Widespread alteration of protein autoinhibition in human cancers.

Autoinhibition is a prevalent allosteric regulatory mechanism in signaling proteins. Reduced autoinhibition underlies the tumorigenic effect of some known cancer drivers, but whether autoinhibition is altered generally in cancer remains elusive. Here, we demonstrate that cancer-associated missense mutations, in-frame insertions/deletions, and fusion breakpoints are enriched within inhibitory allosteric switches (IASs) across all cancer types. Selection for IASs that are recurrently mutated in cancers identifies established and unknown cancer drivers. Recurrent missense mutations in IASs of these drivers are associated with distinct, cancer-specific changes in molecular signaling. For the specific case of PPP3CA, the catalytic subunit of calcineurin, we provide insights into the molecular mechanisms of altered autoinhibition by cancer mutations using biomolecular simulations, and demonstrate that such mutations are associated with transcriptome changes consistent with increased calcineurin signaling. Our integrative study shows that autoinhibition-modulating genetic alterations are positively selected for by cancer cells.

The signalling pathways, calcineurin B-like protein 5 (CBL5)-CBL-interacting protein kinase 8 (CIPK8)/CIPK24-salt overly sensitive 1 (SOS1), transduce salt signals in seed germination in Arabidopsis.

For plant growth under salt stress, sensing and transducing salt signals are central to cellular Na+ homoeostasis. The calcineurin B-like protein (CBL)-CBL-interacting protein kinase (CIPK) complexes play critical roles in transducing salt signals in plants. Here, we show that CBL5, an ortholog of CBL4 and CBL10 in Arabidopsis, interacts with and recruits CIPK8/CIPK24 to the plasma membrane. Yeast cells coexpressing CBL5, CIPK8/CIPK24 and SOS1 demonstrated lesser Na+ accumulation and a better growth phenotype than the untransformed or SOS1 transgenic yeast cells under salinity. Overexpression of CBL5 improved the growth of the cipk8 or cipk24 single mutant but not the cipk8 cipk24 double mutant under salt stress, suggesting that CIPK8 and CIPK24 were the downstream targets of CBL5. Interestingly, seed germination in cbl5 was severely inhibited by NaCl, which was recovered by the overexpression of CBL5. Furthermore, CBL5 was mainly expressed in the cotyledons and hypocotyls, which are essential to seed germination. Na+ efflux activity in the hypocotyls of cbl5 was reduced relative to the wild-type under salt stress, enhancing Na+ accumulation. These findings indicate that CBL5 functions in seed germination and protects seeds and germinating seedlings from salt stress through the CBL5-CIPK8/CIPK24-SOS1 pathways.

Calcineurin-dependent contributions to fitness in the opportunistic pathogen Candida glabrata.

The protein phosphatase calcineurin is vital for the virulence of the opportunistic fungal pathogen Candida glabrata. The host-induced stresses that activate calcineurin signaling are unknown, as are the targets of calcineurin relevant to virulence. To potentially shed light on these processes, millions of transposon insertion mutants throughout the genome of C. glabrata were profiled en masse for fitness defects in the presence of FK506, a specific inhibitor of calcineurin. Eighty-seven specific gene deficiencies depended on calcineurin signaling for full viability in vitro both in wild-type and pdr1∆ null strains lacking pleiotropic drug resistance. Three genes involved in cell wall biosynthesis (FKS1, DCW1, FLC1) possess co-essential paralogs whose expression depended on calcineurin and Crz1 in response to micafungin, a clinical antifungal that interferes with cell wall biogenesis. Interestingly, 80% of the FK506-sensitive mutants were deficient in different aspects of vesicular trafficking, such as endocytosis, exocytosis, sorting, and biogenesis of secretory proteins in the endoplasmic reticulum (ER). In response to the experimental antifungal manogepix that blocks GPI-anchor biosynthesis in the ER, calcineurin signaling increased and strongly prevented cell death independent of Crz1, one of its major targets. Comparisons between manogepix, micafungin, and the ER-stressing tunicamycin reveal a correlation between the degree of calcineurin signaling and the degree of cell survival. These findings suggest that calcineurin plays major roles in mitigating stresses of vesicular trafficking. Such stresses may arise during host infection and in response to antifungal therapies.IMPORTANCECalcineurin plays critical roles in the virulence of most pathogenic fungi. This study sheds light on those roles in the opportunistic pathogen Candida glabrata using a genome-wide analysis in vitro. The findings could lead to antifungal developments that also avoid immunosuppression.

Calcineurin contributes to RNAi-mediated transgene silencing and small interfering RNA production in the human fungal pathogen Cryptococcus neoformans.

Adaptation to external environmental challenges at the cellular level requires rapid responses and involves relay of information to the nucleus to drive key gene expression changes through downstream transcription factors. Here, we describe an alternative route of adaptation through a direct role for cellular signaling components in governing gene expression via RNA interference-mediated small RNA production. Calcium-calcineurin signaling is a highly conserved signaling cascade that plays central roles in stress adaptation and virulence of eukaryotic pathogens, including the human fungal pathogen Cryptococcus neoformans. Upon activation in C. neoformans, calcineurin localizes to P-bodies, membraneless organelles that are also the site for RNA processing. Here, we studied the role of calcineurin and its substrates in RNAi-mediated transgene silencing. Our results reveal that calcineurin regulates both the onset and the reversion of transgene silencing. We found that some calcineurin substrates that localize to P-bodies also regulate transgene silencing but in opposing directions. Small RNA sequencing in mutants lacking calcineurin or its targets revealed a role for calcineurin in small RNA production. Interestingly, the impact of calcineurin and its substrates was found to be different in genome-wide analysis, suggesting that calcineurin may regulate small RNA production in C. neoformans through additional pathways. Overall, these findings define a mechanism by which signaling machinery induced by external stimuli can directly alter gene expression to accelerate adaptative responses and contribute to genome defense.

Calcineurin B-like protein ZmCBL8-1 promotes salt stress resistance in Arabidopsis.

MAIN CONCLUSION: ZmCBL8-1 enhances salt stress tolerance in maize by improving the antioxidant system to neutralize ROS homeostasis and inducing Na+/H+ antiporter gene expressions of leaves. Calcineurin B-like proteins (CBLs) as plant-specific calcium sensors have been explored for their roles in the regulation of abiotic stress tolerance. Further, the functional variations in ZmCBL8, encoding a component of the salt overly sensitive pathway, conferred the salt stress tolerance in maize. ZmCBL8-1 is a transcript of ZmCBL8 found in maize, but its function in the salt stress response is still unclear. The present study aimed to characterize the protein ZmCBL8-1 that was determined to be composed of 194 amino acids (aa) with three conserved EF hands responsible for binding Ca2+. However, a 20-aa fragment was found to be missing from its C-terminus relative to another transcript of ZmCBL8. Results indicated that it harbored a dual-lipid modification motif MGCXXS at its N-terminus and was located on the cell membrane. The accumulation of ZmCBL8-1 transcripts was high in the roots but relatively lower in the leaves of maize under normal condition. In contrast, its expression was significantly decreased in the roots, while increased in the leaves under NaCl treatment. The overexpression of ZmCBL8-1 resulted in higher salt stress resistance of transgenic Arabidopsis in a Ca2+-dependent manner relative to that of the wild type (WT). In ZmCBL8-1-overexpressing plants exposed to NaCl, the contents of malondialdehyde and hydrogen peroxide were decreased in comparison with those in the WT, and the expression of key genes involved in the antioxidant defense system and Na+/H+ antiporter were upregulated. These results suggested that ZmCBL8-1 played a positive role in the response of leaves to salt stress by inducing the expression of Na+/H+ antiporter genes and enhancing the antioxidant system to neutralize the accumulation of reactive oxygen species. These observations further indicate that ZmCBL8-1 confers salt stress tolerance, suggesting that transcriptional regulation of the ZmCBL8 gene is important for salt tolerance.

Development of a monoclonal antibody specific for a calpain-generated ∆48 kDa calcineurin fragment, a marker of distressed astrocytes.

BACKGROUND: Calcineurin (CN) is a Ca2+/calmodulin-dependent protein phosphatase. In healthy tissue, CN exists mainly as a full-length (∼60 kDa) highly-regulated protein phosphatase involved in essential cellular functions. However, in diseased or injured tissue, CN is proteolytically converted to a constitutively active fragment that has been causatively-linked to numerous pathophysiologic processes. These calpain-cleaved CN fragments (∆CN) appear at high levels in human brain at early stages of cognitive decline associated with Alzheimer's disease (AD). NEW METHOD: We developed a monoclonal antibody to ∆CN, using an immunizing peptide corresponding to the C-terminal end of the ∆CN fragment. RESULTS: We obtained a mouse monoclonal antibody, designated 26A6, that selectively detects ∆CN in Western analysis of calpain-cleaved recombinant human CN. Using this antibody, we screened both pathological and normal human brain sections provided by the University of Kentucky's Alzheimer's Disease Research Center. 26A6 showed low reactivity towards normal brain tissue, but detected astrocytes both surrounding AD amyloid plaques and throughout AD brain tissue. In brain tissue with infarcts, there was considerable concentration of 26A6-positive astrocytes within/around infarcts, suggesting a link with anoxic/ischemia pathways. COMPARISON WITH EXISTING METHOD: The results obtained with the new monoclonal are similar to those obtained with a polyclonal we had previously developed. However, the monoclonal is an abundant tool available to the dementia research community. CONCLUSIONS: The new monoclonal 26A6 antibody is highly selective for the ∆CN proteolytic fragment and labels a subset of astrocytes, and could be a useful tool for marking insidious brain pathology and identifying novel astrocyte phenotypes.

LINC-PINT suppresses breast cancer cell proliferation and migration via MEIS2/PPP3CC/NF-κB pathway by sponging miR-576-5p.

BACKGROUND: Breast cancer (BCa) is the most frequent malignant tumor in women. Long non-coding RNAs (lncRNAs) have been acknowledged to exert critical regulating functions in various cancers. Long intergenic non-protein coding RNA, p53 induced transcript (LINC-PINT) has been reported to be a chemosensitizer and a tumor suppressor in BCa. However, its downstream molecular mechanism contributing to its tumor-suppressing role remains to be explored in BCa. METHODS: LINC-PINT expression in BCa tissues and cells was measured using quantitative real-time polymerase chain reaction (RT-qPCR). The proliferation of transfected BCa cells was examined by counting kit-8 (CCK-8) and EdU assay. The migrating ability of indicate BCa cells was assessed by wound healing assays. Bioinformatics analysis and mechanism experiments such as RNA immunoprecipitation (RIP), RNA pull down assay, and luciferase reporter assay, were applied to demonstrate the downstream targets of LINC-PINT. RESULTS: LINC-PINT was downregulated in BCa tissues and cell lines. Overexpression of LINC-PINT suppressed BCa cell proliferation and migration. LINC-PINT could interact with miR-576-5p to upregulate Meis homeobox 2 (MEIS2) that positively regulated protein phosphatase 3 catalytic subunit gamma (PPP3CC) by inactivating the nuclear factor-κB (NF-κB) pathway. CONCLUSIONS: These findings elucidated the anti-tumor role of LINC-PINT in BCa via the miR-576-5p/MEIS2/PPP3CC/NF-κB axis, which suggested that LINC-PINT might serve as a potential therapeutic target for BCa.

Longitudinal modeling of human neuronal aging reveals the contribution of the RCAN1-TFEB pathway to Huntington's disease neurodegeneration.

Aging is a common risk factor in neurodegenerative disorders. Investigating neuronal aging in an isogenic background stands to facilitate analysis of the interplay between neuronal aging and neurodegeneration. Here we perform direct neuronal reprogramming of longitudinally collected human fibroblasts to reveal genetic pathways altered at different ages. Comparative transcriptome analysis of longitudinally aged striatal medium spiny neurons (MSNs) in Huntington's disease identified pathways involving RCAN1, a negative regulator of calcineurin. Notably, RCAN1 protein increased with age in reprogrammed MSNs as well as in human postmortem striatum and RCAN1 knockdown rescued patient-derived MSNs of Huntington's disease from degeneration. RCAN1 knockdown enhanced chromatin accessibility of genes involved in longevity and autophagy, mediated through enhanced calcineurin activity, leading to TFEB's nuclear localization by dephosphorylation. Furthermore, G2-115, an analog of glibenclamide with autophagy-enhancing activities, reduced the RCAN1-calcineurin interaction, phenocopying the effect of RCAN1 knockdown. Our results demonstrate that targeting RCAN1 genetically or pharmacologically can increase neuronal resilience in Huntington's disease.

Synergistic effects of putative Ca2+-binding sites of calmodulin in fungal development, temperature stress and virulence of Aspergillus fumigatus.

In pathogenic fungi, calcium-calmodulin-dependent serine-threonine-specific phosphatase calcineurin is involved in morphogenesis and virulence. Therefore, calcineurin and its tightly related protein complexes are attractive antifungal drug targets. However, there is limited knowledge available on the relationship between in vivo Ca2+-binding sites of calmodulin (CaM) and its functions in regulating stress responses, morphogenesis, and pathogenesis. In the current study, we demonstrated that calmodulin is required for hyphal growth, conidiation, and virulence in the human fungal pathogen, Aspergillus fumigatus. Site-directed mutations of calmodulin revealed that a single Ca2+-binding site mutation had no significant effect on A. fumigatus hyphal development, but multiple Ca2+-binding site mutations exhibited synergistic effects, especially when cultured at 42 °C, indicating that calmodulin function in response to temperature stress depends on its Ca2+-binding sites. Western blotting implied that mutations in Ca2+-binding sites caused highly degraded calmodulin fragments, suggesting that the loss of Ca2+-binding sites results in reduced protein stability. Moreover, normal intracellular calcium homeostasis and the nuclear translocation of the transcriptional factor CrzA are dependent on Ca2+-binding sites of AfCaM, demonstrating that Ca2+-binding sites of calmodulin are required for calcium signalling and its major transcription factor CrzA. Importantly, in situ mutations for four Ca2+-binding sites of calmodulin resulted in an almost complete loss of virulence in the Galleria mellonella wax moth model. This study shed more light on the functional characterization of putative calcium-binding sites of calmodulin in the morphogenesis and virulence of A. fumigatus, which enhances our understanding of calmodulin biological functions in cells of opportunistic fungal pathogens.

Susceptibility to caspofungin is regulated by temperature and is dependent on calcineurin in Candida albicans.

IMPORTANCE: Echinocandins are the newest antifungal drugs and are first-line treatment option for life-threatening systemic infections. Due to lack of consensus regarding what temperature should be used when evaluating susceptibility of yeasts to echinocandins, typically either 30°C, 35°C, or 37°C is used. However, the impact of temperature on antifungal efficacy of echinocandins is unexplored. In the current study, we demonstrated that Candida albicans laboratory strain SC5314 was more susceptible to caspofungin at 37°C than at 30°C. We also found that calcineurin was required for temperature-modulated caspofungin susceptibility. Surprisingly, the altered caspofungin susceptibility was not due to differential expression of some canonical genes such as FKS, CHS, or CHT genes. The molecular mechanism of temperature-modulated caspofungin susceptibility is undetermined and deserves further investigations.

Calcineurin-mediated dephosphorylation enhances the stability and transactivation of c-Myc.

c-Myc, a transcription factor, induces cell proliferation and is often aberrantly or highly expressed in cancers. However, molecular mechanisms underlying this aberrantly high expression remain unclear. Here, we found that intracellular Ca2+ concentration regulates c-Myc oncoprotein stability. We identified that calcineurin, a Ca2+-dependent protein phosphatase, is a positive regulator of c-Myc expression. Calcineurin depletion suppresses c-Myc targeted gene expression and c-Myc degradation. Calcineurin directly dephosphorylates Thr58 and Ser62 in c-Myc, which inhibit binding to the ubiquitin ligase Fbxw7. Mutations within the autoinhibitory domain of calcineurin, most frequently observed in cancer, may increase phosphatase activity, increasing c-Myc transcriptional activity in turn. Notably, calcineurin inhibition with FK506 decreased c-Myc expression with enhanced Thr58 and Ser62 phosphorylation in a mouse xenograft model. Thus, calcineurin can stabilize c-Myc, promoting tumor progression. Therefore, we propose that Ca2+ signaling dysfunction affects cancer-cell proliferation via increased c-Myc stability and that calcineurin inhibition could be a new therapeutic target of c-Myc-overexpressing cancers.

USP7-mediated JUND suppresses RCAN2 transcription and elevates NFATC1 to enhance stem cell property in colorectal cancer.

Cancer stem cells (CSCs) encompass a subset of highly aggressive tumor cells that are involved in tumor initiation and progression. This study investigates the function of regulator of calcineurin 2 (RCAN2) in the stem cell property in colorectal cancer (CRC). By analyzing four GEO datasets, we obtained RCAN2 as a stemness-related gene in CRC. RCAN2 was poorly expressed in CRC tissues and cells, especially in CSCs. RCAN2 restoration reduced calcineurin activity and promoted phosphorylation and degradation of nuclear factor of activated T cells 1 (NFATC1) protein, leading to reduced stemness of CSCs. JunD proto-oncogene (JUND), whose protein level was increased in CRC samples and CRC stem cells, bound to RCAN2 and suppressed its transcription. The abundant ubiquitin specific peptidase 7 (USP7) in CSCs enhanced JUND protein stability through deubiquitination modification. Lentivirus-mediated knockdown of USP7 or JUND also blocked the calcineurin-NFATC1 signaling and reduced the protein levels of stemness-related proteins. Moreover, the USP7 knockdown weakened the colony/sphere formation ability as well as the tumorigenicity of CSCs, and it reduced the CSC content in xenograft tumors. However, further restoration of JUND rescued the stemness of the CSCs. Overall, this study demonstrates that USP7-mediated JUND suppresses RCAN2 transcription and activates NFATC1 to enhance stem cell property in CRC. 1. RCAN2 is poorly expressed in CRC tissues and cells and especially in CSCs. 2. RCAN2 reduces stemness of CSCs by blocking calcineurin-NFATC1 signal transduction. 3. JUND binds to RCAN2 promoter to suppresses RCAN2 transcription. 4. USP7 enhances JUND protein stability via deubiquitination modification. 5. Downregulation of USP7 or JUND restores RCAN2 level and suppresses stemness of CSCs.

A novel function of the tomato CALCINEURIN-B LIKE 10 gene as a root-located negative regulator of salt stress.

Climate change exacerbates abiotic stresses like salinization, negatively impacting crop yield, so development of strategies, like using salt-tolerant rootstocks, is crucial. The CALCINEURIN B-LIKE 10 (SlCBL10) gene has been previously identified as a positive regulator of salt tolerance in the tomato shoot. Here, we report a different function of SlCBL10 in tomato shoot and root, as disruption of SlCBL10 only induced salt sensitivity when it was used in the scion but not in the rootstock. The use of SlCBL10 silencing rootstocks (Slcbl10 mutant and RNAi line) improved salt tolerance on the basis of fruit yield. These changes were associated with improved Na+ and K+ homoeostasis, as SlCBL10 silencing reduced the Na+ content and increased the K+ content under salinity, not only in the rootstock but also in the shoot. Improvement of Na+ homoeostasis in Slcbl10 rootstock seems to be mainly due to induction of SlSOS1 expression, while the higher K+ accumulation in roots seems to be mainly determined by expression of LKT1 transporter and SlSKOR channel. These findings demonstrate that SlCBL10 is a negative regulator of salt tolerance in the root, so the use of downregulated SlCBL10 rootstocks may provide a suitable strategy to increase tomato fruit production under salinity.

The CALCINEURIN B-LIKE 4/CBL-INTERACTING PROTEIN 3 module degrades repressor JAZ5 during rose petal senescence.

Flower senescence is genetically regulated and developmentally controlled. The phytohormone ethylene induces flower senescence in rose (Rosa hybrida), but the underlying signaling network is not well understood. Given that calcium regulates senescence in animals and plants, we explored the role of calcium in petal senescence. Here, we report that the expression of calcineurin B-like protein 4 (RhCBL4), which encodes a calcium receptor, is induced by senescence and ethylene signaling in rose petals. RhCBL4 interacts with CBL-interacting protein kinase 3 (RhCIPK3), and both positively regulate petal senescence. Furthermore, we determined that RhCIPK3 interacts with the jasmonic acid response repressor jasmonate ZIM-domain 5 (RhJAZ5). RhCIPK3 phosphorylates RhJAZ5 and promotes its degradation in the presence of ethylene. Our results reveal that the RhCBL4-RhCIPK3-RhJAZ5 module mediates ethylene-regulated petal senescence. These findings provide insights into flower senescence, which may facilitate innovations in postharvest technology for extending rose flower longevity.

MoCbp7, a Novel Calcineurin B Subunit-Binding Protein, Is Involved in the Calcium Signaling Pathway and Regulates Fungal Development, Virulence, and ER Homeostasis in Magnaporthe oryzae.

Calcineurin, a key regulator of the calcium signaling pathway, is involved in calcium signal transduction and calcium ion homeostasis. Magnaporthe oryzae is a devastating filamentous phytopathogenic fungus in rice, yet little is known about the function of the calcium signaling system. Here, we identified a novel calcineurin regulatory-subunit-binding protein, MoCbp7, which is highly conserved in filamentous fungi and was found to localize in the cytoplasm. Phenotypic analysis of the MoCBP7 gene deletion mutant (ΔMocbp7) showed that MoCbp7 influenced the growth, conidiation, appressorium formation, invasive growth, and virulence of M. oryzae. Some calcium-signaling-related genes, such as YVC1, VCX1, and RCN1, are expressed in a calcineurin/MoCbp7-dependent manner. Furthermore, MoCbp7 synergizes with calcineurin to regulate endoplasmic reticulum homeostasis. Our research indicated that M. oryzae may have evolved a new calcium signaling regulatory network to adapt to its environment compared to the fungal model organism Saccharomyces cerevisiae.

Calmodulin Regulates Transient Receptor Potential TRPM3 and TRPM8-Induced Gene Transcription.

Calmodulin is a small protein that binds Ca2+ ions via four EF-hand motifs. The Ca2+/calmodulin complex as well as Ca2+-free calmodulin regulate the activities of numerous enzymes and ion channels. Here, we used genetic and pharmacological tools to study the functional role of calmodulin in regulating signal transduction of TRPM3 and TRPM8 channels. Both TRPM3 and TRPM8 are important regulators of thermosensation. Gene transcription triggered by stimulation of TRPM3 or TRPM8 channels was significantly impaired in cells expressing a calmodulin mutant with mutations in all four EF-hand Ca2+ binding motifs. Similarly, incubation of cells with the calmodulin inhibitor ophiobolin A reduced TRPM3 and TRPM8-induced signaling. The Ca2+/calmodulin-dependent protein phosphatase calcineurin was shown to negatively regulate TRPM3-induced gene transcription. Here, we show that TRPM8-induced transcription is also regulated by calcineurin. We propose that calmodulin plays a dual role in regulating TRPM3 and TRPM8 functions. Calmodulin is required for the activation of TRPM3 and TRPM8-induced intracellular signaling, most likely through a direct interaction with the channels. Ca2+ influx through TRPM3 and TRPM8 feeds back to TRPM3 and TRPM8-induced signaling by activation of the calmodulin-regulated enzyme calcineurin, which acts as a negative feedback loop for both TRPM3 and TRPM8 channel signaling.

Nitrate supply regulates tissue calcium abundance and transcript level of Calcineurin B-like (CBL) gene family in wheat.

Calcium ion (Ca2+) is the most ubiquitous signalling molecule and is sensed by different classes of Ca2+ sensor proteins. Recent evidences underscore the role of calcium signalling in plant response to nitrogen/nitrate supply. Recently we found that under nitrate deficiency, a short-term supply of calcium could improve the plant biomass, nitrate assimilation, anthocyanin accumulation and expression of nitrate uptake and signalling genes. Long-term calcium supply, on the other hand, was not beneficial. Calcineurin B-like (CBL) proteins are one of the vital plant Ca2+ sensory protein family which is essential for stress perception and signaling. To understand the dynamics of CBL-mediated stress signalling in bread wheat, we identified CBL genes in bread wheat (Triticum aestivum) and its progenitors, namely Triticum dicoccoides, Triticum urartu and Aegilops tauschii with the aid of newly available whole-genome sequence. The expression of different CBLs and the changes in root Ca2+ localization in response to nitrate provision or deficiency were analysed. Expression of the CBLs were studied in two bread wheat genotypes with comparatively higher (B.T. Schomburgk, BTS) and lower (Gluyas early, GE) nitrate responsiveness and nitrogen use efficiency. High N promoted the expression of CBLs in seedling leaves while in roots the expression was promoted by N deficiency. At the 5 days after anthesis stage, nitrate starvation downregulated the expression of CBLs while nitrate supply enhanced the expression. At anthesis stage, expression of CBL6 was significantly promoted by HN in panicles of both the genotypes, the highest expression was recorded in BTS. Expression of CBL6 was significantly upregulated by short term nitrate treatment also suggesting its role in Primary nitrate response (PNR) in wheat. There was a significant down regulation of CBL6 expression post nitrate starvation, making it a probable regulator of nitrogen starvation response (NSR) as well. In seedling roots, the tissue localization of Ca2+ was increased both by high and low nitrate treatments, albeit at different magnitudes. Our results suggest that calcium signalling might be a major signalling pathway governing nitrogen responsiveness and CBL6 might be playing pivotal role in NSR and PNR in wheat.

Human disease-associated calmodulin mutations alter calcineurin function through multiple mechanisms.

Calmodulin (CaM) is a ubiquitous, calcium-sensing protein that regulates a multitude of processes throughout the body. In response to changes in [Ca2+], CaM modifies, activates, and deactivates enzymes and ion channels, as well as many other cellular processes. The importance of CaM is highlighted by the conservation of an identical amino acid sequence in all mammals. Alterations to CaM amino acid sequence were once thought to be incompatible with life. During the last decade modifications to the CaM protein sequence have been observed in patients suffering from life-threatening heart disease (calmodulinopathy). Thus far, inadequate or untimely interaction between mutant CaM and several proteins (LTCC, RyR2, and CaMKII) have been identified as mechanisms underlying calmodulinopathy. Given the extensive number of CaM interactions in the body, there are likely many consequences for altering CaM protein sequence. Here, we demonstrate that disease-associated CaM mutations alter the sensitivity and activity of the Ca2+-CaM-enhanced serine/threonine phosphatase calcineurin (CaN). Biophysical characterization by circular dichroism, solution NMR spectroscopy, stopped-flow kinetic measurements, and MD simulations provide mechanistic insight into mutation dysfunction as well as highlight important aspects of CaM Ca2+ signal transduction. We find that individual CaM point mutations (N53I, F89L, D129G, and F141L) impair CaN function, however, the mechanisms are not the same. Specifically, individual point mutations can influence or modify the following properties: CaM binding, Ca2+ binding, and/or Ca2+kinetics. Moreover, structural aspects of the CaNCaM complex can be altered in manners that indicate changes to allosteric transmission of CaM binding to the enzyme active site. Given that loss of CaN function can be fatal, as well as evidence that CaN modifies ion channels already associated with calmodulinopathy, our results raise the possibility that altered CaN function contributes to calmodulinopathy.

Dysregulating PHO Signaling via the CDK Machinery Differentially Impacts Energy Metabolism, Calcineurin Signaling, and Virulence in Cryptococcus neoformans.

Fungal pathogens uniquely regulate phosphate homeostasis via the cyclin-dependent kinase (CDK) signaling machinery of the phosphate acquisition (PHO) pathway (Pho85 kinase-Pho80 cyclin-CDK inhibitor Pho81), providing drug-targeting opportunities. Here, we investigate the impact of a PHO pathway activation-defective Cryptococcus neoformans mutant (pho81Δ) and a constitutively activated PHO pathway mutant (pho80Δ) on fungal virulence. Irrespective of phosphate availability, the PHO pathway was derepressed in pho80Δ with all phosphate acquisition pathways upregulated and much of the excess phosphate stored as polyphosphate (polyP). Elevated phosphate in pho80Δ coincided with elevated metal ions, metal stress sensitivity, and a muted calcineurin response, all of which were ameliorated by phosphate depletion. In contrast, metal ion homeostasis was largely unaffected in the pho81Δ mutant, and Pi, polyP, ATP, and energy metabolism were reduced, even under phosphate-replete conditions. A similar decline in polyP and ATP suggests that polyP supplies phosphate for energy production even when phosphate is available. Using calcineurin reporter strains in the wild-type, pho80Δ, and pho81Δ background, we also demonstrate that phosphate deprivation stimulates calcineurin activation, most likely by increasing the bioavailability of calcium. Finally, we show that blocking, as opposed to permanently activating, the PHO pathway reduced fungal virulence in mouse infection models to a greater extent and that this is most likely attributable to depleted phosphate stores and ATP, and compromised cellular bioenergetics, irrespective of phosphate availability. IMPORTANCE Invasive fungal diseases cause more than 1.5 million deaths per year, with an estimated 181,000 of these deaths attributable to Cryptococcal meningitis. Despite the high mortality, treatment options are limited. In contrast to humans, fungal cells maintain phosphate homeostasis via a CDK complex, providing drug-targeting opportunities. To investigate which CDK components are the best targets for potential antifungal therapy, we used strains with a constitutively active (pho80Δ) and an activation-defective (pho81Δ) PHO pathway, to investigate the impact of dysregulated phosphate homeostasis on cellular function and virulence. Our studies suggest that inhibiting the function of Pho81, which has no human homologue, would have the most detrimental impact on fungal growth in the host due to depletion of phosphate stores and ATP, irrespective of phosphate availability in the host.

The Ycx1 protein encoded by the yeast YDL206W gene plays a role in calcium and calcineurin signaling.

Calcium is ubiquitously present in all living cells and plays important regulatory roles in a wide variety of biological processes. In yeast, many effects of calcium are mediated via the action of calcineurin, a calcium/calmodulin-dependent protein phosphatase. Proper signaling of calcium and calcineurin is important in yeast, and the calcineurin pathway has emerged as a valuable target for developing novel antifungal drugs. Here, we report a role of YDL206W in calcium and calcineurin signaling in yeast. YDL206W is an uncharacterized gene in yeast, encoding a protein with two sodium/calcium exchange domains. Disrupting the YDL206W gene leads to a diminished level of calcium-induced activation of calcineurin and a reduced accumulation of cytosolic calcium. Consistent with a role of calcineurin in regulating pheromone and cell wall integrity signaling, the ydl206wΔ mutants display an enhanced growth arrest induced by pheromone treatment and poor growth at elevated temperature. Subcellular localization studies indicate that YDL206W is localized in endoplasmic reticulum and Golgi. Together, our results reveal YDL206W as a new regulator for calcineurin signaling in yeast and suggest a role of the endoplasmic reticulum and Golgi in regulating cytosolic calcium in yeast.