Autophagy of OTUD5 destabilizes GPX4 to confer ferroptosis-dependent kidney injury.

Ferroptosis is an iron-dependent programmed cell death associated with severe kidney diseases, linked to decreased glutathione peroxidase 4 (GPX4). However, the spatial distribution of renal GPX4-mediated ferroptosis and the molecular events causing GPX4 reduction during ischemia-reperfusion (I/R) remain largely unknown. Using spatial transcriptomics, we identify that GPX4 is situated at the interface of the inner cortex and outer medulla, a hyperactive ferroptosis site post-I/R injury. We further discover OTU deubiquitinase 5 (OTUD5) as a GPX4-binding protein that confers ferroptosis resistance by stabilizing GPX4. During I/R, ferroptosis is induced by mTORC1-mediated autophagy, causing OTUD5 degradation and subsequent GPX4 decay. Functionally, OTUD5 deletion intensifies renal tubular cell ferroptosis and exacerbates acute kidney injury, while AAV-mediated OTUD5 delivery mitigates ferroptosis and promotes renal function recovery from I/R injury. Overall, this study highlights a new autophagy-dependent ferroptosis module: hypoxia/ischemia-induced OTUD5 autophagy triggers GPX4 degradation, offering a potential therapeutic avenue for I/R-related kidney diseases.

Inositol possesses antifibrotic activity and mitigates pulmonary fibrosis.

BACKGROUND: Myo-inositol (or inositol) and its derivatives not only function as important metabolites for multiple cellular processes but also act as co-factors and second messengers in signaling pathways. Although inositol supplementation has been widely studied in various clinical trials, little is known about its effect on idiopathic pulmonary fibrosis (IPF). Recent studies have demonstrated that IPF lung fibroblasts display arginine dependency due to loss of argininosuccinate synthase 1 (ASS1). However, the metabolic mechanisms underlying ASS1 deficiency and its functional consequence in fibrogenic processes are yet to be elucidated. METHODS: Metabolites extracted from primary lung fibroblasts with different ASS1 status were subjected to untargeted metabolomics analysis. An association of ASS1 deficiency with inositol and its signaling in lung fibroblasts was assessed using molecular biology assays. The therapeutic potential of inositol supplementation in fibroblast phenotypes and lung fibrosis was evaluated in cell-based studies and a bleomycin animal model, respectively. RESULTS: Our metabolomics studies showed that ASS1-deficient lung fibroblasts derived from IPF patients had significantly altered inositol phosphate metabolism. We observed that decreased inositol-4-monophosphate abundance and increased inositol abundance were associated with ASS1 expression in fibroblasts. Furthermore, genetic knockdown of ASS1 expression in primary normal lung fibroblasts led to the activation of inositol-mediated signalosomes, including EGFR and PKC signaling. Treatment with inositol significantly downregulated ASS1 deficiency-mediated signaling pathways and reduced cell invasiveness in IPF lung fibroblasts. Notably, inositol supplementation also mitigated bleomycin-induced fibrotic lesions and collagen deposition in mice. CONCLUSION: These findings taken together demonstrate a novel function of inositol in fibrometabolism and pulmonary fibrosis. Our study provides new evidence for the antifibrotic activity of this metabolite and suggests that inositol supplementation may be a promising therapeutic strategy for IPF.

IRAK-M suppresses the activation of microglial NLRP3 inflammasome and GSDMD-mediated pyroptosis through inhibiting IRAK1 phosphorylation during experimental autoimmune encephalomyelitis.

The activation of the NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome triggers pyroptosis proinflammatory cell death in experimental autoimmune encephalomyelitis (EAE). However, the underlying mechanisms of the inflammatory processes of microglia in EAE remain unclear. Our previous studies suggested that interleukin-1 receptor-associated kinase (IRAK)-M down-regulates the toll-like receptor 4/interleukin-1 receptor signaling pathway. Here, we used IRAK-M knockout (IRAK-M-/-) mice and their microglia to dissect the role of IRAK-M in EAE. We found that deletion of IRAK-M increased the incidence rate and exacerbated the clinical symptoms in EAE mice. We then found that IRAK-M deficiency promoted the activation of microglia, activated NLRP3 inflammasomes, and enhanced GSDMD-mediated pyroptosis in the microglia of EAE. In contrast, over-expression of IRAK-M exerted inhibitory effects on neuroinflammation, NLRP3 activation, and pyroptosis. Moreover, IRAK-M deficiency enhanced the phosphorylation of IRAK1, while IRAK-M over-expression downregulated the level of phosphorylated IRAK1. Finally, we found upregulated binding of IRAK1 and TNF receptor-associated factor 6 (TRAF6) in IRAK-M-/- EAE mice compared to WT mice, which was blocked in AAVIRAK-M EAE mice. Our study reveals a complex signaling network of IRAK-M, which negatively regulates microglial NLRP3 inflammasomes and pyroptosis by inhibiting IRAK1 phosphorylation during EAE. These findings suggest a potential target for the novel therapeutic approaches of multiple sclerosis (MS)/EAE and NLRP3-related inflammatory diseases.

MTAP deficiency contributes to immune landscape remodelling and tumour evasion.

Methylthioadenosine phosphorylase (MTAP) deficiency occurs in various malignancies and is associated with poor survival in cancer patients. However, the mechanisms underlying tumour progression due to MTAP loss are yet to be elucidated. Utilizing integrated analyses of the transcriptome, proteome and secretome, we demonstrated that MTAP deficiency alters tumour-intrinsic, immune-related pathways and reprograms cytokine profiles towards a tumour-favourable environment. Additionally, MTAP-knockout cells exhibited a marked increase in the immune checkpoint protein PD-L1. Upon co-culturing primary T cells with cancer cells, MTAP loss-mediated PD-L1 upregulation inhibited T cell-mediated killing activity and induced several T cell exhaustion markers. In two xenograft tumour models, we showed a modest increase in average volume of tumours derived from MTAP-deficient cells than that of MTAP-proficient tumours. Surprisingly, a remarkable increase in tumour size was observed in humanized mice bearing MTAP-deficient tumours, as compared to their MTAP-expressing counterparts. Following immunophenotypic characterization of tumour-infiltrating leukocytes by mass cytometry analysis, MTAP-deficient tumours were found to display decreased immune infiltrates with lower proportions of both T lymphocytes and natural killer cells and higher proportions of immunosuppressive cells as compared to MTAP-expressing tumour xenografts. Taken together, our results suggest that MTAP deficiency restructures the tumour immune microenvironment, promoting tumour progression and immune evasion.

A Novel Renoprotective Strategy: Upregulation of PD-L1 Mitigates Cisplatin-Induced Acute Kidney Injury.

The innate and adaptive immunities have been documented to participate in the pathogenesis of nephrotoxic acute kidney injury (AKI); however, the mechanisms controlling these processes have yet to be established. In our cisplatin-induced AKI mouse model, we show pathological damage to the kidneys, with the classical markers elevated, consistent with the response to cisplatin treatment. Through assessments of the components of the immune system, both locally and globally, we demonstrate that the immune microenvironment of injured kidneys was associated with an increased infiltration of CD4+ T cells and macrophages concomitant with decreased Treg cell populations. Our cell-based assays and animal studies further show that cisplatin exposure downregulated the protein levels of programmed death-ligand 1 (PD-L1), an immune checkpoint protein, in primary renal proximal tubular epithelial cells, and that these inhibitions were dose-dependent. After orthotopic delivery of PD-L1 gene into the kidneys, cisplatin-exposed mice displayed lower levels of both serum urea nitrogen and creatinine upon PD-L1 expression. Our data suggest a renoprotective effect of the immune checkpoint protein, and thereby provide a novel therapeutic strategy for cisplatin-induced AKI.

MARCKS cooperates with NKAP to activate NF-kB signaling in smoke-related lung cancer.

Rationale: Cigarette smoking is a major risk factor for lung cancer development and progression; however, the mechanism of how cigarette smoke activates signaling pathways in promoting cancer malignancy remains to be established. Herein, we aimed to determine the contribution of a signaling protein, myristoylated alanine-rich C kinase substrate (MARCKS), in smoke-mediated lung cancer. Methods: We firstly examined the levels of phosphorylated MARCKS (phospho-MARCKS) in smoke-exposed human lung cancer cells and specimens as well as non-human primate airway epithelium. Next, the MARCKS-interactome and its gene networks were identified. We also used genetic and pharmacological approaches to verify the functionality and molecular mechanism of smoke-induced phospho-MARCKS. Results: We observed that MARCKS becomes activated in airway epithelium and lung cancer cells in response to cigarette smoke. Functional proteomics revealed MARCKS protein directly binds to NF-κB-activating protein (NKAP). Following MARCKS phosphorylation at ser159 and ser163, the MARCKS-NKAP interaction was inhibited, leading to the activation of NF-κB signaling. In a screen of two cohorts of lung cancer patients, we confirmed that phospho-MARCKS is positively correlated with phospho-NF-κB (phospho-p65), and poor survival. Surprisingly, smoke-induced phospho-MARCKS upregulated the expression of pro-inflammatory cytokines, epithelial-mesenchymal transition, and stem-like properties. Conversely, targeting of MARCKS phosphorylation with MPS peptide, a specific MARCKS phosphorylation inhibitor, suppressed smoke-mediated NF-κB signaling activity, pro-inflammatory cytokines expression, aggressiveness and stemness of lung cancer cells. Conclusion: Our results suggest that phospho-MARCKS is a novel NF-kB activator in smoke-mediated lung cancer progression and provide a promising molecular model for developing new anticancer strategies.

Targeting the AXL Receptor in Combating Smoking-related Pulmonary Fibrosis.

Tobacco smoking is a well-known risk factor for both fibrogenesis and fibrotic progression; however, the mechanisms behind these processes remain enigmatic. RTKs (receptor tyrosine kinases) have recently been reported to drive profibrotic phenotypes in fibroblasts during pulmonary fibrosis (PF). Using a phospho-RTK array screen, we identified the RTK AXL as a top upregulated RTK in response to smoke. Both expression and signaling activity of AXL were indeed elevated in lung fibroblasts exposed to tobacco smoke, whereas no significant change to the levels of a canonical AXL ligand, Gas6 (growth arrest-specific 6), was seen upon smoke treatment. Notably, we found that smoke-exposed human lung fibroblasts exhibited highly proliferative and invasive activities and were capable of inducing fibrotic lung lesions in mice. Conversely, genetic suppression of AXL in smoke-exposed fibroblasts cells led to suppression of AXL downstream pathways and aggressive phenotypes. We further demonstrated that AXL interacted with MARCKS (myristoylated alanine-rich C kinase substrate) and cooperated with MARCKS in regulating downstream signaling activity and fibroblast invasiveness. Pharmacological inhibition of AXL with AXL-specific inhibitor R428 showed selectivity for smoke-exposed fibroblasts. In all, our data suggest that AXL is a potential marker for smoke-associated PF and that targeting of the AXL pathway is a potential therapeutic strategy in treating tobacco smoking-related PF.

Combinatorial interactions of the LEC1 transcription factor specify diverse developmental programs during soybean seed development.

The LEAFY COTYLEDON1 (LEC1) transcription factor is a central regulator of seed development, because it controls diverse biological programs during seed development, such as embryo morphogenesis, photosynthesis, and seed maturation. To understand how LEC1 regulates different gene sets during development, we explored the possibility that LEC1 acts in combination with other transcription factors. We identified and compared genes that are directly transcriptionally regulated by ABA-RESPONSIVE ELEMENT BINDING PROTEIN3 (AREB3), BASIC LEUCINE ZIPPER67 (bZIP67), and ABA INSENSITIVE3 (ABI3) with those regulated by LEC1. We showed that LEC1 operates with specific sets of transcription factors to regulate different gene sets and, therefore, distinct developmental processes. Thus, LEC1 controls diverse processes through its combinatorial interactions with other transcription factors. DNA binding sites for the transcription factors are closely clustered in genomic regions upstream of target genes, defining cis-regulatory modules that are enriched for DNA sequence motifs that resemble sequences known to be bound by these transcription factors. Moreover, cis-regulatory modules for genes regulated by distinct transcription factor combinations are enriched for different sets of DNA motifs. Expression assays with embryo cells indicate that the enriched DNA motifs are functional cis elements that regulate transcription. Together, the results suggest that combinatorial interactions between LEC1 and other transcription factors are mediated by cis-regulatory modules containing clustered cis elements and by physical interactions that are documented to occur between the transcription factors.

Approximate Mortality Risks between Hyperuricemia and Diabetes in the United States.

Aim: This study aimed to compare mortality risks across uric acid (UA) levels between non-diabetes adults and participants with diabetes and to investigate the association between hyperuricemia and mortality risks in low-risk adults. Methods: We analyzed data from adults aged >18 years without coronary heart disease and chronic kidney disease (n = 29,226) from the National Health and Nutrition Examination Survey (1999-2010) and the associated mortality data (up to December 2011). We used the Cox proportional hazards models to examine the risk of all-cause and cause-specific (cardiovascular disease (CVD) and cancer) mortality at different UA levels between adults with and without diabetes. Results: Over a median follow-up of 6.6 years, 2069 participants died (495 from CVD and 520 from cancers). In non-diabetes adults at UA ≥ 5 mg/dL, all-cause and CVD mortality risks increased across higher UA levels (p-for-trend = 0.037 and 0.058, respectively). The lowest all-cause mortality risk in participants with diabetes was at the UA level of 5-7 mg/dL. We set the non-diabetes participants with UA levels of <7 mg/dL as a reference group. Without considering the effect of glycemic control, the all-cause mortality risk in non-diabetes participants with UA levels of ≥7 mg/dL was equivalent to risk among diabetes adults with UA levels of <7 mg/dL (hazard ratio = 1.44 vs. 1.57, p = 0.49). A similar result was shown in CVD mortality risk (hazard ratio = 1.80 vs. 2.06, p = 0.56). Conclusion: Hyperuricemia may be an indicator to manage multifaceted cardiovascular risk factors in low-risk adults without diabetes, but further studies and replication are warranted.

A naïve Bayesian classifier for identifying plant microRNAs.

MicroRNAs (miRNAs) are important regulatory molecules in eukaryotic organisms. Existing methods for the identification of mature miRNA sequences in plants rely extensively on the search for stem-loop structures, leading to high false negative rates. Here, we describe a probabilistic method for ranking putative plant miRNAs using a naïve Bayes classifier and its publicly available implementation. We use a number of properties to construct the classifier, including sequence length, number of observations, existence of detectable predicted miRNA* sequences, the distribution of nearby reads and mapping multiplicity. We apply the method to small RNA sequence data from soybean, peach, Arabidopsis and rice and provide experimental validation of several predictions in soybean. The approach performs well overall and strongly enriches for known miRNAs over other types of sequences. By utilizing a Bayesian approach to rank putative miRNAs, our method is able to score miRNAs that would be eliminated by other methods, such as those that have low counts or lack detectable miRNA* sequences. As a result, we are able to detect several soybean miRNA candidates, including some that are 24 nucleotides long, a class that is almost universally eliminated by other methods.

Genomic dissection of the seed.

Seeds play an integral role in the global food supply and account for more than 70% of the calories that we consume on a daily basis. To meet the demands of an increasing population, scientists are turning to seed genomics research to find new and innovative ways to increase food production. Seed genomics is evolving rapidly, and the information produced from seed genomics research has exploded over the past two decades. Advances in modern sequencing strategies that profile every molecule in every cell, tissue, and organ and the emergence of new model systems have provided the tools necessary to unravel many of the biological processes underlying seed development. Despite these advances, the analyses and mining of existing seed genomics data remain a monumental task for plant biologists. This review summarizes seed region and subregion genomic data that are currently available for existing and emerging oilseed models. We provide insight into the development of tools on how to analyze large-scale datasets.

Identification of the tapetum/microspore-specific promoter of the pathogenesis-related 10 gene and its regulation in the anther of Lilium longiflorum.

A tapetum/microspore-specific pathogenesis-related (PR) 10 gene was previously identified in lily (Lilium longiflorum Thunb.) anthers. In situ hybridization and RNA blot analysis indicated that the lily PR10 genes are expressed specifically and differentially in the tapetum of the anther wall and in microspores during anther development. The accumulation of PR10 transcripts was exogenously induced by gibberellic acid (GA) and was suppressed by ethylene. Studies using inhibitors of GA and ethylene revealed that the lily PR10 is modulated by an antagonistic interaction between GA and ethylene. The treatment of norbornadien, an ethylene inhibitor, caused the tapetum to become densely cytoplasmic and highly polarized, whereas uniconazole, an inhibitor of GA biosynthesis, arrested tapetal development to a status close to that of control. The expression of the lily PR10g promoter in transgenic Arabidopsis was determined using the ß-glucuronidase (GUS) reporter gene indicated that the decisive fragment required for anther specificity is located -1183 bp to -880 bp upstream of the transcription start site. The PR10gPro::barnase transgenic lines exhibited complete male sterility because of the disruption of the tapetum and the deformation of microspore/pollen. The anther specificity of lily PR10 highlights the importance of the tapetum/microspore-specific PR10g promoter for future biotechnological and agricultural applications.

New insights into desiccation-associated gene regulation by Lilium longiflorum ASR during pollen maturation and in transgenic Arabidopsis.

LLA23, a member of the abscisic acid-, stress-, and ripening-induced (ASR) protein family, was previously isolated from lily (Lilium longiflorum) pollen. The lily ASR is induced through desiccation-associated ABA signaling transduction in the pollen. ASRs are highly hydrophilic and intrinsically unstructured proteins with molecular masses generally less than 18 kDa. LLA23 is abundant in the cytoplasm and nuclei of both vegetative and generative cells of pollen grains. The protein in the nucleus and in the cytoplasm is partly regulated by dehydration. A dual role is proposed for LLA23, as a regulator and a protective molecule, upon exposure to water deficits. This chapter reviews the current state of literature on Asr genes, protein structure, function, and their responses to various stresses. In a study, a genome-wide microarray was used to monitor the expression of LLA23-regulated genes, focusing on the relationship between ASR-, glucose-, and drought-inducible genes, and outlined the difference and cross talk of gene expression among these signaling networks. A strong association was observed in the expression of stress-responsive genes and found 25 genes that respond to all three treatments. Highly inducible genes were also found in each specific stress treatment. Promoter sequence analysis of LLA23-inducible genes enabled us not only to identify possible known cis-acting elements in the promoter regions but also to expect the existence of novel cis-acting elements involved in ASR-responsive gene expression. ASR can be used to improve crops and economically important plants against various environmental stresses.

Lily Cdc42/Rac-interactive binding motif-containing protein, a Rop target, involves calcium influx and phosphoproteins during pollen germination and tube growth.

We report unique desiccation-associated ABA signaling transduction through which the Rop (Rho GTPase of plants) and its target LLP12-2 are regulated during the stage of pollen maturation and tube growth. Overexpression of LLP12-2 drastically inhibited pollen germination and tube growth. Studies on the germination inhibitors, Ca (2+) influx blocking agents LaCl 3 and EGTA and an actin-depolymerizing drug, latrunculin B (LatB), revealed that the LLP12-2-induced inhibition of germination and tube growth is significantly suppressed by LaCl 3 and EGTA in the LLP12-2-overexpressing pollen but not by LatB. These results suggested that LLP12-2 is associated with Ca (2+) influx in the cytoplasm and may be not with actin assembly. With the addition of LaCl 3 and EGTA, LLP12-2-overexpressing pollen increased germination and tube growth compared with the one without addition, whereas pollen expressing GFP decreased germination and tube growth. Thus, an optimum level of [Ca (2+) ]cyt influx is crucial for normal germination and tube growth. Studies on the inhibitors, staurosporine and okadaic acid in the LLP12-2-overexpressing pollen, showed no appreciable increase in germination when compared with the one without addition, suggesting that staurosporine-sensitive protein kinases and dephosphorylation of phosphoproteins may be not involved in the LLP12-2 mediated germination. However, the LLP12-2-induced inhibition of tube length was slightly but significantly suppressed by staurosporine, suggesting that staurosporine-sensitive protein kinases involve in the LLP12-2-induced inhibition of tube growth.

Rop GTPase and its target Cdc42/Rac-interactive-binding motif-containing protein genes respond to desiccation during pollen maturation.

Here, we report unique desiccation-associated ABA signaling transduction through which the Rop (Rho GTPase of plants) gene is regulated during the stage of pollen maturation. A gene encoding Rho GTPase was identified in lily (Lilium longiflorum Thunb.) pollen. Phylogenetic tree analysis of lily LLP-Rop1 revealed that the protein shares greatest similarity with Group 4 Rops. The LLP-Rop1 gene was spatially and temporally regulated in lily plants during anther development. Accumulation of the LLP-Rop1 transcript decreased its level of accumulation while LLP-12-2, a Rop-interactive CRIB motif-containing (RIC) transcript increased either by premature drying of developing anther/pollen or by the exogenous application of various concentrations of abscisic acid (ABA) during pollen maturation and tube growth. Application of norflurazon, an ABA biosynthesis inhibitor, also resulted in the downregulation of the LLP-Rop1 gene while LLP-12-2 was upregulated by ABA. Furthermore, an increase in ABA in the maturing pollen correlated with desiccation that occurred in the anther prior to anthesis. LLP-Rop1 overexpression inhibited tube elongation, and caused tube expansion and the formation of a ballooned tip. CFP-LLP-Rop1 was localized to the cytoplasm having a greater intensity along the tube plasma membrane. Fluorescence resonance energy transfer analysis of lily pollen tubes coexpressing CFP-LLP-Rop1 and YFP-LLP-12-2 demonstrated that LLP-12-2 is a target RIC protein of active LLP-Rop1, but the interaction between LLP-Rop1 and LLP-12-2 proteins is probably irrelevant of dehydration in the dried pollen.

Expression and regulation of two novel anther-specific genes in Lilium longiflorum.

Two stage-specific genes have been isolated from a subtractive cDNA library constructed from developing anthers of lily (Lilium longiflorum). The proteins encoded by the two genes have a strong hydrophobic region at the N-terminus, indicating the presence of a signal peptide. The deduced LLA-67 is a new type of small cysteine-rich protein whose sequence exhibits four consecutive CX(3)CX(6-10) repeats that could form signal-receiving finger motifs, while the deduced LLA-115 protein shows significant similarities to a rice unknown protein, and putative cell wall proteins of Medicago truncatula and Arabidopsis. The transcripts of LLA-67 and LLA-115 were anther specific and differentially detected at the phase of microspore development. In situ hybridization with antisense riboprobes of the two genes in the anther showed strong signals localized to the tapetal layer of the anther wall. The LLA-67 mRNA was also detected in the microspore at the phase of microspore development but the LLA-115 mRNA was not. The LLA-115 gene can be exogenously induced by gibberellin (GA), whereas the LLA-67 gene cannot be induced. Studies with the GA biosynthesis inhibitor uniconazole and an inhibitor of ethylene activity, 2,5-norbornadien (NBD), revealed that the two genes were negatively regulated by ethylene and a cross-talk between GA and ethylene was involved in the regulation of the two genes occurring in young anthers. The treatment of NBD caused the tapetum to become densely cytoplasmic and highly polarized, whereas uniconazole arrested tapetal development to a status close to that of control. DNA blots of lily genomic DNA indicated that the two genes were encoded by a small gene family. The different actions of hormones on gene expression and the possible function of the gene products in young anthers are discussed.