Small Heat Shock Proteins (sHSP) are critical molecular chaperones that function to maintain protein homeostasis (proteostasis) and prevent the aggregation of other proteins during cellular stress. Any disruption in the process of proteostasis can lead to prevalent diseases ranging from cancer and cataract to cardiovascular and Alzheimer's disease. CryAB (αB-crystallin, HspB5) is one of ten known human sHSP that is abundant in the lens, skeletal, and cardiac muscle. This protein is required for cardiac function and muscle cell integrity. When the cell experiences physiological stress, including heat shock, CryAB moves to the cytoskeleton to act as a chaperone and prevent aggregation of its protein clientele. This research is designed to investigate the molecular role of CryAB in cell proteostasis through the identification of putative protein clientele and chaperone activity analysis. We have identified over twenty CryAB-binding partners through combined yeast two-hybrid (Y2H) and co-purification approaches, including interactions with myofibril proteins. Previously reported disease-associated CryAB missense variants were analyzed in comparison to wild type CryAB through Y2H binding assays. The characterization of the similarities and differences in binding specificities of these variants provide a foundation to better understand the chaperone pathways of CryAB and how these changes in molecular function result in the development of disparate diseases such as cataract, cancer, and various myopathies.

In this study, possible involvement of the small heat shock proteins (sHsps) from a thermoacidophilic archaeon, Thermoplasma (Tp) volcanium in the stress response was investigated. Our results showed that heterologous, high level expression of TVN0775/sHsp gene in E.coli increased its thermotolerance at 53&deg / C for two hours. But, the second sHsp of the Tp. volcanium, TVN0984/sHsp was not effective in improvement of the thermal resistance of the mesophilic bacterium (i. e., E.coli). The expression of the TVN0775/sHsp and TVN0984/sHsp genes increased about 3 fold after heat-shock at 65&deg / C, as revealed by Real-Time PCR analysis. Although expression of the both genes was induced at 70&deg / C, TVN0984/sHsp gene expression was increased higher (about 5 fold) than that of the TVN0775/sHsp gene expression (about 1.5 fold). Tp. volcanium cells were exposed to high pH (pH: 3.5, pH: 4.0, pH: 4.5, pH: 5.0), and the change in the sHsp genes&rsquo / expression profile were analyzed. The results showed that TVN0775/sHsp gene expression was more sensitive to increased pH than TVN0984/sHsp gene expression. The TVN0775/sHsp gene transcription induced at most 2.5 fold at pH 4.0 and the gene expression either reduced or did not change at higher pH values (i.e., pH 4.5 and 5.0). On the other hand, TVN0984/sHsp gene expression did not change at pH 4.0 but significantly reduced at higher pH values. The effect of oxidative stress on the expression of TVN0775 and TVN0984 genes was investigated by treatment of Tp. volcanium cells with 0.01 mM, 0.02 mM, 0,03 mM and 0.05 mM H2O2. For both sHsp genes, transcription was induced at lower concentrations of H2O2 (0.01 mM and 0.02 mM). At higher concentrations of H2O2 expression of both genes&rsquo / transcription either did not changed or down regulated. Lastly, in this study we have purified the recombinant TVN0775/sHsp, as an Nterminal 6x his-tag fusion to homogeneity on Ni-NTA affinity column. Purified protein samples were used in the chaperone activity assays using bovine glutamate dehydrogenase enzyme (boGDH) as substrate. We have found that the recovery of glutamate dehydrogenase activity at 45&deg / C, 50&deg / C and 53&deg / C in the presence of the Tp. volcanium sHsps was higher than that of spontaneous refolding. Also, TVN0775/sHsp increased the recovery of the boGDH enzyme that was denatured at 2.5 M GdnHCl concentrations for 30 min.

HSP27, the Drosophila homolog of mammalian HspB1, is a nuclear sHsp that is both stress induced and developmentally regulated with a conserved cyto-protective function. It is multiply phosphorylated in vivo through an unconfirmed mechanism at unidentified residues. The effect of phosphorylation on its localization, oligomerization, and function is also not well understood. Here we report a genetic investigation into the role of Hsp27 in Drosophila development, and a preliminary investigation into the effect of phosphorylation on HSP27 localization and function in Drosophila S2 cells. Through a proteomic screen, a pro-apoptotic role for Hsp27 in embryonic developmentally regulated programmed cell death was suggested and supported by RNAi experiments, but not replicated using Hsp27null mutant stocks. These stocks were complicated by the intriguing appearance of multiple background mutations. Specific developmental defects in transgenic lines overexpressing phospho-mutant isoforms were then investigated. These too were subject to multiple independent incidences of background genetic mutation, which we believe may be related to Hsp27 mis-expression. We also studied the endogenous expression and localization pattern of HSP27 in stressed and unstressed Drosophila S2 cells. We found evidence that wild-type protein localization is influenced by stress. Finally, we took a first step toward understanding how phosphorylation might regulate HSP27 localization by examining the effect of targeted mutations of serine residues (S58, S71, and S75) on the localization pattern of exogenous HSP27. By characterizing the expression of endogenous and overexpressed HSP27 in Drosophila cells, we provide a foundation for future investigation into the regulated localization and function of HSP27 that can be extended to address the regulatory mechanisms that govern the protective capacities and oligomeric properties of phosphorylated HSP27 in Drosophila.

A diapause upregulated cDNA clone was isolated from a cDNA library generated from brain mRNA of diapausing Sarcophaga crassipalpis pupae. The clone hybridized to a 1600 bp transcript on a northern blot. The insert is 823 bp in length, has a tentative open reading frame of 615 bp, and codes for a 23 kDa protein. The clone has a high level of identity at the amino acid level with the four small heat shock proteins of Drosophila melanogaster. Northern analysis revealed no detectable expression of the transcript in diapause- or nondiapause-programmed wandering larvae, and only trace expression in nondiapausing pupae. But, the transcript was highly expressed beginning at the onset of diapause and continuing throughout diapause. Expression promptly decreased when diapause was terminated. In nondiapausing individuals the transcript was highly expressed in response to cold shock or heat shock, but temperature stress did not cause greater expression in diapausing pupae. The results imply that expression of this small heat shock protein, a response elicited by temperature stress in nondiapausing individuals, is a normal component of the diapause syndrome. The upregulation of this gene during diapause suggests that it plays an essential role during this overwintering developmental arrest.

HspB2 is a small heat shock protein encoded on human chromosome 11. Less than 1000 base pairs away from HSPB2 and situated in a head-to-head orientation lies the gene encoding another small heat shock protein, CRYAB. Because they are uncommonly close to one another they share regulatory elements. In addition, they share protein homology as sHSPs, suggesting that they perhaps perform aimilar functions. SHSPs such as HspB2 and CryAB are traditionally thought to provide protective effects to cells in response to a variety of stress inducers. In response to stress they form complexes around misfolded proteins or proteins in danger of denaturation. HspB2 has been shown to exhibit protective effects during cellular stress and to localize to the Z-line of skeletal muscle. It has also been implicated in cardiac energetics, specifically in the production of ATP, however little is known about its molecular targets. Here I report the use of yeast two-hybrid screening to uncover the molecular targets of HspB2. I also detail the process by which the screens are performed as well as the verification steps, including co-precipitation experiments in mammalian cells. Through these studies we identify many novelbinding partners of HspB2, including CryAB as well as multiple muscle and mitochondrial proteins. Proteins discovered to bind to HspB2 include such proteins as actin and myosin, enzymes catalyzing various steps of glycolysis and the electron transport chain, as well as redox-, small heat shock protein-, kinase-, and electrolyte-related proteins, among others. Studies of the binding partners of HspB2 in cardiac tissue will provide important information clarifying the involvement of HspB2 in cardiac muscle maintenance and metabolism.

Small heat shock proteins (sHSPs) are a ubiquitous family of molecular chaperones that play a vital role in maintaining protein homeostasis in cells. They are the first line of defence against the detrimental effects of cellular stress conditions like fluctuations in temperature, pH, oxidative and osmotic potentials, heavy metal toxicity, drought and anoxia. Many sHSPs are also constitutively expressed during developmental stages of different plant tissues. Members of this family are ATP-independent chaperones, with monomeric masses varying from 12-40 kDa. A characteristic feature of sHSPs is their ability to assemble into large oligomers, ranging from dimers to 48-mers. Under stress conditions, these oligomers dissociate and/or undergo drastic conformational changes to facilitate their binding to misfolded substrate proteins in the cell. This interaction prevents the substrate from aggregating during stress. When physiological conditions are restored, the substrates are transferred to other ATP-dependent heat shock proteins for refolding. Thus sHSPs do not refold their substrates, but instead prevent them from aggregating and maintain them in a „folding-competent‟ state. The clientele of sHSPs includes proteins with a wide range of molecular masses, secondary structures and pIs. This promiscuity has led to sHSPs occupying key positions in the protein quality control network. As molecular chaperones that protect proteins, sHSPs prevent disease. Concomitantly, mutations in sHSPs have also been linked to various human diseases. Till date, high resolution crystal structures are available only for 3 sHSP oligomers. This insufficiency of structural information has hindered our understanding of the mechanism of chaperone function, the link between the oligomeric status and chaperone activity, identification of substrate binding sites and the role of the flexible terminal segments in mediating both the oligomerization and chaperone function. We undertook structural and functional characterization of plant and bacterial sHSPs in order to address some of these questions. Chapter 1 of this thesis gives an overview of the sHSP family, with special emphasis on the oligomeric assemblies of sHSPs of known structures. We highlight what we know about this family through mutational studies, what is as yet unknown, and why it is important to study this family. Chapter 2 describes our efforts at structural and functional characterization of 5 sHSPS in rice, each targeted to a different organelle. We probed the role played by the N-terminal region in mediating oligomer assembly and in the chaperone activity of the protein. Rice sHSPs displayed a wide range of hydrodynamic radii, from 4 nm to 14 nm, suggesting that their oligomeric assemblies are likely to be diverse. In chapter 3, we discuss our attempts at the structural characterization of a bacterial sHSP, Aggregation suppressing protein A, or AgsA from Salmonella enterica. We obtained a high resolution crystal structure of the dimer of the core sHSP domain. We compared this dimer with other known sHSP dimers, reported the deviations that we observed and analysed the structure to account for these differences. We used this dimer structure to successfully obtain solutions for low resolution X-ray diffraction data for oligomers of different truncated constructs of AgsA. We observed that a C-terminal truncated construct formed an octahedral 24¬mer (4.5 Å resolution), whereas a construct truncated at both termini formed a triangular bipyramidal 18-mer (7.7 Å resolution), an assembly hitherto unobserved for any sHSP. A similar 18-mer was obtained when the C-terminal truncated construct was incubated with a dipeptide prior to crystallisation (6.7 Å resolution). The cryo-EM map of the wild type protein (12 Å resolution) could be fitted with a different 18-mer. The low resolution of the data pre-empted an atomic-level description of the interfaces of the assemblies. However, our work highlights the structural plasticity of this protein and probes the sensitivity of the oligomeric assembly to minor differences in construct length.

<p>Postmortem aging has been extensively practiced as value-adding process due to the beneficial impacts on meat palatability. Meat tenderization occurred through proteolytic fragmentation of myofibrillar structural proteins via endogenous protease systems, which is considered as the primary drive to enhance major palatability attributes including tenderness, juiciness, and flavor. Recent theoretical framework proposes apoptosis, or programmed cell death, as the preceding step that initiates postmortem proteolysis. Whereas small heat shock proteins have been consistently recognized as meat quality biomarkers, probably due to their protective activities against proteolysis through anti-stress, anti-apoptotic, and chaperoning functionalities. To shed light on detailed mechanisms controlling postmortem proteolysis and consequential impacts on the development of fresh meat quality characteristics, postmortem proteolytic changes of small heat shock proteins, apoptotic factors, and myofibrillar structural proteins were profiled in postmortem skeletal muscles under different metabolic backgrounds and across species. </p> <p>In beef, three muscles, <i>longissimus lumborum</i> (LL), <i>semimembranosus</i> (SM), and <i>psoas major</i> (PM), have been selected to represent glycolytic, intermediate, and oxidative muscle types. Tenderness and water - holding capacity were determined, and proteolysis, apoptotic features, and small heat shock proteins were measured in 8 beef carcasses at 1, 2, 9, 16, and 23 days of aging. PM exhibited limited aging potential in quality developments shown by lower extents of shear force, water-holding capacity, and proteolytic changes, including calpain 1 autolysis, troponin T, and HSP27 compared to LL and SM. Conversely, LL had an increase in tenderization and water-holding capacity, which was accompanied with more extended calpain 1 autolysis, proteolysis and HSP27 degradation, compared with other muscles. The results of this study suggest that postmortem proteolytic changes of myofibrillar proteins, small HSPs and apoptotic factors occur in a muscle-specific manner, which is likely attributed to different rate and extent of meat quality developments of each muscle during aging. </p> <p>Callipyge lambs are a unique genetic background showing calpastatin over-expression, muscle hypertrophy in loin and hindquarter area, substantially compromised meat tenderization potential, and a shift of muscle fiber composition towards fast-glycolytic directions. Proteome and metabolome changes in muscles from callipyge mutation (+/C) and non-callipyge phenotype (+/+, C/+, and C/C) lambs were profiled to provide insight into the biochemical changes affecting meat quality attributes. M. longissimus thoracis from lambs with all four possible callipyge genotype (n = 4, C/+, C/C, +/C, and +/+) were collected after 3d aging and analyzed using mass-spectrometry based platforms. Among identified proteomes, cytochrome c (pro-apoptotic protein) was detected with significantly lower abundances in +/C. Anti-apoptotic HSP70, BAG3, and PARK7 were over-abundant in +/C, which could result in delayed apoptosis and possibly attributed to tougher meat in callipyge lambs. Eight glycolysis enzymes were overabundant in +/C lambs, whereas 3 enzymes involved in TCA cycle were overabundant in non-callipyge ones (C/C and/or C/+). Twenty-five metabolites were affected by genotypes (P < 0.05), including metabolic co-factors, polyphenols, and AA/short peptides.</p> <p>Pig production is facing increased public pressure regarding antibiotic usage restriction. Recently, dietary L-glutamine at cost effective level (0.2%) was identified as an effective antibiotic alternative in post-transport nursery pig diets. To evaluate carcass and meat quality characteristics in market-ready pigs when 0.2% dietary L-glutamine was applied as for early-life post-weaning and transport recovery, pigs (N=480) were weaned and transported in two replication trials in SPRING (April of 2017) vs. SUMMER (July of 2016), fed 3 different diets (Non: no antibiotic, Anti: 441 ppm chlortetracycline and + 38.6 ppm tiamulin, Gln: 0.20% L-glutamine) for 14 days after transport, and fed basal diet until reaching market weight. Pairs of <i>longissimus dorsi</i> (LD) and <i>psoas major</i> (PM) muscles from each carcass (n=10/diet/trial) were separated at 1 d and 7 d postmortem, respectively. Carcass yield and meat physical and quality attributes were evaluated. Overall impacts of Gln on physical attributes of carcasses and porcine muscles were minimal. No dietary effects were found in carcass, proximate composition, water-holding capacity, or shear force. Significant difference between trials were found in terms of productivity and pork/carcass qualities, where SPRING replicates showed increased body weight, faster pH decline, paler surface color, higher intra-muscular fat deposition, and improved tenderness and water-holding capacity as indicated by lower shear force values, thaw-purge loss, and cooking loss (P < 0.05).</p> <p>The pork and carcass quality results give rise to a postulation that different metabolism and animal growth might have been occured between the two production trials, consequentially differentiated meat quality development. In this regard, myofibrillar proteolysis, small heat shock proteins, and apoptotic factors were characterized during 7 d postmortem aging in porcine LD and PM muscles from both seasonal trials, combined with metabolomics profiles of 1d samples using the GC-TOF-MS/MS platform. Compared to SUMMER counterparts, SPRING muscles showed concurrence of more extended apoptosis, further calpain 1 autolysis, and increased structural protein degradation (P<0.05). SPRING muscles showed more ATP catabolism compounds and increase in carbohydrates, branched-chain amino acids, and 16-18 carbon fatty acids, which could be chemistry fingerprints of increased cellular oxidative stress, consequentially favoring onset of apoptosis and proteolysis. Meanwhile, SUMMER pigs showed increased stress-defending metabolites, such as ascorbic acid, antioxidant amino acids, and decreased inhibitory neuro-transmitter GABA, which may indicate elevated stress-defending activity in SUMMER pigs that possibly inhibited apoptosis and proteolysis. </p>

Small Heat Shock Proteins (sHSP) are molecular chaperones that play protective roles in cell survival and have been shown to possess chaperone activity. As such, mutations in this family of proteins result in a wide variety of diseases from cancers to cardiomyopathies. The sHSPs Beta-2 (HspB2) and alpha-beta crystalline (CryAB) are two of the ten human sHSPs and are both expressed in cardiac and skeletal muscle cells. A heart that cannot properly recover or defend against stressors such as extreme heat or cold, oxidative/reductive stress, and heavy metal-induced stress will constantly struggle to maintain efficient function. Accordingly, CryAB is required for myofibril recovery from ischemia/reperfusion (I/R) and HspB2 is required I/R recovery as well as efficient cardiac ATP production. Despite these critical roles, little is known about the molecular function of these chaperones. We have identified over two hundred HspB2-binding partners through both yeast two-hybrid and copurification approaches, including interactions with myofibril and mitochondrial proteins. There is remarkable overlap between the two approaches (80%) suggesting a high confidence level in our findings. The sHSP, CryAB, only binds a subset of the HspB2 interactome, showing that the HspB2 interactome is specific to HspB2 and supporting non-redundant roles for sHSPs. We have confirmed a subset of these binding partners as HspB2 clients via in vitro chaperone activity assays. In addition, comparing the binding patterns and activity of sHSP variants in comparison to wild type can help to elucidate how variants participate in causing disease. Accordingly, we have used Y2H and in vitro chaperone activity assays to compare the disease-associated human variants R120GCryAB and A177PHspB2 to wild type and have identified differences in binding and chaperone function. These results not only provide the first molecular evidence for non-redundancy of the sHSPs, but provides a useful resource for the study of sHSPs in mitochondrial and myofibril function.

Las proteínas pequeñas de choque térmico (sHSP) responden a una amplia variedad de estreses ambientales, estabilizando proteínas parcialmente desplegadas y evitando su agregación irreversible, en forma independiente de ATP. En plantas, las sHSPs son especialmente diversas siendo las sHSPs de organelas una característica única de las plantas. La estructura primaria de las sHSP incluye una secuencia N-terminal no conservada de longitud variable, un dominio α-cristalino conservado (ACD) y una secuencia C-terminal corta no conservada. El dominio ACD representa una característica conservada presente en todas las sHSPs, sin embargo, no todas las proteínas que contienen un dominio ACD son sHSP. Las sHSPs pertenecen a una gran superfamilia, siendo su importancia funcional y fisiológica en gran parte desconocida. El objetivo de este trabajo fue dilucidar el rol de sHSPs de localización mitocondrial (sHSPs-M) en Arabidopsis thaliana y Solanum lycopersicum en situaciones de estrés ambiental, y caracterizar probables promotores bidireccionales que regulan la expresión de genes codificantes de proteínas con dominio ACD con orientación cabeza a cabeza en el genoma de Arabidopsis. Este trabajo cubre aspectos desde la organización genómica y la función de sHSPs-M en Arabidopsis hasta el rol de las sHSPs-M en la respuesta al estrés por frío de frutos de tomate. Para ello, se generaron plantas mutantes de Arabidopsis y de tomate utilizando la tecnología de silenciamiento génico por micro ARNs artificiales. Las plantas mutantes fueron analizadas en su proteoma, metaboloma y lipidoma en distintas condiciones de estrés. A continuación, se presenta un resumen de los resultados obtenidos. En la primera parte de este trabajo, se realizó la caracterización funcional de genes con orientación cabeza a cabeza en el genoma de Arabidopsis, que codifican para proteínas con ACD y las regiones intergénicas correspondientes. Se lograron identificar y caracterizar cuatro distintos promotores bidireccionales, entre ellos el promotor del gen At5g51440 que codifica una sHSP de localización mitocondrial (sHSP23.5). Los resultados obtenidos sugieren que el promotor bidireccional contenido en el par At5g51430-At5g51440 es fuertemente inducido por altas temperaturas en una dirección, pero no así en la dirección opuesta. El promotor contenido en el par At1g06460-At1g06470 mostró una actividad alta en ambas direcciones, teniendo por ello, un alto potencial de aplicación en ingeniería genética. Los dos promotores restantes mostraron mayor actividad en una dirección y, por lo tanto, pueden ser considerados como promotores bidireccionales asimétricos. El estudio funcional de los promotores seleccionados reveló el potencial biotecnológico de los mismos ya que pueden ser inducidos específicamente en una determinada condición (como altas temperaturas) en una o en ambas direcciones cuando sea necesario. En la segunda parte, se presenta la caracterización funcional de sHSP mitocondriales en condiciones de estrés y durante el desarrollo de Arabidopsis. Se identificaron tres genes parálogos en el genoma de Arabidopsis (At5g51440, At4g25200, y At1g52560), y se diseñaron microARN artificiales específicos para la obtención de plantas mutantes por silenciamiento (amiR simple, doble y triple). Las plantas amiR simples y dobles (para sHSP23.5 y sHSP23.6) no mostraron un fenotipo evidentemente afectado, probablemente debido a la compensación o redundancia funcional de las sHSP mitocondriales. Por otro lado, las plantas triple mutante amiR23.5/23.6/26.5 muestran un fenotipo alterado en las etapas vegetativa y reproductiva. Estas plantas presentan hojas pequeñas, células epidérmicas con áreas reducidas, pero no reducción en el número de células epidérmicas por hoja. Además, exhiben hojas cloróticas, raíces cortas y menor producción de semillas en comparación con las plantas Col-0. Las plantas triple amiR son considerablemente pequeñas debido a una alteración en el proceso de expansión celular, pero no en la proliferación celular, lo que indica una profunda alteración en el programa de desarrollo de la planta. En el análisis proteómico de las mutantes amiR se observó un aumento significativo de proteínas implicadas en el metabolismo, y alteración en la abundancia de varias proteínas relacionadas con la traducción, y con el funcionamiento y la estructura de los ribosomas. La triple mutante exhibió un mayor número de proteínas con abundancia alterada involucradas en estos procesos en comparación con las mutantes simples y doble amiR23.5/23.6. Estos cambios tan amplios observados en proteínas relacionadas con el funcionamiento de los ribosomas sugieren una posible alteración en la función normal de los mismos. Los resultados presentados en este trabajo proporcionan evidencias sobre el importante rol de las sHSPs-M no sólo en la respuesta a las altas temperaturas, sino también durante el desarrollo de la planta de Arabidopsis. Los resultados indican que una compensación funcional podría ser responsable del fenotipo observado en las plantas mutantes con niveles reducidos de cada sHSPs-M individual. Sin embargo, la reducción simultanea de las tres sHSPs-M causó una profunda alteración en la función normal de mitocondrias y ribosomas, afectando gravemente el metabolismo energético y la homeostasis celular, lo que llevó a alteraciones en el desarrollo correcto de la planta. En la última parte de este trabajo, las consecuencias de la disminución de la proteína sHSP23.8 de localización mitocondrial en frutos de tomate fueron investigadas. Los frutos fueron analizados en su fenotipo y en la susceptibilidad al daño por frío. Los frutos de las plantas mutantes amiR23.8 que fueron conservados en frío mostraron mayor pérdida de agua y de electrolitos en el tejido pericárpico en comparación con frutos WT. El deterioro observado en los frutos amiR23.8 indica que estos frutos mutantes son mayormente susceptibles al estrés por frío desarrollando síntomas de daño por frío. El lipidoma de los frutos amiR23.8 frigoconservados mostró cantidades alteradas de glicerolípidos, y los niveles de lípidos saturados en amiR23.8 disminuyeron luego del tratamiento con frío, pero no por debajo de los niveles encontrados en frutos WT en condiciones normales. Lo opuesto se encontró en el porcentaje relativo de lípidos insaturados, con niveles significativamente más bajos de insaturaciones en frutos amiR23.8 en condiciones normales y después del enfriamiento. Los resultados presentados indican una degradación diferencial de lípidos extraplastídicos y plastídicos en frutos amiR23.8, y alteraciones en la remodelación del lipidoma luego del estrés por frío, lo que podría conducir a una mayor sensibilidad al daño por frío. Los resultados discutidos aquí indican que la proteína sHSP23.8 podría estar directamente involucrada en los mecanismos de protección contra el estrés por frío en frutos de tomate.

Prokaryotic and eukaryotic organisms respond to various stresses with the production of heat shock proteins (HSPs). HSPs are molecular chaperones that bind to unfolded proteins and inhibit their aggregation as well as maintaining their solubility until they can be refolded to their original conformation. Stress-inducible hsp gene transcription is mediated by the heat shock element (HSE), which interacts with heat shock transcription factor (HSF). In this study, we examined the effect of KNK437 (N-formyl-3,4-methylenedioxy-benzylidene-g-butyrolactam), a benzylidene lactam compound, on heat shock, sodium arsenite, cadmium chloride and herbimycin A-induced hsp gene expression in Xenopus laevis A6 kidney epithelial cells. In studies limited to mammalian cultured cells, KNK437 has been shown to inhibit HSE-HSF1 binding activity and stress-induced hsp gene expression. In the present study, western and northern blot analysis revealed that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP30 protein and hsp30 mRNA, respectively. Western blot analysis also determined that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP70 protein. Pre-treatment of A6 cells with 100 µM KNK437 inhibited stress-induced hsp30 mRNA as well as HSP30 and HSP70 protein accumulation. Immunocytochemistry and confocal microscopy were used to confirm the results gained from western blot analysis as well as determine the localization of HSP30 accumulation in A6 cells. A 2 h heat shock at 33°C resulted in the accumulation of HSP30 in the mostly in the cytoplasm with a small amount in the nucleus. Heat shock at 35°C resulted in substantial HSP30 accumulation in the nucleus. This is in contrast with A6 cells treated for 14 h with 10 µM sodium arsenite, 100 µM cadmium chloride and 1 µg/mL herbimycin A, where HSP30 accumulation was found only in the cytoplasm and not in the nucleus. A 6 h pre-treatment with 100 µM KNK437 completely inhibited the accumulation of HSP30 in A6 cells heat shocked at 33 or 35°C as well as cells treated with 1 µg/mL herbimycin A. The same pre-treatment with KNK437 resulted in a 97-100% decrease in HSP30 accumulation in A6 cells treated with 10 µM sodium arsenite or 100 µM cadmium chloride. These results show that KNK437 is effective at inhibiting both heat shock and chemical induced hsp gene expression in amphibian cells.