Identification of amino compounds synthesized and translocated in symbiotic Parasponia

We investigated the synthesis and translocation of amino compounds in Parasponia, a genus of the Ulmaceae that represents the only non-legumes known to form a root nodule symbiosis with rhizohia. In the xylem sap of P. andersonii we identified asparagine. aspartate. glutamine, glutamated significant quantities of a non-protein amino acid. 4-methylglutamte(2-amino-4-methylpentanedioic acid). This identification was confirmed by two methods, capillary gas chromatography (GC) electron ionization (El) mass spectrometry (MS) and reverse phase high pressure liquid chromatography (HPLC) analysis of derivatized compounds. In leaf, root and nodule samples from P. andersonii and P. parviflora we also identified the related compounds 4-methyleneglutamate and 4-methyleneglulamine. Using ¹⁵N₂ labelling and GC-Ms analysis of root nodule extracts we followed N₂ fixation and ammonia assimilation in P. andersonii root nodules and observed Label initially in glutamine and subsequently in glutamate, suggesting operation of the glutamine synthetase/glutamine:2-oxoglutarate aminotransferase (GS/GOGAT) pathway. Importantly, we observed the incorporation of significant quantities of ¹⁵N into 4-methylglutamate in nodules, demonstrating the de nova synthesis of this non protein amino acid and suggesting a role in the translation of N in symbioticParasponia.     

VEGF-C attenuates renal damage in salt-sensitive hypertension

Salt-sensitive hypertension is a major risk factor for renal impairment leading to chronic kidney disease. High-salt diet leads to hypertonic skin interstitial volume retention enhancing the activation of the tonicity-responsive enhancer-binding protein (TonEBP) within macrophages leading to vascular endothelial growth factor C (VEGF-C) secretion and NOS3 modulation. This promotes skin lymphangiogenesis and blood pressure regulation. Whether VEGF-C administration enhances renal and skin lymphangiogenesis and attenuates renal damage in salt-sensitive hypertension remains to be elucidated. Hypertension was induced in BALB/c mice by a high-salt diet. VEGF-C was administered subcutaneously to high-salt-treated mice as well as control animals. Analyses of kidney injury, inflammation, fibrosis, and biochemical markers were performed in vivo. VEGF-C reduced plasma inflammatory markers in salt-treated mice. In addition, VEGF-C exhibited a renal anti-inflammatory effect with the induction of macrophage M2 phenotype, followed by reductions in interstitial fibrosis. Antioxidant enzymes within the kidney as well as urinary RNA/DNA damage markers were all revelatory of abolished oxidative stress under VEGF-C. Furthermore, VEGF-C decreased the urinary albumin/creatinine ratio and blood pressure as well as glomerular and tubular damages. These improvements were associated with enhanced TonEBP, NOS3, and lymphangiogenesis within the kidney and skin. Our data show that VEGF-C administration plays a major role in preserving renal histology and reducing blood pressure. VEGF-C might constitute an interesting potential therapeutic target for improving renal remodeling in salt-sensitive hypertension.     

A novel regulatory role of TRAPPC9 in L-plastin-mediated osteoclast actin ring formation

Trafficking protein particle complex 9 (TRAPPC9) is a major subunit of the TRAPPII complex. TRAPPC9 has been reported to bind nuclear factor κB kinase subunit β (IKKβ) and NF-kB-inducing kinase (NIK) where it plays a role in the canonical and noncanonical of nuclear factor-κB (NF-kB) signaling pathways, receptively. The role of TRAPPC9 in protein trafficking and cytoskeleton organization in osteoclast (OC) has not been studied yet. In this study, we examined the mRNA expression of TRAPPC9 during OC differentiation. Next, we examined the colocalization of TRAPPC9 with cathepsin-K, known to mediate OC resorption suggesting that TRAPPC9 mediates the trafficking pathway within OC. To identify TRAPPC9 protein partners important for OC-mediated cytoskeleton re-organization, we conducted immunoprecipitation of TRAPPC9 in mature OCs followed by mass spectrometry analysis. Our data showed that TRAPPC9 binds various protein partners. One protein with high recovery rate is L-plastin (LPL). LPL localizes at the podosomes and reported to play a crucial role in actin aggregation thereby actin ring formation and OC function. Although the role of LPL in OC-mediated bone resorption has not fully reported in detail. Here, first, we confirmed the binding of LPL to TRAPPC9 and, then, we investigated the potential regulatory role of TRAPPC9 in LPL-mediated OC cytoskeleton reorganization. We assessed the localization of TRAPPC9 and LPL in OC and found that TRAPPC9 is colocalized with LPL at the periphery of OC. Next, we determined the effect of TRAPPC9 overexpression on LPL recruitment to the actin ring using a viral system. Interestingly, our data showed that TRAPPC9 overexpression promotes the recruitment of LPL to the actin ring when compared with control cultures. In addition, we observed that TRAPPC9 overexpression reorganizes actin clusters/aggregates and regulates vinculin recruitment into the OC periphery to initiate podosome formation.     

DIA-Based Proteomics Identifies IDH2 as a Targetable Regulator of Acquired Drug Resistance in Chronic Myeloid Leukemia

Drug resistance is a critical obstacle to effective treatment in patients with chronic myeloid leukemia. To understand the underlying resistance mechanisms in response to imatinib mesylate (IMA) and adriamycin (ADR), the parental K562 cells were treated with low doses of IMA or ADR for 2 months to generate derivative cells with mild, intermediate, and severe resistance to the drugs as defined by their increasing resistance index. PulseDIA-based (DIA [data-independent acquisition]) quantitative proteomics was then employed to reveal the proteome changes in these resistant cells. In total, 7082 proteins from 98,232 peptides were identified and quantified from the dataset using four DIA software tools including OpenSWATH, Spectronaut, DIA-NN, and EncyclopeDIA. Sirtuin signaling pathway was found to be significantly enriched in both ADR-resistant and IMA-resistant K562 cells. In particular, isocitrate dehydrogenase (NADP(+)) 2 was identified as a potential drug target correlated with the drug resistance phenotype, and its inhibition by the antagonist AGI-6780 reversed the acquired resistance in K562 cells to either ADR or IMA. Together, our study has implicated isocitrate dehydrogenase (NADP(+)) 2 as a potential target that can be therapeutically leveraged to alleviate the drug resistance in K562 cells when treated with IMA and ADR.     

Homodimerization as a molecular switch between low and high efficiency PrPC cell surface delivery and neuroprotective activity

PrP^C is associated with a variety of functions, and its ability to interact with a multitude of partners, including itself, may largely explain PrP multifunctionality and the lack of consensus on the genuine physiological function of the protein in vivo. In contrast, there is a consensus in the literature that alterations in PrP^C trafficking and intracellular retention result in neuronal degeneration. In addition, a proteolytic modification in the late secretory pathway termed the α-cleavage induces the secretion of PrPN1, a PrP^C-derived metabolite with fascinating neuroprotective activity against toxic oligomeric Aβ molecules implicated in Alzheimer disease. Thus, studies focusing on understanding the regulation of PrP^C trafficking to the cell surface and the modulation of α-cleavage are essential. The objective of this commentary is to highlight recent evidences that PrP^C homodimerization stimulates trafficking of the protein to the cell surface and results in high levels of PrPN1 secretion. We also discuss a hypothetical model for these results and comment on future challenges and opportunities.     

Application of Dolichos biflorus in immunoassay detection of kidney collecting duct biomarkers

Currently there are no biomarkers for detecting collecting duct damage in man. Antibodies to several collecting duct-specific antigens exist but sandwich assays have been difficult to establish due to the need for two different antibodies to the same protein. We hypothesized that a collecting duct-specific lectin could be used in combination with a collecting duct-specific antibody to negate the need for two different antibodies. The collecting duct specificity of selected antibodies (NiCa II 13C2, Pap XI 3C7, HuPaP VII 2B11 and aquaporin 2), was verified by immunohistochemistry. Aquaporin 2 and Pap XI 3C7 were used successfully in setting up assays with the lectin Dolichos biflorus, using the Meso Scale Discovery (MSD) platform. Antigen expression was highest in the papillae of rat and human kidney (corresponding to the greatest density of collecting ducts) and was also present in normal urine. We propose that further qualification and validation would lead to an assay for detecting collecting duct damage in man.     

ZMYND8-regulated IRF8 transcription axis is an acute myeloid leukemia dependency

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Effect of retinyl acetate on transglutaminase 2 activity in carcinogen treated rat liver

Transglutaminase 2 (TG2) has been implicated in wound healing, cellular differentiation, apoptosis and cell survival. TG2 activity increases following acute and chronic liver injury; however, the role of TG2 in tumors, is controversial. TG2 is a retinoid-inducible enzyme. We investigated the effects of retinyl acetate (RA) on the activity and levels of TG2 during the initiation and promotion stages of liver cancer. p-Dimethylaminoazobenzene (p-DAB) was used as initiator and 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) was used as promoter in our model of carcinogenesis. Rats were divided into four groups of 24: control, corn oil control, p-DAB + TCDD, and p-DAB + TCDD + RA. Six rats from each group were sacrificed at days 30, 60, 90 and 120. TG2 activity decreased in the p-DAB + TCDD treated group, but TG2 immunostaining scores did not change by days 90 and 120. Neither TG2 enzyme activity nor the immunostaining score of TG2 protein changed in the tissues of the p-DAB + TCDD + RA group by days 90 and 120. TG2 activity was not be ameliorated by RA during the initiation or promotion stages of carcinogen induced liver cancer.