, we have studied the blood concentration of essential (Cr, Co, Cu, Fe, Mn, Mo, Ni, Se, Zn) and nonessential (As, Cd, Sb, Sn, V) trace elements in HD patients.We found increased levels of the essential elements iron, chromium, selenium and zinc and of the nonessential element arsenic in the blood of HD patients.Since alteration in metals homeostasis may contribute to the pathogenesis of neurodegenerative disease and could eventually constitute a target for therapy, we may suggest the utilize of the blood metal profile as a further in vivo tool to study and characterize Huntington disease.     

Upper extremity kinematic and kinetic adaptations during a fatiguing repetitive task

Repetitive low-force contractions are common in the workplace and yet can lead to muscle fatigue and work-related musculoskeletal disorders. The current study aimed to investigate potential motion adaptations during a simulated repetitive light assembly work task designed to fatigue the shoulder region, focusing on changes over time and age-related group differences. Ten younger and ten older participants performed four 20-min task sessions separated by short breaks. Mean and variability of joint angles and scapular elevation, joint net moments for the shoulder, elbow, and wrist were calculated from upper extremity kinematics recorded by a motion tracking system. Results showed that joint angle and joint torque decreased across sessions and across multiple joints and segments. Increased kinematic variability over time was observed in the shoulder joint; however, decreased kinematic variability over time was seen in the more distal part of the upper limb. The changes of motion adaptations were sensitive to the task-break schedule. The results suggested that kinematic and kinetic adaptations occurred to reduce the biomechanical loading on the fatigued shoulder region. In addition, the kinematic and kinetic responses at the elbow and wrist joints also changed, possibly to compensate for the increased variability caused by the shoulder joint while still maintaining task requirements. These motion strategies in responses to muscle fatigue were similar between two age groups although the older group showed more effort in adaptation than the younger in terms of magnitude and affected body parts.