Altered Subcellular Localization Research Articles

Lansoprazole (LPZ) reverses multidrug resistance (MDR) in cancer through impeding ATP-binding cassette (ABC) transporter-mediated chemotherapeutic drug efflux and lysosomal sequestration

AimsLansoprazole is one of the many proton pump inhibitors (PPIs) that acts more strongly with ABCB1 and ABCG2. The present study is to investigate the potential of lansoprazole on reversal of ABCB1/G2-mediated MDR in cancer, in vitro and in vivo. MethodsReversal studies and combination evaluation were conducted to determine the synergistic anti-MDR effects on lansoprazole. Lysosomal staining was used to determination of lansoprazole on ABCB1-mediated lysosomal sequestration. Substrate accumulation and efflux assays, ATPase activity, and molecular docking were conducted to evaluate lansoprazole on ABCB1/G2 functions. Western blot and immunofluorescence were used to detect lansoprazole on ABCB1/G2 expression and subcellular localization. MDR nude mice models were established to evaluate the effects of lansoprazole on MDR in vivo. ResultsLansoprazole attenuated ABCB1/G2-mediated MDR and exhibited synergistic effects with substrate drugs in MDR cells. In vivo experiments demonstrated that lansoprazole attenuated ABCB1/G2-mediated MDR and exhibited synergistic effects that augmented the sensitivity of substrate anticancer drugs in ABCB1/G2-mediated settings without obvious toxicity. Lansoprazole impeded lysosomal sequestration mediated by ABCB1, leading to a substantial increase in intracellular accumulation of substrate drugs. The effects of lansoprazole were not attributable to downregulation or alterations in subcellular localization of ABCB1/G2. Lansoprazole promoted the ATPase activity of ABCB1/G2 and competitively bound to the substrate-binding region of ABCB1/G2. ConclusionsThese findings present novel therapeutic avenues whereby the combination of lansoprazole and chemotherapeutic agents mitigates MDR mediated by ABCB1/G2 overexpression.

Exploring Syndecan-4 and MLP and Their Interaction in Primary Cardiomyocytes and H9c2 Cells.

The transmembrane proteoglycan syndecan-4 is known to be involved in the hypertrophic response to pressure overload. Although multiple downstream signaling pathways have been found to be involved in this response in a syndecan-4-dependent manner, there are likely more signaling components involved. As part of a larger syndecan-4 interactome screening, we have previously identified MLP as a binding partner to the cytoplasmic tail of syndecan-4. Interestingly, many human MLP mutations have been found in patients with hypertrophic (HCM) and dilated cardiomyopathy (DCM). To gain deeper insight into the role of the syndecan-4-MLP interaction and its potential involvement in MLP-associated cardiomyopathy, we have here investigated the syndecan-4-MLP interaction in primary adult rat cardiomyocytes and the H9c2 cell line. The binding of syndecan-4 and MLP was analyzed in total lysates and subcellular fractions of primary adult rat cardiomyocytes, and baseline and differentiated H9c2 cells by immunoprecipitation. MLP and syndecan-4 localization were determined by confocal microscopy, and MLP oligomerization was determined by immunoblotting under native conditions. Syndecan-4-MLP binding, as well as MLP self-association, were also analyzed by ELISA and peptide arrays. Our results showed that MLP-WT and syndecan-4 co-localized in many subcellular compartments; however, their binding was only detected in nuclear-enriched fractions of isolated adult cardiomyocytes. In vitro, syndecan-4 bound to MLP at three sites, and this binding was reduced in some HCM-associated MLP mutations. While MLP and syndecan-4 also co-localized in many subcellular fractions of H9c2 cells, these proteins did not bind at baseline or after differentiation into cardiomyocyte-resembling cells. Independently of syndecan-4, mutated MLP proteins had an altered subcellular localization in H9c2 cells, compared to MLP-WT. The DCM- and HCM-associated MLP mutations, W4R, L44P, C58G, R64C, Y66C, K69R, G72R, and Q91L, affected the oligomerization of MLP with an increase in monomeric at the expense of trimeric and tetrameric recombinant MLP protein. Lastly, two crucial sites for MLP self-association were identified, which were reduced in most MLP mutations. Our data indicate that the syndecan-4-MLP interaction was present in nuclear-enriched fractions of isolated adult cardiomyocytes and that this interaction was disrupted by some HCM-associated MLP mutations. MLP mutations were also linked to changes in MLP oligomerization and self-association, which may be essential for its interaction with syndecan-4 and a critical molecular mechanism of MLP-associated cardiomyopathy.

Molecular and cellular consequences of mevalonate kinase deficiency

Mevalonate kinase deficiency (MKD) is an autosomal recessive metabolic disorder associated with recurrent autoinflammatory episodes. The disorder is caused by bi-allelic loss-of-function variants in the MVK gene, which encodes mevalonate kinase (MK), an early enzyme in the isoprenoid biosynthesis pathway. To identify molecular and cellular consequences of MKD, we studied primary fibroblasts from severely affected patients with mevalonic aciduria (MKD-MA) and more mildly affected patients with hyper IgD and periodic fever syndrome (MKD-HIDS). As previous findings indicated that the deficient MK activity in MKD impacts protein prenylation in a temperature-sensitive manner, we compared the subcellular localization and activation of the small Rho GTPases RhoA, Rac1 and Cdc42 in control, MKD-HIDS and MKD-MA fibroblasts cultured at physiological and elevated temperatures. This revealed a temperature-induced altered subcellular localization and activation in the MKD cells. To study if and how the temperature-induced ectopic activation of these signalling proteins affects cellular processes, we performed comparative transcriptome analysis of control and MKD-MA fibroblasts cultured at 37 °C or 40 °C. This identified cell cycle and actin cytoskeleton organization as respectively most down- and upregulated gene clusters. Further studies confirmed that these processes were affected in fibroblasts from both patients with MKD-MA and MKD-HIDS. Finally, we found that, similar to immune cells, the MK deficiency causes metabolic reprogramming in MKD fibroblasts resulting in increased expression of genes involved in glycolysis and the PI3K/Akt/mTOR pathway. We postulate that the ectopic activation of small GTPases causes inappropriate signalling contributing to the molecular and cellular aberrations observed in MKD.

GRP78 Contributes to the Beneficial Effects of SGLT2 Inhibitor on Proximal Tubular Cells in DKD.

Beneficial effects of SGLT2 inhibitors on kidney function are well-known; however, their molecular mechanisms are not fully understood. We focused on 78 kDa glucose-regulated protein (GRP78) and its interaction with SGLT2 and Integrin ß1 beyond the chaperone property of GRP78. In STZinduced diabetic mouse kidneys, GRP78, SGLT2, and Integrin ß1 increased in the plasma membrane fraction, while they were suppressed by canagliflozin. The altered subcellular localization of GRP78/Integrin ß1 in STZ mice promoted epithelial mesenchymal transition (EMT) and fibrosis, which were mitigated by canagliflozin. High glucose conditions reduced intracellular GRP78, increased its secretion, and caused EMT-like changes in cultured HK2 cells, which were again inhibited by canagliflozin. Urinary GRP78 increased in STZ mice, and in vitro experiments with recombinant GRP78 suggested that inflammation spread to surrounding tubular cells and canagliflozin reversed this effect. Under normal glucose culture, canagliflozin maintained SERCA activity, promoted endoplasmic reticulum (ER) robustness, reduced ER stress response impairment, and protected proximal tubular cells. In conclusion, canagliflozin restored subcellular localization of GRP78, SGLT2 and Integrin ß1 and inhibited EMT and fibrosis in DKD. In non-diabetic CKD, canagliflozin promoted ER robustness by maintaining SERCA activity and preventing ER stress response failure, and contribute to tubular protection.

Ciliopathy due to POC1A deficiency: clinical and metabolic features, and cellular modeling.

SOFT syndrome (MIM#614813), denoting Short stature, Onychodysplasia, Facial dysmorphism, and hypoTrichosis, is a rare primordial dwarfism syndrome caused by biallelic variants in POC1A, encoding a centriolar protein. SOFT syndrome, characterized by severe growth failure of prenatal onset and dysmorphic features, was recently associated with insulin resistance. This study aims to further explore its endocrinological features and pathophysiological mechanisms. We present clinical, biochemical, and genetic features of 2 unrelated patients carrying biallelic pathogenic POC1A variants. Cellular models of the disease were generated using patients' fibroblasts and POC1A-deleted human adipose stem cells. Both patients present with clinical features of SOFT syndrome, along with hyperinsulinemia, diabetes or glucose intolerance, hypertriglyceridemia, liver steatosis, and central fat distribution. They also display resistance to the effects of IGF-1. Cellular studies show that the lack of POC1A protein expression impairs ciliogenesis and adipocyte differentiation, induces cellular senescence, and leads to resistance to insulin and IGF-1. An altered subcellular localization of insulin receptors and, to a lesser extent, IGF1 receptors could also contribute to resistance to insulin and IGF1. Severe growth retardation, IGF-1 resistance, and centripetal fat repartition associated with insulin resistance-related metabolic abnormalities should be considered as typical features of SOFT syndrome caused by biallelic POC1A null variants. Adipocyte dysfunction and cellular senescence likely contribute to the metabolic consequences of POC1A deficiency. SOFT syndrome should be included within the group of monogenic ciliopathies with metabolic and adipose tissue involvement, which already encompasses Bardet-Biedl and Alström syndromes.

Tpr Misregulation in Hippocampal Neural Stem Cells in Mouse Models of Alzheimer's Disease.

Nuclear pore complexes (NPCs) are highly dynamic macromolecular protein structures that facilitate molecular exchange across the nuclear envelope. Aberrant NPC functioning has been implicated in neurodegeneration. The translocated promoter region (Tpr) is a critical scaffolding nucleoporin (Nup) of the nuclear basket, facing the interior of the NPC. However, the role of Tpr in adult neural stem/precursor cells (NSPCs) in Alzheimer's disease (AD) is unknown. Using super-resolution (SR) and electron microscopy, we defined the different subcellular localizations of Tpr and phospho-Tpr (P-Tpr) in NSPCs in vitro and in vivo. Elevated Tpr expression and reduced P-Tpr nuclear localization accompany NSPC differentiation along the neurogenic lineage. In 5xFAD mice, an animal model of AD, increased Tpr expression in DCX+ hippocampal neuroblasts precedes increased neurogenesis at an early stage, before the onset of amyloid-β plaque formation. Whereas nuclear basket Tpr interacts with chromatin modifiers and NSPC-related transcription factors, P-Tpr interacts and co-localizes with cyclin-dependent kinase 1 (Cdk1) at the nuclear chromatin of NSPCs. In hippocampal NSPCs in a mouse model of AD, aberrant Tpr expression was correlated with altered NPC morphology and counts, and Tpr was aberrantly expressed in postmortem human brain samples from patients with AD. Thus, we propose that altered levels and subcellular localization of Tpr in CNS disease affect Tpr functionality, which in turn regulates the architecture and number of NSPC NPCs, possibly leading to aberrant neurogenesis.

Upregulation of LRRK2 following traumatic brain injury does not directly phosphorylate Thr175 tau.

Phosphorylated microtubule-associated protein tau (tau) aggregates are a pathological hallmark of various neurodegenerative diseases, including chronic traumatic encephalopathy and amyotrophic lateral sclerosis with cognitive impairment. While there are many residues phosphorylated on tau, phosphorylation of threonine 175 (pThr175 tau) has been shown to initiate fibril formation in vitro and is present in pathological tau aggregates in vivo. Given this, preventing Thr175 tau phosphorylation presents a potential approach to reduce fibril formation; however, the kinase(s) acting on Thr175 are not yet fully defined. Using a single controlled cortical impact rodent model of traumatic brain injury (TBI), which rapidly induces Thr175 tau phosphorylation, we observed an upregulation and alteration in subcellular localization of leucine-rich repeat kinase 2 (LRRK2), a kinase that has been implicated in tau phosphorylation. LRRK2 upregulation was evident by one-day post-injury and persisted to day 10. The most notable changes were observed in microglia at the site of injury in the cortex. To determine if the appearance of pThr175 tau was causally related to the upregulation of LRRK2 expression, we examined the ability of LRRK2 to phosphorylate Thr175in vitro by co-transfecting 2N4R human WT-tau with either LRRK2-WT, constitutively-active LRRK2-G2019S or inactive LRRK2-3XKD. We found no significant difference in the level of pThr175 tau between the overexpression of LRRK2-WT, -G2019S or -3XKD, suggesting LRRK2 does not phosphorylate tau at Thr175. Further, downstream events known to follow Thr175 phosphorylation and known to be associated with pathological tau fibril formation (pSer9-GSK3β and pThr231 tau induction) also remained unchanged. We conclude that while LRRK2 expression is altered in TBI, it does not contribute directly to pThr175 tau generation.

Caspase cleaves Drosophila BubR1 to modulate spindle assembly checkpoint function and lifespan of the organism.

Caspases cleave over 1500 substrates in the human proteome in both lethal and non-lethal scenarios. However, reports of the physiological consequences of substrate cleavage are limited. Additionally, the manner in which caspase cleaves only a subset of substrates in the non-lethal scenario remains to be elucidated. BubR1, a spindle assembly checkpoint component, is a caspase substrate in humans, the physiological function of which remains unclear. Here, we found that caspases, especially Drice, cleave Drosophila BubR1 between the N-terminal KEN box motif and C-terminal kinase domain. By using proximity labelling, we found that Drice, but not Dcp-1, is in proximity to BubR1, suggesting that protein proximity facilitates substrate preference. The cleaved fragments displayed altered subcellular localization and protein-protein interactions. Flies that harboured cleavage-resistant BubR1 showed longer duration of BubR1 localization to the kinetochore upon colchicine treatment. Furthermore, these flies showed extended lifespan. Thus, we propose that the caspase-mediated cleavage of BubR1 limits spindle assembly checkpoint and organismal lifespan. Our results highlight the importance of the individual analysis of substrates in vivo to determine the biological significance of caspase-dependent non-lethal cellular processes.

Abstract 1491: Delineating the oncogenic mechanisms of NAT10 in hepatocellular carcinoma

Abstract Protein synthesis is often dysregulated in cancer, highlighting the regulatory mechanisms of translation as potential targets for cancer treatment. With more than 140 chemical modifications of RNA, the epitranscriptome is a central mechanism in regulating protein synthesis. Remarkably, the epitranscriptome is often altered in cancerous tumors and under stress conditions such as reactive oxygen species (ROS) and anti-tumor therapies. For instance, N-acetyltransferase 10 (NAT10), the enzyme responsible for acetylating cytidines (N4-acetylcytidine or ac4C) in RNA, is highly overexpressed in cancers such as Hepatocellular Carcinoma (HCC). Of significant relevance to cancer, NAT10 is considered an oncogenic driver in fatty acid-induced HCC. Hence, this project aims to characterize the underlying oncogenic mechanisms of the NAT10/ac4C axis. Our data indicate that NAT10 and RNA acetylation levels are altered in response to fatty acids, ROS, and chemotherapeutic drugs. Likewise, we observed altered subcellular localization of NAT10 and ac4C in response to ROS and chemotherapeutic drugs. Building on our previous findings that NAT10-catalyzed ac4C promotes translation efficiency, our model stipulates that subcellular redistribution of the RNA acetyltransferase complexes enhances cell cycle progression, cancer cell proliferation, and cellular resilience by fine-tuning the translation of cancer driver genes. Collectively, our findings advance our understanding of how NAT10 promotes cancer growth while providing the mechanistic basis to target NAT10 for therapeutic purposes. Citation Format: Emmely A. Patrasso, Sweta P. Raikundalia, Daniel Arango. Delineating the oncogenic mechanisms of NAT10 in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1491.

The length of FOXE1 polyalanine tract in congenital hypothyroidism: Evidence for a pathogenic role from familial, molecular and cohort studies.

FOXE1 is required for thyroid function and its homozygous mutations cause a rare syndromic form of congenital hypothyroidism (CH). FOXE1 has a polymorphic polyalanine tract whose involvement in thyroid pathology is controversial. Starting from genetic studies in a CH family, we explored the functional role and involvement of FOXE1 variations in a large CH population. We applied NGS screening to a large CH family and a cohort of 1752 individuals and validated these results by in silico modeling and in vitro experiments. A new heterozygous FOXE1 variant segregated with 14-Alanine tract homozygosity in 5 CH siblings with athyreosis. The p.L107V variant demonstrated to significantly reduce the FOXE1 transcriptional activity. The 14-Alanine-FOXE1 displayed altered subcellular localization and significantly impaired synergy with other transcription factors, when compared with the more common 16-Alanine-FOXE1. The CH group with thyroid dysgenesis was largely and significantly enriched with the 14-Alanine-FOXE1 homozygosity. We provide new evidence that disentangle the pathophysiological role of FOXE1 polyalanine tract, thereby significantly broadening the perspective on the role of FOXE1 in the complex pathogenesis of CH. FOXE1 should be therefore added to the group of polyalanine disease-associated transcription factors.

Functional characterization of GATA6 genetic variants associated with mild congenital heart defects

Damaging GATA6 variants can cause moderate congenital heart defects. With the application of next-generation sequencing approaches, various novel GATA6 variants with unknown significance have been identified from a broad spectrum of congenital heart defects. However, functional assessment for distinct GATA6 variants from different severity of congenital heart defects, especially from mild defects, is lacking, which hinders our understanding of the genotype-phenotype correlations and underlying mechanisms.Here, we assessed the functional consequences of nine rare GATA6 variants, which had been implicated as the most significant variants associated with mild congenital heart defects using the largest case and control cohort. We examined the effects of these variants on subcellular localization, transcriptional activity, and protein interactions in 293T or AC16 cells and their ability to rescue heart malformation in gata6 zebrafish mutant.We found that two of these nine variants, Q120X and S424I, significantly decreased transcriptional activity. Additionally, Q120X altered subcellular localization. Consistent with the in vitro results, the in vivo results showed that Q120X and S424I lost their potency to rescue ventricular malformation in gata6 −/− embryos.The results indicated that Q120X and S424I are pathogenic in mild congenital heart defects. Further, the inconsistence of severely impaired Q120X function and mild CHDs phenotype suggested the complexity of the genotype-phenotype correlation between the GATA6 variant and heart phenotype, which may help to inform prenatal genetic counseling and pre-implantation genotyping for congenital heart defects.

Low aerobic capacity in McArdle disease: A role for mitochondrial network impairment?

BackgroundMcArdle disease is caused by myophosphorylase deficiency and results in complete inability for muscle glycogen breakdown. A hallmark of this condition is muscle oxidation impairment (e.g., low peak oxygen uptake (VO2peak)), a phenomenon traditionally attributed to reduced glycolytic flux and Krebs cycle anaplerosis. Here we hypothesized an additional role for muscle mitochondrial network alterations associated with massive intracellular glycogen accumulation. MethodsWe analyzed in depth mitochondrial characteristics-content, biogenesis, ultrastructure-and network integrity in skeletal-muscle from McArdle/control mice and two patients. We also determined VO2peak in patients (both sexes, N = 145) and healthy controls (N = 133). ResultsBesides corroborating very poor VO2peak values in patients and impairment in muscle glycolytic flux, we found that, in McArdle muscle: (a) damaged fibers are likely those with a higher mitochondrial and glycogen content, which show major disruption of the three main cytoskeleton components-actin microfilaments, microtubules and intermediate filaments-thereby contributing to mitochondrial network disruption in skeletal muscle fibers; (b) there was an altered subcellular localization of mitochondrial fission/fusion proteins and of the sarcoplasmic reticulum protein calsequestrin-with subsequent alteration in mitochondrial dynamics/function; impairment in mitochondrial content/biogenesis; and (c) several OXPHOS-related complex proteins/activities were also affected. ConclusionsIn McArdle disease, severe muscle oxidative capacity impairment could also be explained by a disruption of the mitochondrial network, at least in those fibers with a higher capacity for glycogen accumulation. Our findings might pave the way for future research addressing the potential involvement of mitochondrial network alterations in the pathophysiology of other glycogenoses.

Different Murine Models of Kidney Injury Reveal a Common Pattern of Dysregulation within the Polyamine System in Favour of its Catabolic Pathways

The polyamines putrescine, spermidine and spermine are organic polycations that regulate many cell functions including proliferation and differentiation. It is known that certain genes of the polyamine system are dysregulated after kidney ischemia reperfusion injury. Here we examined the hypothesis that different forms of acute and chronic kidney injury lead to similar changes in the expression patterns of the polyamine system.In different models of acute and chronic kidney injury expression of genes involved in polyamine homeostasis were analyzed by RT‐qPCR and RNAScope. In these models, expression of catabolic enzymes (Aoc1 and Sat1) was upregulated, and the anabolic enzymes (Odc1, Sms) were downregulated. The putrescine‐degrading enzyme AOC1 exhibits the most striking changes. Interestingly, it can act together with ODC1 as gatekeepers of the polyamine system. The detected increase of Aoc1 takes place in the injured but regenerating proximal tubules. As a screening for stimuli of increased Aoc1 expression, we used mouse embryonic kidney explants. Here we observed changes of Aoc1expression under hypoxia and hyperosmotic conditions. These changes were further examined in mouse models of hypoxia. However, in vivo, hypoxia did not lead to changes of Aoc1 expression. Hyperosmolarity was confirmed as a stimulus by using the kidney cell lines M15 and 209/MDCT as well as cultured primary proximal tubules. Using reporter gene and RNA‐stability assays, we could show that the increase in Aoc1 expression is based on mRNA‐stabilization and transcriptional activation of one certain isoform. The activated isoform contains an additional set of 22 amino acids N‐terminally that lead to an altered subcellular localization.In conclusion, different models of kidney injury exhibit a similar pattern of dysregulation of the polyamine system with the most striking change being the upregulation of Aoc1 in proximal tubules. Using hyperosmolarity as a stimulus, we provide first insights into the regulation of Aoc1 under harmful conditions.

Enhanced Ribonucleoprotein Immunoprecipitation (RIP) Technique for the Identification of mRNA Species in Ribonucleoprotein Complexes.

RNA binding proteins (RBPs) are critical regulators of cellular phenotypes, and dysregulated RBP expression is implicated in various diseases including cancer. A single RBP can bind to and regulate the expression of many RNA molecules via a variety of mechanisms, including translational suppression, prevention of RNA degradation, and alteration in subcellular localization. To elucidate the role of a specific RBP within a given cellular context, it is essential to first identify the group of RNA molecules to which it binds. This has traditionally been achieved using cross-linking-based assays in which cells are first exposed to agents that cross-link RBPs to nucleic acids and then lysed to extract and purify the RBP-nucleic acid complexes. The nucleic acids within the mixture are then released and analyzed via conventional means (e.g., microarray analysis, qRT-PCR, RNA sequencing, or Northern blot). While cross-linking-based ribonucleoprotein immunoprecipitation (RIP) has proven its utility within some contexts, it is technically challenging, inefficient, and suboptimal given the amount of time and resources (e.g., cells and antibodies) required. Additionally, these types of studies often require the use of over-expressed versions of proteins, which can introduce artifacts. Here, we describe a streamlined version of RIP that utilizes exclusion-based purification technologies. This approach requires significantly less starting material and resources compared to traditional RIP approaches, takes less time, which is tantamount given the labile nature of RNA, and can be used with endogenously expressed proteins. The method described here can be used to study RNA-protein interactions in a variety of cellular contexts. Graphical abstract.

Dynamically expressed single ELAV/Hu orthologue elavl2 of bees is required for learning and memory

Changes in gene expression are a hallmark of learning and memory consolidation. Little is known about how alternative mRNA processing, particularly abundant in neuron-specific genes, contributes to these processes. Prototype RNA binding proteins of the neuronally expressed ELAV/Hu family are candidates for roles in learning and memory, but their capacity to cross-regulate and take over each other’s functions complicate substantiation of such links. Honey bees Apis mellifera have only one elav/Hu family gene elavl2, that has functionally diversified by increasing alternative splicing including an evolutionary conserved microexon. RNAi knockdown demonstrates that ELAVL2 is required for learning and memory in bees. ELAVL2 is dynamically expressed with altered alternative splicing and subcellular localization in mushroom bodies, but not in other brain regions. Expression and alternative splicing of elavl2 change during memory consolidation illustrating an alternative mRNA processing program as part of a local gene expression response underlying memory consolidation.

Attenuated SIRT1 Activity Leads to PER2 Cytoplasmic Localization and Dampens the Amplitude of Bmal1 Promoter-Driven Circadian Oscillation.

The circadian clock possesses robust systems to maintain the rhythm approximately 24 h, from cellular to organismal levels, whereas aging is known to be one of the risk factors linked to the alternation of circadian physiology and behavior. The amount of many metabolites in the cells/body is altered with the aging process, and the most prominent metabolite among them is the oxidized form of nicotinamide adenine dinucleotide (NAD+), which is associated with posttranslational modifications of acetylation and poly-ADP-ribosylation status of circadian clock proteins and decreases with aging. However, how low NAD+ condition in cells, which mimics aged or pathophysiological conditions, affects the circadian clock is largely unknown. Here, we show that low NAD+ in cultured cells promotes PER2 to be retained in the cytoplasm through the NAD+/SIRT1 axis, which leads to the attenuated amplitude of Bmal1 promoter-driven luciferase oscillation. We found that, among the core clock proteins, PER2 is mainly affected in its subcellular localization by NAD+ amount, and a higher cytoplasmic PER2 localization was observed under low NAD+ condition. We further found that NAD+-dependent deacetylase SIRT1 is the regulator of PER2 subcellular localization. Thus, we anticipate that the altered PER2 subcellular localization by low NAD+ is one of the complex changes that occurs in the aged circadian clock.

Reduced erythrocytic CHCHD2 mRNA is associated with brain pathology of Parkinson\u2019s disease

Peripheral biomarkers indicative of brain pathology are critically needed for early detection of Parkinson’s disease (PD). In this study, using NanoString and digital PCR technologies, we began by screening for alterations in genes associated with PD or atypical Parkinsonism in erythrocytes of PD patients, in which PD-related changes have been reported, and which contain ~ 99% of blood α-synuclein. Erythrocytic CHCHD2 mRNA was significantly reduced even at the early stages of the disease. A significant reduction in protein and/or mRNA expression of CHCHD2 was confirmed in PD brains collected at autopsy as well as in the brains of a PD animal model overexpressing α-synuclein, in addition to seeing a reduction of CHCHD2 in erythrocytes of the same animals. Overexpression of α-synuclein in cellular models of PD also resulted in reduced CHCHD2, via mechanisms likely involving altered subcellular localization of p300 histone acetyltransferase. Finally, the utility of reduced CHCHD2 mRNA as a biomarker for detecting PD, including early-stage PD, was validated in a larger cohort of 205 PD patients and 135 normal controls, with a receiver operating characteristic analysis demonstrating > 80% sensitivity and specificity.

FAM3C: an emerging biomarker and potential therapeutic target for cancer.

FAM3C is a member of the FAM3 family. Recently, overexpression of FAM3C has been reported in numerous types of cancer, including breast and colon cancer. Increasing evidence suggests that elevated FAM3C and its altered subcellular localization are closely associated with tumor formation, invasion, metastasis and poor survival. Moreover, FAM3C has been found to be the regulator of various proteins that associate with cancer, including Ras, STAT3, TGF-β and LIFR. This review summarizes the current knowledge regarding FAM3C, including its structure, expression patterns, regulation, physiological roles and regulatory functions in various malignancies. These findings highlight the importance of FAM3C in cancer development and provide evidence that FAM3C is a novel biomarker and potential therapeutic target for various cancers.

Nuclear receptor SHP dampens transcription function and abrogates mitotic chromatin association of PXR and ERα via intermolecular interactions

Mitosis is a cellular process that produces two identical progenies. Genome-wide transcription is believed to be silenced during mitosis. However, some transcription factors have been reported to associate with the mitotic chromatin to uphold a role in ‘gene-bookmarking’. Here, we investigated the dynamic role of nuclear receptor SHP during cell cycle, and observed intermolecular interactions with PXR and ERα. This was reflected in altered subcellular localization, transcription function and mitotic chromatin behavior of these receptors. Subsequently, by in silico and live cell imaging approaches we identified the minimal domain(s) and crucial amino-acid residues required for such receptor-receptor interactions. It was apparent that both PXR/ERα interact with SHP to translocate cytoplasmic RFP-tagged SHP into the nucleus. In addition, during mitosis SHP interacted with some of the key nuclear receptors, altering partners, as well as, its own relationship with mitotic chromatin. SHP displaced a major fraction of PXR and ERα from the mitotic chromatin while promoted its own weak association reflected in its binding. Since SHP lacks DBD this association is attributed to receptor-receptor interactions rather than SHP-DNA interactions. The abrogation of PXR and ERα from the mitotic chromatin by SHP implies potential implications in regulation of gene bookmarking events in cellular development. Overall, it is concluded that intermolecular interactions between SHP and partner PXR/ERα result in attenuation of target promoter activities. It is proposed that SHP may act as an indirect physiological regulator and functions in a hog-tie manner by displacing the interacting transcription factor from gene regulatory sites.

NFAT5 promotes oral squamous cell carcinoma progression in a hyperosmotic environment

AbstractEpidermal growth factor receptor (EGFR) is highly expressed in several types of cancer cells including oral squamous cell carcinoma (OSCC). EGF/EGFR signaling is recognized as an important molecular target in cancer therapy. However, cancer cells often become tolerant to EGF/EGFR signaling-targeted therapies. In the tumor microenvironment, the tumor incites inflammation and the inflammation-derived cytokines make a considerable impact on cancer development. In addition, hyperosmolarity is also induced, but the role of osmotic stress in cancer development has not been fully understood. This study demonstrates molecular insights into hyperosmolarity effect on OSCC development and shows that NFAT5 transcription factor plays an important functional role in enhancing the oral cancer cell proliferation by inducing the EGFR translocation from the endoplasmic reticulum to the plasma membrane through increase the expression of DPAGT1, an essential enzyme for catalyzing the first committed step of N-linked protein glycosylation. These results suggest that hyperosmolarity-induced intra-nuclear translocation of NFAT5 essential for DPAGT1 activation and EGFR subcellular translocation responsible for OSCC tumor progression.This study demonstrates that NFAT5 promotes oral squamous cell carcinoma progression in the hyperosmotic environment through increased expression of DPAGT1, an essential enzyme for protein glycosylation, and altered EGFR subcellular localization from the cytoplasm to the plasma membrane in tumor cells.