CCAR-1 works together with the U2AF large subunit UAF-1 to regulate alternative splicing.

The Cell Division Cycle and Apoptosis Regulator (CCAR) protein family members have recently emerged as regulators of alternative splicing and transcription, as well as having other key physiological functions. For example, mammalian CCAR2/DBC1 forms a complex with the zinc factor protein ZNF326 to integrate alternative splicing with RNA polymerase II transcriptional elongation in AT-rich regions of the DNA. Additionally, Caenorhabditis elegans CCAR-1, a homolog to mammalian CCAR2, facilitates the alternative splicing of the perlecan unc-52 gene. However, much about the CCAR family's role in alternative splicing is unknown. Here, we have examined the role of CCAR-1 in genome-wide alternative splicing in Caenorhabditis elegans and have identified new alternative splicing targets of CCAR-1 using RNA sequencing. Also, we found that CCAR-1 interacts with the spliceosome factors UAF-1 and UAF-2 using mass spectrometry, and that knockdown of ccar-1 affects alternative splicing patterns, motility, and proteostasis of UAF-1 mutant worms. Collectively, we demonstrate the role of CCAR-1 in regulating global alternative splicing in C. elegans and in conjunction with UAF-1.

CBP-HSF2 structural and functional interplay in Rubinstein-Taybi neurodevelopmental disorder.

Patients carrying autosomal dominant mutations in the histone/lysine acetyl transferases CBP or EP300 develop a neurodevelopmental disorder: Rubinstein-Taybi syndrome (RSTS). The biological pathways underlying these neurodevelopmental defects remain elusive. Here, we unravel the contribution of a stress-responsive pathway to RSTS. We characterize the structural and functional interaction between CBP/EP300 and heat-shock factor 2 (HSF2), a tuner of brain cortical development and major player in prenatal stress responses in the neocortex: CBP/EP300 acetylates HSF2, leading to the stabilization of the HSF2 protein. Consequently, RSTS patient-derived primary cells show decreased levels of HSF2 and HSF2-dependent alteration in their repertoire of molecular chaperones and stress response. Moreover, we unravel a CBP/EP300-HSF2-N-cadherin cascade that is also active in neurodevelopmental contexts, and show that its deregulation disturbs neuroepithelial integrity in 2D and 3D organoid models of cerebral development, generated from RSTS patient-derived iPSC cells, providing a molecular reading key for this complex pathology.

The CBP-1/p300 Lysine Acetyltransferase Regulates the Heat Shock Response in C. elegans.

The decline of proteostasis is a hallmark of aging that is, in part, affected by the dysregulation of the heat shock response (HSR), a highly conserved cellular response to proteotoxic stress in the cell. The heat shock transcription factor HSF-1 is well-studied as a key regulator of proteostasis, but mechanisms that could be used to modulate HSF-1 function to enhance proteostasis during aging are largely unknown. In this study, we examined lysine acetyltransferase regulation of the HSR and HSF-1 in C. elegans. We performed an RNA interference screen of lysine acetyltransferases and examined mRNA expression of the heat-shock inducible gene hsp-16.2, a widely used marker for HSR activation. From this screen, we identified one acetyltransferase, CBP-1, the C. elegans homolog of mammalian CREB-binding protein CBP/p300, as a negative regulator of the HSR. We found that while knockdown of CBP-1 decreases the overall lifespan of the worm, it also enhances heat shock protein production upon heat shock and increases thermotolerance of the worm in an HSF-1 dependent manner. Similarly, we examined a hallmark of HSF-1 activation, the formation of nuclear stress bodies (nSBs). In analyzing the recovery rate of nSBs, we found that knockdown of CBP-1 enhanced the recovery and resolution of nSBs after stress. Collectively, our studies demonstrate a role of CBP-1 as a negative regulator of HSF-1 activity and its physiological effects at the organismal level upon stress.

HSF-1 displays nuclear stress body formation in multiple tissues in Caenorhabditis elegans upon stress and following the transition to adulthood.

The transcription factor heat shock factor-1 (HSF-1) regulates the heat shock response (HSR), a cytoprotective response induced by proteotoxic stresses. Data from model organisms has shown that HSF-1 also has non-stress biological roles, including roles in the regulation of development and longevity. To better study HSF-1 function, we created a C. elegans strain containing HSF-1 tagged with GFP at its endogenous locus utilizing CRISPR/Cas9-guided transgenesis. We show that the HSF-1::GFP CRISPR worm strain behaves similarly to wildtype worms in response to heat and other stresses, and in other physiological processes. HSF-1 was expressed in all tissues assayed. Immediately following the initiation of reproduction, HSF-1 formed nuclear stress bodies, a hallmark of activation, throughout the germline. Upon the transition to adulthood, of HSF-1 nuclear stress bodies appeared in most somatic cells. Genetic loss of the germline suppressed nuclear stress body formation with age, suggesting that the germline influences HSF-1 activity. Interestingly, we found that various neurons did not form nuclear stress bodies after transitioning to adulthood. Therefore, the formation of HSF-1 nuclear stress bodies upon the transition to adulthood does not occur in a synchronous manner in all cell types. In sum, these studies enhance our knowledge of the expression and activity of the aging and proteostasis factor HSF-1 in a tissue-specific manner with age.

CCAR-1 is a negative regulator of the heat-shock response in Caenorhabditis elegans.

Defects in protein quality control during aging are central to many human diseases, and strategies are needed to better understand mechanisms of controlling the quality of the proteome. The heat-shock response (HSR) is a conserved survival mechanism mediated by the transcription factor HSF1 which functions to maintain proteostasis. In mammalian cells, HSF1 is regulated by a variety of factors including the prolongevity factor SIRT1. SIRT1 promotes the DNA-bound state of HSF1 through deacetylation of the DNA-binding domain of HSF1, thereby enhancing the HSR. SIRT1 is also regulated by various factors, including negative regulation by the cell-cycle and apoptosis regulator CCAR2. CCAR2 negatively regulates the HSR, possibly through its inhibitory interaction with SIRT1. We were interested in studying conservation of the SIRT1/CCAR2 regulatory interaction in Caenorhabditis elegans, and in utilizing this model organism to observe the effects of modulating sirtuin activity on the HSR, longevity, and proteostasis. The HSR is highly conserved in C. elegans and is mediated by the HSF1 homolog, HSF-1. We have uncovered that negative regulation of the HSR by CCAR2 is conserved in C. elegans and is mediated by the CCAR2 ortholog, CCAR-1. This negative regulation requires the SIRT1 homolog SIR-2.1. In addition, knockdown of CCAR-1 via ccar-1 RNAi works through SIR-2.1 to enhance stress resistance, motility, longevity, and proteostasis. This work therefore highlights the benefits of enhancing sirtuin activity to promote the HSR at the level of the whole organism.

Coffee extract and caffeine enhance the heat shock response and promote proteostasis in an HSF-1-dependent manner in Caenorhabditis elegans.

As the population ages, there is a critical need to uncover strategies to combat diseases of aging. Studies in the soil-dwelling nematode Caenorhabditis elegans have demonstrated the protective effects of coffee extract and caffeine in promoting the induction of conserved longevity pathways including the insulin-like signaling pathway and the oxidative stress response. We were interested in determining the effects of coffee and caffeine treatment on the regulation of the heat shock response. The heat shock response is a highly conserved cellular response that functions as a cytoprotective mechanism during stress, mediated by the heat shock transcription factor HSF-1. In the worm, HSF-1 not only promotes protection against stress but is also essential for development and longevity. Induction of the heat shock response has been suggested to be beneficial for diseases of protein conformation by preventing protein misfolding and aggregation, and as such has been proposed as a therapeutic target for age-associated neurodegenerative disorders. In this study, we demonstrate that coffee is a potent, dose-dependent, inducer of the heat shock response. Treatment with a moderate dose of pure caffeine was also able to induce the heat shock response, indicating caffeine as an important component within coffee for producing this response. The effects that we observe with both coffee and pure caffeine on the heat shock response are both dependent on HSF-1. In a C. elegans Huntington's disease model, worms treated with caffeine were protected from polyglutamine aggregates and toxicity, an effect that was also HSF-1-dependent. In conclusion, these results demonstrate caffeinated coffee, and pure caffeine, as protective substances that promote proteostasis through induction of the heat shock response.

Spheroid growth in ovarian cancer alters transcriptome responses for stress pathways and epigenetic responses.

Ovarian cancer is the most lethal gynecological cancer, with over 200,000 women diagnosed each year and over half of those cases leading to death. These poor statistics are related to a lack of early symptoms and inadequate screening techniques. This results in the cancer going undetected until later stages when the tumor has metastasized through a process that requires the epithelial to mesenchymal transition (EMT). In lieu of traditional monolayer cell culture, EMT and cancer progression in general is best characterized through the use of 3D spheroid models. In this study, we examine gene expression changes through microarray analysis in spheroid versus monolayer ovarian cancer cells treated with TGFß to induce EMT. Transcripts that included Coiled-Coil Domain Containing 80 (CCDC80), Solute Carrier Family 6 (Neutral Amino Acid Transporter), Member 15 (SLC6A15), Semaphorin 3E (SEMA3E) and PIF1 5'-To-3' DNA Helicase (PIF1) were downregulated more than 10-fold in the 3D cells while Inhibitor Of DNA Binding 2, HLH Protein (ID2), Regulator Of Cell Cycle (RGCC), Protease, Serine 35 (PRSS35), and Aldo-Keto Reductase Family 1, Member C1 (AKR1C1) were increased more than 50-fold. Interestingly, EMT factors, stress responses and epigenetic processes were significantly affected by 3D growth. The heat shock response and the oxidative stress response were also identified as transcriptome responses that showed significant changes upon 3D growth. Subnetwork enrichment analysis revealed that DNA integrity (e.g. DNA damage, genetic instability, nucleotide excision repair, and the DNA damage checkpoint pathway) were altered in the 3D spheroid model. In addition, two epigenetic processes, DNA methylation and histone acetylation, were increased with 3D growth. These findings support the hypothesis that three dimensional ovarian cell culturing is physiologically different from its monolayer counterpart.

HSF-1 is a regulator of miRNA expression in Caenorhabditis elegans.

The ability of an organism to sense and adapt to environmental stressors is essential for proteome maintenance and survival. The highly conserved heat shock response is a survival mechanism employed by all organisms, including the nematode Caenorhabditis elegans, upon exposure to environmental extremes. Transcriptional control of the metazoan heat shock response is mediated by the heat shock transcription factor HSF-1. In addition to regulating global stress-responsive genes to promote stress-resistance and survival, HSF-1 has recently been shown to regulate stress-independent functions in controlling development, metabolism, and longevity. However, the indirect role of HSF-1 in coordinating stress-dependent and -independent processes through post-transcriptional regulation is largely unknown. MicroRNAs (miRNAs) have emerged as a class of post-transcriptional regulators that control gene expression through translational repression or mRNA degradation. To determine the role of HSF-1 in regulating miRNA expression, we have performed high-throughput small RNA-sequencing in C. elegans grown in the presence and absence of hsf-1 RNAi followed by treatment with or without heat shock. This has allowed us to uncover the miRNAs regulated by HSF-1 via heat-dependent and -independent mechanisms. Integrated miRNA/mRNA target-prediction analyses suggest HSF-1 as a post-transcriptional regulator of development, metabolism, and longevity through regulating miRNA expression. This provides new insight into the possible mechanism by which HSF-1 controls these processes. We have also uncovered oxidative stress response factors and insulin-like signaling factors as a common link between processes affected by HSF-1-regulated miRNAs in stress-dependent and -independent mechanisms, respectively. This may provide a role for miRNAs in regulating cross-talk between various stress responses. Our work therefore uncovers an interesting potential role for HSF-1 in post-transcriptionally controlling gene expression in C. elegans, and suggests a mechanism for cross-talk between stress responses.

Loss of function CHCHD10 mutations in cytoplasmic TDP-43 accumulation and synaptic integrity.

Although multiple CHCHD10 mutations are associated with the spectrum of familial and sporadic frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS) diseases, neither the normal function of endogenous CHCHD10 nor its role in the pathological milieu (that is, TDP-43 pathology) of FTD/ALS have been investigated. In this study, we made a series of observations utilizing Caenorhabditis elegans models, mammalian cell lines, primary neurons and mouse brains, demonstrating that CHCHD10 normally exerts a protective role in mitochondrial and synaptic integrity as well as in the retention of nuclear TDP-43, whereas FTD/ALS-associated mutations (R15L and S59L) exhibit loss of function phenotypes in C. elegans genetic complementation assays and dominant negative activities in mammalian systems, resulting in mitochondrial/synaptic damage and cytoplasmic TDP-43 accumulation. As such, our results provide a pathological link between CHCHD10-associated mitochondrial/synaptic dysfunction and cytoplasmic TDP-43 inclusions.

The Heat Shock Transcription Factor HSF1 Induces Ovarian Cancer Epithelial-Mesenchymal Transition in a 3D Spheroid Growth Model.

Ovarian cancer is the most lethal gynecological cancer, with over 200,000 women diagnosed each year and over half of those cases leading to death. The proteotoxic stress-responsive transcription factor HSF1 is frequently overexpressed in a variety of cancers and is vital to cellular proliferation and invasion in some cancers. Upon analysis of various patient data sets, we find that HSF1 is frequently overexpressed in ovarian tumor samples. In order to determine the role of HSF1 in ovarian cancer, inducible HSF1 knockdown cell lines were created. Knockdown of HSF1 in SKOV3 and HEY ovarian cancer cell lines attenuates the epithelial-to-mesenchymal transition (EMT) in cells treated with TGFß, as determined by western blot and quantitative RT-PCR analysis of multiple EMT markers. To further explore the role of HSF1 in ovarian cancer EMT, we cultured multicellular spheroids in a non-adherent environment to simulate early avascular tumors. In the spheroid model, cells more readily undergo EMT; however, EMT inhibition by HSF1 becomes more pronounced in the spheroid model. These findings suggest that HSF1 is important in the ovarian cancer TGFß response and in EMT.

Subcellular and in-vivo Nano-Endoscopy.

Analysis of individual cells at the subcellular level is important for understanding diseases and accelerating drug discovery. Nanoscale endoscopes allow minimally invasive probing of individual cell interiors. Several such instruments have been presented previously, but they are either too complex to fabricate or require sophisticated external detectors because of low signal collection efficiency. Here we present a nanoendoscope that can locally excite fluorescence in labelled cell organelles and collect the emitted signal for spectral analysis. Finite Difference Time Domain (FDTD) simulations have shown that with an optimized nanoendoscope taper profile, the light emission and collection was localized within ~100 nm. This allows signal detection to be used for nano-photonic sensing of the proximity of fluorophores. Upon insertion into the individual organelles of living cells, the nanoendoscope was fabricated and resultant fluorescent signals collected. This included the signal collection from the nucleus of Acridine orange labelled human fibroblast cells, the nucleus of Hoechst stained live liver cells and the mitochondria of MitoTracker Red labelled MDA-MB-231 cells. The endoscope was also inserted into a live organism, the yellow fluorescent protein producing nematode Caenorhabditis elegans, and a fluorescent signal was collected. To our knowledge this is the first demonstration of in vivo, local fluorescence signal collection on the sub-organelle level.

The genome-wide role of HSF-1 in the regulation of gene expression in Caenorhabditis elegans.

BACKGROUND: The heat shock response, induced by cytoplasmic proteotoxic stress, is one of the most highly conserved transcriptional responses. This response, driven by the heat shock transcription factor HSF1, restores proteostasis through the induction of molecular chaperones and other genes. In addition to stress-dependent functions, HSF1 has also been implicated in various stress-independent functions. In C. elegans, the HSF1 homolog HSF-1 is an essential protein that is required to mount a stress-dependent response, as well as to coordinate various stress-independent processes including development, metabolism, and the regulation of lifespan. In this work, we have performed RNA-sequencing for C. elegans cultured in the presence and absence of hsf-1 RNAi followed by treatment with or without heat shock. This experimental design thus allows for the determination of both heat shock-dependent and -independent biological targets of HSF-1 on a genome-wide level. RESULTS: Our results confirm that C. elegans HSF-1 can regulate gene expression in both a stress-dependent and -independent fashion. Almost all genes regulated by HS require HSF-1, reinforcing the central role of this transcription factor in the response to heat stress. As expected, major categories of HSF-1-regulated genes include cytoprotection, development, metabolism, and aging. Within both the heat stress-dependent and -independent gene groups, significant numbers of genes are upregulated as well as downregulated, demonstrating that HSF-1 can both activate and repress gene expression either directly or indirectly. Surprisingly, the cellular process most highly regulated by HSF-1, both with and without heat stress, is cuticle structure. Via network analyses, we identify a nuclear hormone receptor as a common link between genes that are regulated by HSF-1 in a HS-dependent manner, and an epidermal growth factor receptor as a common link between genes that are regulated by HSF-1 in a HS-independent manner. HSF-1 therefore coordinates various physiological processes in C. elegans, and HSF-1 activity may be coordinated across tissues by nuclear hormone receptor and epidermal growth factor receptor signaling. CONCLUSION: This work provides genome-wide HSF-1 regulatory networks in C. elegans that are both heat stress-dependent and -independent. We show that HSF-1 is responsible for regulating many genes outside of classical heat stress-responsive genes, including genes involved in development, metabolism, and aging. The findings that a nuclear hormone receptor may coordinate the HS-induced HSF-1 transcriptional response, while an epidermal growth factor receptor may coordinate the HS-independent response, indicate that these factors could promote cell non-autonomous signaling that occurs through HSF-1. Finally, this work highlights the genes involved in cuticle structure as important HSF-1 targets that may play roles in promoting both cytoprotection as well as longevity.

Interlaboratory comparison of in vitro bioassays for screening of endocrine active chemicals in recycled water.

In vitro bioassays have shown promise as water quality monitoring tools. In this study, four commercially available in vitro bioassays (GeneBLAzer(®) androgen receptor (AR), estrogen receptor-alpha (ER), glucocorticoid receptor (GR) and progesterone receptor (PR) assays) were adapted to screen for endocrine active chemicals in samples from two recycled water plants. The standardized protocols were used in an interlaboratory comparison exercise to evaluate the reproducibility of in vitro bioassay results. Key performance criteria were successfully achieved, including low background response, standardized calibration parameters and high intra-laboratory precision. Only two datasets were excluded due to poor calibration performance. Good interlaboratory reproducibility was observed for GR bioassay, with 16-26% variability among the laboratories. ER and PR bioactivity was measured near the bioassay limit of detection and showed more variability (21-54%), although interlaboratory agreement remained comparable to that of conventional analytical methods. AR bioassay showed no activity for any of the samples analyzed. Our results indicate that ER, GR and PR, were capable of screening for different water quality, i.e., the highest bioactivity was observed in the plant influent, which also contained the highest concentrations of endocrine active chemicals measured by LC-MS/MS. After advanced treatment (e.g., reverse osmosis), bioactivity and target chemical concentrations were both below limits of detection. Comparison of bioassay and chemical equivalent concentrations revealed that targeted chemicals accounted for ≤5% of bioassay activity, suggesting that detection limits by LC-MS/MS for some chemicals were insufficient and/or other bioactive compounds were present in these samples. Our study demonstrated that in vitro bioassays responses were reproducible, and can provide information to complement conventional analytical methods for a more comprehensive water quality assessment.

Mechanisms of amino acid-mediated lifespan extension in Caenorhabditis elegans.

BACKGROUND: Little is known about the role of amino acids in cellular signaling pathways, especially as it pertains to pathways that regulate the rate of aging. However, it has been shown that methionine or tryptophan restriction extends lifespan in higher eukaryotes and increased proline or tryptophan levels increase longevity in C. elegans. In addition, leucine strongly activates the TOR signaling pathway, which when inhibited increases lifespan. RESULTS: Therefore each of the 20 proteogenic amino acids was individually supplemented to C. elegans and the effects on lifespan were determined. All amino acids except phenylalanine and aspartate extended lifespan at least to a small extent at one or more of the 3 concentrations tested with serine and proline showing the largest effects. 11 of the amino acids were less potent at higher doses, while 5 even decreased lifespan. Serine, proline, or histidine-mediated lifespan extension was greatly inhibited in eat-2 worms, a model of dietary restriction, in daf-16/FOXO, sir-2.1, rsks-1 (ribosomal S6 kinase), gcn-2, and aak-2 (AMPK) longevity pathway mutants, and in bec-1 autophagy-defective knockdown worms. 8 of 10 longevity-promoting amino acids tested activated a SKN-1/Nrf2 reporter strain, while serine and histidine were the only amino acids from those to activate a hypoxia-inducible factor (HIF-1) reporter strain. Thermotolerance was increased by proline or tryptophan supplementation, while tryptophan-mediated lifespan extension was independent of DAF-16/FOXO and SKN-1/Nrf2 signaling, but tryptophan and several related pyridine-containing compounds induced the mitochondrial unfolded protein response and an ER stress response. High glucose levels or mutations affecting electron transport chain (ETC) function inhibited amino acid-mediated lifespan extension suggesting that metabolism plays an important role. Providing many other cellular metabolites to C. elegans also increased longevity suggesting that anaplerosis of tricarboxylic acid (TCA) cycle substrates likely plays a role in lifespan extension. CONCLUSIONS: Supplementation of C. elegans with 18 of the 20 individual amino acids extended lifespan, but lifespan often decreased with increasing concentration suggesting hormesis. Lifespan extension appears to be caused by altered mitochondrial TCA cycle metabolism and respiratory substrate utilization resulting in the activation of the DAF-16/FOXO and SKN-1/Nrf2 stress response pathways.

Fluorodeoxyuridine enhances the heat shock response and decreases polyglutamine aggregation in an HSF-1-dependent manner in Caenorhabditis elegans.

The heat shock response (HSR) protects cells from protein-denaturing stress through the induction of chaperones. The HSR is conserved in all organisms and is mediated by the transcription factor HSF-1. We show here that a compound commonly used to prevent larval development in Caenorhabditis elegans, 5-fluoro-2'-deoxyuridine (FUdR), can enhance heat shock induction of hsp mRNA in an HSF-1-dependent manner. Treatment with FUdR can also decrease age-dependent polyglutamine aggregation in a Huntington's disease model, and this effect depends on HSF-1 as well. Therefore, FUdR treatment can modulate the HSR and proteostasis, and should be used with caution when used to inhibit reproduction.

Benchmarking organic micropollutants in wastewater, recycled water and drinking water with in vitro bioassays.

Thousands of organic micropollutants and their transformation products occur in water. Although often present at low concentrations, individual compounds contribute to mixture effects. Cell-based bioassays that target health-relevant biological endpoints may therefore complement chemical analysis for water quality assessment. The objective of this study was to evaluate cell-based bioassays for their suitability to benchmark water quality and to assess efficacy of water treatment processes. The selected bioassays cover relevant steps in the toxicity pathways including induction of xenobiotic metabolism, specific and reactive modes of toxic action, activation of adaptive stress response pathways and system responses. Twenty laboratories applied 103 unique in vitro bioassays to a common set of 10 water samples collected in Australia, including wastewater treatment plant effluent, two types of recycled water (reverse osmosis and ozonation/activated carbon filtration), stormwater, surface water, and drinking water. Sixty-five bioassays (63%) showed positive results in at least one sample, typically in wastewater treatment plant effluent, and only five (5%) were positive in the control (ultrapure water). Each water type had a characteristic bioanalytical profile with particular groups of toxicity pathways either consistently responsive or not responsive across test systems. The most responsive health-relevant endpoints were related to xenobiotic metabolism (pregnane X and aryl hydrocarbon receptors), hormone-mediated modes of action (mainly related to the estrogen, glucocorticoid, and antiandrogen activities), reactive modes of action (genotoxicity) and adaptive stress response pathway (oxidative stress response). This study has demonstrated that selected cell-based bioassays are suitable to benchmark water quality and it is recommended to use a purpose-tailored panel of bioassays for routine monitoring.

DBC1/CCAR2 and CCAR1 Are Largely Disordered Proteins that Have Evolved from One Common Ancestor.

Deleted in breast cancer 1 (DBC1, CCAR2, KIAA1967) is a large, predominantly nuclear, multidomain protein that modulates gene expression by inhibiting several epigenetic modifiers, including the deacetylases SIRT1 and HDAC3, and the methyltransferase SUV39H1. DBC1 shares many highly conserved protein domains with its paralog cell cycle and apoptosis regulator 1 (CCAR1, CARP-1). In this study, we examined the full-length sequential and structural properties of DBC1 and CCAR1 from multiple species and correlated these properties with evolution. Our data shows that the conserved domains shared between DBC1 and CCAR1 have similar domain structures, as well as similar patterns of predicted disorder in less-conserved intrinsically disordered regions. Our analysis indicates similarities between DBC1, CCAR1, and the nematode protein lateral signaling target 3 (LST-3), suggesting that DBC1 and CCAR1 may have evolved from LST-3. Our data also suggests that DBC1 emerged later in evolution than CCAR1. DBC1 contains regions that show less conservation across species as compared to the same regions in CCAR1, suggesting a continuously evolving scenario for DBC1. Overall, this study provides insight into the structure and evolution of DBC1 and CCAR1, which may impact future studies on the biological functions of these proteins.

Stress Inducibility of SIRT1 and Its Role in Cytoprotection and Cancer.

Cells must continuously respond to stressful insults via the upregulation of cytoprotective pathways. The longevity factor and deacetylase SIRT1 plays a critical role in coordinating this cellular response to stress. SIRT1 activity and levels are regulated by cellular stressors, including metabolic, genotoxic, oxidative, and proteotoxic stress. As a stress sensor, SIRT1 impacts cell survival by deacetylating substrate proteins to drive the cell towards a cytoprotective pathway. Extreme stress conditions, however, can cause SIRT1 to lead cells down an apoptotic pathway instead. SIRT1 is frequently dysregulated in cancer cells and has been characterized to have a dual role as both an oncogene and a tumor suppressor, likely due to its pivotal function in regulating cytoprotection. Recently, the ability of SIRT1 to regulate HSF1-dependent induction of the heat shock response has highlighted another pathway through which SIRT1 can modulate cytoprotection. Activation of HSF1 results in the production of cytoprotective chaperones that can facilitate the transformed phenotype of cancer cells. In this review, we discuss the stress-dependent regulation of SIRT1. We highlight the role of SIRT1 in stress management and cytoprotection and emphasize SIRT1-dependent activation of HSF1 as a potential mechanism for cancer promotion.

The SIRT1 modulators AROS and DBC1 regulate HSF1 activity and the heat shock response.

The heat shock response, the cellular response to protein damaging stress, is critical in maintaining proteostasis. The heat shock response is regulated by the transcription factor HSF1, which is activated upon heat shock and other stresses to induce the expression of molecular chaperones. SIRT1 has previously been shown to activate HSF1 by deacetylating it, leading to increased DNA binding ability. We have investigated how the heat shock response may be controlled by factors influencing SIRT1 activity. We found that heat shock results in an increase in the cellular NAD(+)/NADH ratio and an increase in recruitment of SIRT1 to the hsp70 promoter. Furthermore, we found that the SIRT1 modulators AROS and DBC1 have an impact on hsp70 transcription, HSF1 acetylation status, and HSF1 recruitment to the hsp70 promoter. Therefore, AROS and DBC1 are now two new targets available for therapeutic regulation of the heat shock response.