Rapid kinetics of calcium dissociation from plant calmodulin and calmodulin-like proteins and effect of target peptides.

Calcium (Ca2+) signaling represents a universal information code in plants, playing crucial roles spanning developmental processes to stress responses. Ca2+ signals are decoded into defined plant adaptive responses by different Ca2+ sensing proteins, including calmodulin (CaM) and calmodulin-like (CML) proteins. Although major advances have been achieved in describing how these Ca2+ decoding proteins interact and regulate downstream target effectors, the molecular details of these processes remain largely unknown. Herein, the kinetics of Ca2+ dissociation from a conserved CaM and two CML isoforms from A. thaliana has been studied by fluorescence stopped-flow spectroscopy. Kinetic data were obtained for the isolated Ca2+-bound proteins as well as for the proteins complexed with different target peptides. Moreover, the lobe specific interactions between the Ca2+ sensing proteins and their targets were characterized by using a panel of protein mutants deficient in Ca2+ binding at the N-lobe or C-lobe. Results were analyzed and discussed in the context of the Ca2+-decoding and Ca2+-controlled target binding mechanisms in plants.

SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana.

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp-CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein-peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.

Phosphoinositide phosphatase Sac3 regulates the cell surface expression of scavenger receptor A and formation of lipid droplets in macrophages.

The findings of this study suggest that the phosphoinositide phosphatase Sac3 maintains the protein level of scavenger receptor A (SR-A) and regulates foam cell formation. RAW264.7 macrophages were transfected with short hairpin RNAs that target Sac3. The knockdown decreased the level of the cell surface SR-A and suppressed the acetylated low density lipoprotein-induced foam cell formation. The associated regulator of PIKfyve (ArPIKfyve) is a scaffold protein that protects Sac3 from proteasome-dependent degradation. The knockdown of ArPIKfyve decreased Sac3, cell surface SR-A, and foam cell formation. The knockdown of PIKfyve had no effect on SR-A protein levels. These results suggest that the ArPIKfyve-Sac3 complex regulates SR-A protein levels independently of its effect on PIKfyve activity.

The Protein Complex of Neurodegeneration-related Phosphoinositide Phosphatase Sac3 and ArPIKfyve Binds the Lewy Body-associated Synphilin-1, Preventing Its Aggregation.

The 5-phosphoinositide phosphatase Sac3, in which loss-of-function mutations are linked to neurodegenerative disorders, forms a stable cytosolic complex with the scaffolding protein ArPIKfyve. The ArPIKfyve-Sac3 heterodimer interacts with the phosphoinositide 5-kinase PIKfyve in a ubiquitous ternary complex that couples PtdIns(3,5)P2 synthesis with turnover at endosomal membranes, thereby regulating the housekeeping endocytic transport in eukaryotes. Neuron-specific associations of the ArPIKfyve-Sac3 heterodimer, which may shed light on the neuropathological mechanisms triggered by Sac3 dysfunction, are unknown. Here we conducted mass spectrometry analysis for brain-derived interactors of ArPIKfyve-Sac3 and unraveled the α-synuclein-interacting protein Synphilin-1 (Sph1) as a new component of the ArPIKfyve-Sac3 complex. Sph1, a predominantly neuronal protein that facilitates aggregation of α-synuclein, is a major component of Lewy body inclusions in neurodegenerative α-synucleinopathies. Modulations in ArPIKfyve/Sac3 protein levels by RNA silencing or overexpression in several mammalian cell lines, including human neuronal SH-SY5Y or primary mouse cortical neurons, revealed that the ArPIKfyve-Sac3 complex specifically altered the aggregation properties of Sph1-GFP. This effect required an active Sac3 phosphatase and proceeded through mechanisms that involved increased Sph1-GFP partitioning into the cytosol and removal of Sph1-GFP aggregates by basal autophagy but not by the proteasomal system. If uncoupled from ArPIKfyve elevation, overexpressed Sac3 readily aggregated, markedly enhancing the aggregation potential of Sph1-GFP. These data identify a novel role of the ArPIKfyve-Sac3 complex in the mechanisms controlling aggregate formation of Sph1 and suggest that Sac3 protein deficiency or overproduction may facilitate aggregation of aggregation-prone proteins, thereby precipitating the onset of multiple neuronal disorders.

Conduction and validation of a novel mitotic spindle assembly related signature in hepatocellular carcinoma: prognostic prediction, tumor immune microenvironment and drug susceptibility.

Introduction: We have developed a risk-scoring model using gene expression levels related to mitotic spindle assembly (MSA) to predict the prognosis of liver cancer. Methods and results: Initially, we identified 470 genes related to MSA from public databases. Subsequently, through analysis of sequencing data from liver cancer patient samples in online databases, we identified 7 genes suitable for constructing the risk-scoring model. We validated the predictive accuracy and clinical utility of the model. Through drug sensitivity analysis, we identified SAC3D1 as a gene sensitive to the most common anti-tumor drugs among these 7 genes. We propose SAC3D1 as a significant target for future clinical treatment. Furthermore, we conducted in vivo and in vitro experiments to validate the relevance of SAC3D1 to MSA and found its significant impact on the PI3K/Akt signaling pathway and spindle function. Conclusion: Our research introduces a novel risk-scoring model that accurately predicts liver cancer prognosis. Additionally, our findings suggest SAC3D1 as a promising therapeutic target for hepatocellular carcinoma, potentially revealing new mechanisms underlying liver cancer development.

The PIKfyve-ArPIKfyve-Sac3 triad in human breast cancer: Functional link between elevated Sac3 phosphatase and enhanced proliferation of triple negative cell lines.

The phosphoinositide 5-kinase PIKfyve and 5-phosphatase Sac3 are scaffolded by ArPIKfyve in the PIKfyve-ArPIKfyve-Sac3 (PAS) regulatory complex to trigger a unique loop of PtdIns3P-PtdIns(3,5)P2 synthesis and turnover. Whereas the metabolizing enzymes of the other 3-phosphoinositides have already been implicated in breast cancer, the role of the PAS proteins and the PtdIns3P-PtdIns(3,5)P2 conversion is unknown. To begin elucidating their roles, in this study we monitored the endogenous levels of the PAS complex proteins in cell lines derived from hormone-receptor positive (MCF7 and T47D) or triple-negative breast cancers (TNBC) (BT20, BT549 and MDA-MB-231) as well as in MCF10A cells derived from non-tumorigenic mastectomy. We report profound upregulation of Sac3 and ArPIKfyve in the triple negative vs. hormone-sensitive breast cancer or non-tumorigenic cells, with BT cell lines showing the highest levels. siRNA-mediated knockdown of Sac3, but not that of PIKfyve, significantly inhibited proliferation of BT20 and BT549 cells. In these cells, knockdown of ArPIKfyve had only a minor effect, consistent with a primary role for Sac3 in TNBC cell proliferation. Intriguingly, steady-state levels of PtdIns(3,5)P2 in BT20 and T47D cells were similar despite the 6-fold difference in Sac3 levels between these cell lines. However, steady-state levels of PtdIns3P and PtdIns5P, both regulated by the PAS complex, were significantly reduced in BT20 vs. T47D or MCF10A cell lines, consistent with elevated Sac3 affecting directly or indirectly the homeostasis of these lipids in TNBC. Together, our results uncover an unexpected role for Sac3 phosphatase in TNBC cell proliferation. Database analyses, discussed herein, reinforce the involvement of Sac3 in breast cancer pathogenesis.

SAC3D1 activates Wnt/ß­catenin signalling in hepatocellular carcinoma.

ß­catenin accumulates in hepatocellular carcinoma (HCC); therefore, understanding the mechanism of Wnt/ß­catenin pathway activation is important for HCC therapy. SAC3 domain containing 1 (SAC3D1) is involved in numerous types of cancer, such as gastric cancer. To the best of our knowledge, however, the role of SAC3D1 in HCC has not yet been elucidated. Here, the expression of SAC3D in HCC was examined by quantitative PCR, western blotting and immunohistochemistry. The function of SAC3D1 in HCC were examined using Cell Counting Kit­8 and anchorage­independent growth assay. It was found that the levels of SAC3D1 mRNA and protein were upregulated in HCC. When SAC3D1 was overexpressed, the proliferation of HCC cells was promoted; when the expression of SAC3D1 was disrupted, HCC cell growth was inhibited. When the molecular mechanism was investigated using immunoprecipitation, it was found that SAC3D1 interacted with axin, inhibiting ubiquitination of ß­catenin and elevating protein levels of ß­catenin. In summary, the present study revealed the promoting function of SAC3D1 in the progression of HCC. SAC3D1 may be a promising target for HCC therapy.

Upregulated expression of SAC3D1 is associated with progression in gastric cancer.

SAC3 domain containing 1 (SAC3D1) has been reported to be involved in numerous types of cancer. However, the role of SAC3D1 in GC has not yet been elucidated. In the present study, the mRNA expression level of SAC3D1 between GC and normal tissues were assessed with a continuous variable meta­analysis based on multiple datasets from public databases. The protein expression level of SAC3D1 in GC and normal tissues was assessed by an in­house immunohistochemistry (IHC). The association between SAC3D1 expression and some clinical parameters was assessed based on the TCGA and IHC data. Survival analysis was performed to assess the association between SAC3D1 expression and the survival of GC patients. The co­expressed genes of SAC3D1 were determined by integrating three online tools, and the enrichment analyses were performed to determine SAC3D1­related pathways and hub co­expressed genes. SAC3D1 was significantly upregulated in GC tumor tissues in comparison to normal tissues with the SMD being 0.45 (0.12, 0.79). The IHC results also indicated that SAC3D1 protein expression in GC tissues was markedly higher than in normal tissues. The SMD following the addition of the IHC data was 0.59 (0.11, 1.07). The protein levels of SAC3D1 were positively associated with the histological grade, T stage and N stage of GC (P<0.001). The TCGA data also revealed that the SAC3D1 mRNA level was significantly associated with the N stage (P<0.001). Moreover, prognosis analysis indicated that SAC3D1 was closely associated with the prognosis of patients with GC. Moreover, 410 co­expressed genes of SAC3D1 were determined, and these genes were mainly enriched in the cell cycle. In total, 4 genes (CDK1, CCNB1, CCNB2 and CDC20) were considered key co­expressed genes. On the whole, these findings demonstrate that SAC3D1 is highly expressed in GC and may be associated with the progression of GC.

Severe Consequences of SAC3/FIG4 Phosphatase Deficiency to Phosphoinositides in Patients with Charcot-Marie-Tooth Disease Type-4J.

Charcot-Marie-Tooth disease type-4J (CMT4J), an autosomal recessively inherited peripheral neuropathy characterized by neuronal degeneration, segmental demyelination, and limb muscle weakness, is caused by compound heterozygous mutations in the SAC3/FIG4 gene, resulting in SAC3/FIG4 protein deficiency. SAC3/FIG4 is a phosphatase that not only turns over PtdIns(3,5)P2 to PtdIns3P but also promotes PtdIns(3,5)P2 synthesis by activating the PIKFYVE kinase that also makes PtdIns5P. Whether CMT4J patients have alterations in PtdIns(3,5)P2, PtdIns5P or in other phosphoinositides (PIs), and if yes, in what direction these changes might be, has never been examined. We performed PI profiling in primary fibroblasts from a cohort of CMT4J patients. Subsequent to myo-[2-3H]inositol cell labeling to equilibrium, steady-state levels of PIs were quantified by HPLC under conditions concurrently detecting PtdIns5P, PtdIns(3,5)P2, and the other PIs. Immunoblotting verified SAC3/FIG4 depletion in CMT4J fibroblasts. Compared to normal human controls (n = 9), both PtdIns(3,5)P2 and PtdIns5P levels were significantly decreased in CMT4J fibroblasts (n = 13) by 36.4 ± 3.6% and 43.1 ± 4.4%, respectively (p < 0.0001). These reductions were independent of patients' gender or disease onset. Although mean values for PtdIns3P in the CMT4J cohort remained unchanged, there were high variations in PtdIns3P among individual patients. Aberrant endolysosomal vacuoles, typically seen under PtdIns(3,5)P2 reduction, were apparent but not in fibroblasts from all patients. The subset of patients without aberrant vacuoles exhibited especially low PtdIns3P levels. Concomitant decreases in PtdIns5P and PtdIns(3,5)P2 and the link between PtdIns3P levels and cellular vacuolization are novel insights shedding further light into the molecular determinants in CMT4J polyneuropathy.

The mRNA export factor Sac3 maintains nuclear homeostasis and regulates cytoskeleton organization in Candida albicans.

AIM: In eukaryotes, the nuclear export of mRNAs is essential for gene expression and regulations of numerous cellular processes. This study aimed to identify the mRNA export factor Sac3 in Candida albicans. MATERIALS & METHODS: A sac3Δ/Δ mutant was obtained using PCR-mediated homologous recombination. RESULTS: Disruption of SAC3 caused abnormal accumulation of mRNA in the nuclei. Further investigations revealed that sac3Δ/Δ mutant exhibited a severely growth defect, which was related to abnormal aggregation of microtubules. Moreover, loss of Sac3 caused a defect in hyphal polarized growth, which was associated with depolarization of actin cytoskeleton. In addition, the virulence of sac3Δ/Δ mutant was seriously attenuated. CONCLUSION: Our findings provide new insights into the mRNA export factor Sac3 in C. albicans.

PRP4KA, a Putative Spliceosomal Protein Kinase, Is Important for Alternative Splicing and Development in Arabidopsis thaliana.

Splicing of precursor messenger RNAs (pre-mRNAs) is an essential step in the expression of most eukaryotic genes. Both constitutive splicing and alternative splicing, which produces multiple messenger RNA (mRNA) isoforms from a single primary transcript, are modulated by reversible protein phosphorylation. Although the plant splicing machinery is known to be a target for phosphorylation, the protein kinases involved remain to be fully defined. We report here the identification of pre-mRNA processing 4 (PRP4) KINASE A (PRP4KA) in a forward genetic screen based on an alternatively spliced GFP reporter gene in Arabidopsis thaliana (Arabidopsis). Prp4 kinase is the first spliceosome-associated kinase shown to regulate splicing in fungi and mammals but it has not yet been studied in plants. In the same screen we identified mutants defective in SAC3A, a putative mRNA export factor that is highly coexpressed with PRP4KA in Arabidopsis Whereas the sac3a mutants appear normal, the prp4ka mutants display a pleiotropic phenotype featuring atypical rosettes, late flowering, tall final stature, reduced branching, and lowered seed set. Analysis of RNA-sequencing data from prp4ka and sac3a mutants identified widespread and partially overlapping perturbations in alternative splicing in the two mutants. Quantitative phosphoproteomic profiling of a prp4ka mutant detected phosphorylation changes in several serine/arginine-rich proteins, which regulate constitutive and alternative splicing, and other splicing-related factors. Tests of PRP4KB, the paralog of PRP4KA, indicated that the two genes are not functionally redundant. The results demonstrate the importance of PRP4KA for alternative splicing and plant phenotype, and suggest that PRP4KA may influence alternative splicing patterns by phosphorylating a subset of splicing regulators.

Phosphatidylinositol-3,5-bisphosphate: metabolism and physiological functions.

Phosphatidylinositol (PtdIns) is a membrane phospholipid composed of diacylglycerol and a D-myo-inositol head group. In mammals, the hydroxyl groups at the D3, D4 and D5 positions of the inositol ring can be phosphorylated to yield seven phosphoinositide derivatives. PtdIns-3,5-bisphosphate [PtdIns(3,5)P2] is the most recently discovered species of phosphoinositide that is generated by the phosphorylation of PtdIns(3)P at the D5 position by PtdIns phosphate kinase and catabolized through the dephosphorylation by myotubularin family of phosphatases. Genetic and biochemical analyses of the enzymes metabolizing PtdIns(3,5)P2 have revealed that this phospholipid is involved in the control of endolysosomal systems and plays crucial roles in various mammalian tissues. In this article, we review the current state of knowledge of the metabolic/physiological functions of PtdIns(3,5)P2, and describe how disruption of these functions may contribute to human diseases.

Molecular Genetics of Autosomal-Recessive Demyelinating Charcotmarie-Tooth Disease (Review Article) / Монголын Анагаах Ухаан

Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogenous group of disorders. Useful classifi cation is still clinical and electrophysiological classifi cation that divides CMT into CMT type 1 - demyelinating form and CMT type 2 - axonal form. An intermediate type is also increasingly being determined. Inheritance can be autosomal dominant, X-linked and autosomal recessive (AR). In this review, we will focus on the clinical and/or electrophysiological findings and molecular genetics of ARCMT1 (CMT4). Ten genes, GDAP1, MTMR2, MTMR13, SH3TC2, NDRG1, EGR2, PRX, CTDP1, FGD4 and SAC3 have been identifi ed in the CMT4A, CMT4B1, CMT4B2, CMT4C, CMT4D, CMT4E, CMT4F, CCFDN, CMT4H and CMT4J types, respectively. In addition, susceptibility locus on chromosome 10q23 has been found for CMT4G disease. Molecular genetics of demyelinating ARCMT are large disabilities of proteins in Schwann cells and their functions (transcriptional factor, protein transport, protein sorting, intra/extra cellular compartments, signal transduction, cell division, and cell differentiation). It has been rising necessary requirements to defi ne clinical and genetic subtypes of the ARCMT1, prevent from disease, give reproductive and genetic counselling, and develop methods for reducing and clear disease risk factor.