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1. The β subunit of yeast AMP-activated protein kinase directs substrate specificity in response to alkaline stress

   https://doi.org/10.1016/j.cellsig.2016.08.016  

   Chandrashekarappa DG, McCartney RR (August 2016, Cellular signalling)

   Saccharomyces cerevisiae express three isoforms of Snf1 kinase that differ by which β subunit is present, Gal83, Sip1 or Sip2. Here we investigate the abundance, activation, localization and signaling specificity of the three Snf1 isoforms. The relative abundance of these isoforms was assessed by quantitative immunoblotting using two different protein extraction methods and by fluorescence microscopy. The Gal83 containing isoform is the most abundant in all assays while the abundance of the Sip1 and Sip2 isoforms is typically underestimated especially in glass-bead extractions. Earlier studies to assess Snf1 isoform function utilized gene deletions as a means to inactivate specific isoforms. Here we use point mutations in Gal83 and Sip2 and a 17 amino acid C-terminal truncation of Sip1 to inactivate specific isoforms without affecting their abundance or association with the other subunits. The effect of low glucose and alkaline stresses was examined for two Snf1 phosphorylation substrates, the Mig1 and Mig2 proteins. Any of the three isoforms was capable of phosphorylating Mig1 in response to glucose stress. In contrast, the Gal83 isoform of Snf1 was both necessary and sufficient for the phosphorylation of the Mig2 protein in response to alkaline stress. Alkaline stress led to the activation of all three isoforms yet only the Gal83 isoform translocates to the nucleus and phosphorylates Mig2. Deletion of the SAK1 gene blocked nuclear translocation of Gal83 and signaling to Mig2. These data strongly support the idea that Snf1 signaling specificity is mediated by localization of the different Snf1 isoforms. 

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2. Lipid Signaling Requires ROS Production to Elicit Actin Cytoskeleton Remodeling during Plant Innate Immunity

   https://doi.org/10.3390/ijms23052447  

   Cao, Lingyan; Wang, Wenyi; Zhang, Weiwei; Staiger, Christopher J. (March 2022, International Journal of Molecular Sciences)

   In terrestrial plants a basal innate immune system, pattern-triggered immunity (PTI), has evolved to limit infection by diverse microbes. The remodeling of actin cytoskeletal arrays is now recognized as a key hallmark event during the rapid host cellular responses to pathogen attack. Several actin binding proteins have been demonstrated to fine tune the dynamics of actin filaments during this process. However, the upstream signals that stimulate actin remodeling during PTI signaling remain poorly characterized. Two second messengers, reactive oxygen species (ROS) and phosphatidic acid (PA), are elevated following pathogen perception or microbe-associated molecular pattern (MAMP) treatment, and the timing of signaling fluxes roughly correlates with actin cytoskeletal rearrangements. Here, we combined genetic analysis, chemical complementation experiments, and quantitative live-cell imaging experiments to test the role of these second messengers in actin remodeling and to order the signaling events during plant immunity. We demonstrated that PHOSPHOLIPASE Dβ (PLDβ) isoforms are necessary to elicit actin accumulation in response to flg22-associated PTI. Further, bacterial growth experiments and MAMP-induced apoplastic ROS production measurements revealed that PLDβ-generated PA acts upstream of ROS signaling to trigger actin remodeling through inhibition of CAPPING PROTEIN (CP) activity. Collectively, our results provide compelling evidence that PLDβ/PA functions upstream of RBOHD-mediated ROS production to elicit actin rearrangements during the innate immune response in Arabidopsis. 

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3. Hyperglycemia Mediated Changes In A Human Cardiomyocyte Cell Model

   Joddar_B; Singh_I; Chattopadhyay_C; Thakur_V (November 2022, Wolters Kluwer Health, Inc)

   Introduction: Hyperglycemia-mediated cardiac dysfunction is a critical initiator in the development of vascular complications, which, in turn, leads to cardiac fibrosis. In this study, we investigated the role of the Hippo signaling pathway in cardiomyocytes to study the complex signaling network of YAP1/TAZ on fibrotic and vascular inflammatory mediators in hyperglycemic condition. This in-vitro study demonstrated that YAP1/TAZ signaling is highly activated in the hyperglycemic cardiomyocytes. To further investigate the differentially expressed genes that are related to inflammation and fibrosis, RNA-sequencing studies were employed. Methods: To investigate the effects of hyperglycemia-mediated changes in cardiomyocytes, we used human AC16 cells cultured in-vitro under normoglycemic (5 mM D-Glucose) and hyperglycemic (50 mM D-glucose) conditions. After 24-hours of hyperglycemic insult, cells were collected and processed for RNA-seq studies. Furthermore, we also performed Western Blot analysis to evaluate the protein expression of YAP1/TAZ under hyperglycemia induced stress conditions. Results: Our study showed a significant upregulation of the protein expression of the YAP1/TAZ pathway in hyperglycemic cardiomyocytes. RNA-seq studies revealed differentially expressed genes (DEG) in the hyperglycemic condition in comparison with the normoglycemic condition. Among the extracellular matrix proteins, the following ECM and related markers were significantly upregulated including MMP3, TNC, TGF-beta1 and 2, COL4A1, FN1 and FGF-2. Altered expression of inflammatory mediators included the following markers, IL-6, CXCL10, CXCL12, CCL2 and VEGF-C. In addition, the following transcriptional co-activators were also significantly upregulated, including EPHA2 and MYOCD. Conclusions: This study suggests that changes in YAP1/TAZ signaling increases vascular inflammation in response to hyperglycemia. This also leads to increased expression of inflammatory mediators as shown by our results. Thus, the inhibition of the YAP1/TAZ pathway may prevent and improve hyperglycemia associated vascular damage and inflammation. 

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4. A mathematical model of glomerular fibrosis in diabetic kidney disease to predict therapeutic efficacy

   https://doi.org/10.3389/fphar.2024.1481768  

   Thomas, Haryana Y; Ford_Versypt, Ashlee N (October 2024, Frontiers in Pharmacology)

   BackgroundGlomerular fibrosis is a tissue damage that occurs within the kidneys of chronic and diabetic kidney disease patients. Effective treatments are lacking, and the mechanism of glomerular damage reversal is poorly understood. MethodsA mathematical model suitable for hypothesis-driven systems pharmacology of glomerular fibrosis in diabetes was developed from a previous model of interstitial fibrosis. The adapted model consists of a system of ordinary differential equations that models the complex disease etiology and progression of glomerular fibrosis in diabetes. ResultsWithin the scope of the mechanism incorporated, advanced glycation end products (AGE)—matrix proteins that are modified due to high blood glucose—were identified as major contributors to the delay in the recovery from glomerular fibrosis after glucose control. The model predicted that inhibition of AGE production is not an effective approach for accelerating the recovery from glomerular fibrosis. Further, the model predicted that treatment breaking down accumulated AGE is the most productive at reversing glomerular fibrosis. The use of the model led to the identification that glucose control and aminoguanidine are ineffective treatments for reversing advanced glomerular fibrosis because they do not remove accumulated AGE. Additionally, using the model, a potential explanation was generated for the lack of efficacy of alagebrium in treating advanced glomerular fibrosis, which is due to the inability of alagebrium to reduce TGF- $\mathrm{\beta }$. ImpactUsing the mathematical model, a mechanistic understanding of disease etiology and complexity of glomerular fibrosis in diabetes was illuminated, and then hypothesis-driven explanations for the lack of efficacy of different pharmacological agents for treating glomerular fibrosis were provided. This understanding can enable the development of more efficacious therapeutics for treating kidney damage in diabetes. 

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5. Proteasome inhibition triggers tissue-specific immune responses against different pathogens in C. elegans

   https://doi.org/10.1371/journal.pbio.3002543  

   Grover, Manish; Gang, Spencer S; Troemel, Emily R; Barkoulas, Michalis (March 2024, PLOS Biology)

   Simon, Hans-Uwe (Ed.)

   Protein quality control pathways play important roles in resistance against pathogen infection. For example, the conserved transcription factor SKN-1/NRF up-regulates proteostasis capacity after blockade of the proteasome and also promotes resistance against bacterial infection in the nematodeCaenorhabditis elegans. SKN-1/NRF has 3 isoforms, and the SKN-1A/NRF1 isoform, in particular, regulates proteasomal gene expression upon proteasome dysfunction as part of a conserved bounce-back response. We report here that, in contrast to the previously reported role of SKN-1 in promoting resistance against bacterial infection, loss-of-function mutants inskn-1aand its activating enzymesddi-1andpng-1show constitutive expression of immune response programs against natural eukaryotic pathogens ofC.elegans. These programs are the oomycete recognition response (ORR), which promotes resistance against oomycetes that infect through the epidermis, and the intracellular pathogen response (IPR), which promotes resistance against intestine-infecting microsporidia. Consequently,skn-1amutants show increased resistance to both oomycete and microsporidia infections. We also report that almost all ORR/IPR genes induced in common between these programs are regulated by the proteasome and interestingly, specific ORR/IPR genes can be induced in distinct tissues depending on the exact trigger. Furthermore, we show that increasing proteasome function significantly reduces oomycete-mediated induction of multiple ORR markers. Altogether, our findings demonstrate that proteasome regulation keeps innate immune responses in check in a tissue-specific manner against natural eukaryotic pathogens of theC.elegansepidermis and intestine. 

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