RESUMEN
BACKGROUND: The encystation of Acanthamoeba castellanii has important ecological and medical significance. Blocking encystation is the key to preventing transmission and curing infections caused by A. castellanii. The formation of autophagosomes is one of the most important changes that occur during the encystation of Acanthamoeba. Our previous studies have shown that the heat shock protein 20 of A. castellanii (Ac-HSP20) is involved in its encystation. This study aimed to determine the role and mechanism of Ac-HSP20 in regulating autophagy involved in the encystation of A. castellanii. METHODS: Immunofluorescence assay, western blotting and transmission electron microscopy were used to analyze the dynamic changes in autophagy during the initiation and continuation of encystation. The knockdown of Ac-HSP20 was performed to clarify its regulation of encystation and autophagy and to elucidate the molecular mechanism by which Ac-HSP20 participates in autophagy to promote cyst maturation. RESULTS: The encystation rates and autophagosomes were significantly decreased by treatment with the autophagy inhibitor 3-MA. The autophagy marker LC3B and autophagic lysosomes increased with the induced duration of encystation and reached the maximum at 48 h. The encystation rate, LC3B expression and autophagosomes decreased when Ac-HSP20 was knocked down by siRNA transfection. In addition, the expression levels of Ac-HSP20 and LC3B increased and the expressions of p-AKT and p-mTOR decreased after 48 h of encystation without knockdown. However, the expressions of p-AKT and p-mTOR increased while the expression of LC3B decreased under the knockdown of Ac-HSP20. Furthermore, the protein expression of LC3B increased when the PI3K/AKT/mTOR signaling pathway was inhibited but decreased when the pathway was activated. CONCLUSIONS: The results demonstrated that autophagy is positively correlated with the encystation of A. castellanii, and Ac-HSP20 regulates autophagy to maintain the homeostasis of A. castellanii by inhibiting the PI3K /AKT /mTOR signaling pathway, thus promoting the maturation and stability of encystation.
Asunto(s)
Acanthamoeba castellanii , Autofagia , Proteínas del Choque Térmico HSP20 , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Serina-Treonina Quinasas TOR , Serina-Treonina Quinasas TOR/metabolismo , Serina-Treonina Quinasas TOR/genética , Acanthamoeba castellanii/fisiología , Acanthamoeba castellanii/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas del Choque Térmico HSP20/metabolismo , Proteínas del Choque Térmico HSP20/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Enquistamiento de Parásito/fisiología , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Autofagosomas/metabolismoRESUMEN
Heat shock protein 20 (Hsp20) is a small molecule heat shock protein that plays an important role in plant growth, development, and stress resistance. Little is known about the function of Hsp20 family genes in apple (Malus domestica). Here, we performed a genome-wide analysis of the apple Hsp20 gene family, and a total of 49 Hsp20s genes were identified from the apple genome. Phylogenetic analysis revealed that the 49 genes were divided into 11 subfamilies, and MdHsp18.2b, a member located in the CI branch, was selected as a representative member for functional characterization. Treatment with NaCl and Botryosphaeria dothidea (B. dothidea), the causal agent of apple ring rot disease, significantly induced MdHsp18.2b transcription level. Further analysis revealed that overexpressing MdHsp18.2b reduced the resistance to salt stress but enhanced the resistance to B. dothidea infection in apple calli. Moreover, MdHsp18.2b positively regulated anthocyanin accumulation in apple calli. Physiology assays revealed that MdHsp18.2b promoted H2O2 production, even in the absence of stress factors, which might contribute to its functions in response to NaCl and B. dothidea infection. Hsps usually function as homo- or heterooligomers, and we found that MdHsp18.2b could form a heterodimer with MdHsp17.9a and MdHsp17.5, two members from the same branch with MdHsp18.2b in the phylogenetic tree. Therefore, we identified 49 Hsp20s genes from the apple genome and found that MdHsp18.2b was involved in regulating plant resistance to salt stress and B. dothidea infection, as well as in regulating anthocyanin accumulation in apple calli.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Proteínas del Choque Térmico HSP20 , Malus , Filogenia , Enfermedades de las Plantas , Proteínas de Plantas , Malus/genética , Malus/microbiología , Malus/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/genética , Proteínas del Choque Térmico HSP20/genética , Proteínas del Choque Térmico HSP20/metabolismo , Ascomicetos/fisiología , Ascomicetos/genética , Ascomicetos/patogenicidad , Familia de Multigenes , Resistencia a la Enfermedad/genética , Antocianinas/metabolismoRESUMEN
HSP20 emerges as a novel regulator of autophagy in the heart. Nonetheless, the detailed function of HSP20 in the liver and its effect on autophagy remain unknown. Here, we observed that HSP20 expression is increased in liver tissues from mice and patients with metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as nonalcoholic fatty liver disease. Liver-specific downregulation of HSP20 mitigates hepatic steatosis and insulin resistance in obese mice, while upregulating HSP20 promotes lipid deposition and hepatocyte cell death. Mechanistically, liquid chromatography-tandem mass spectrometry revealed that HSP20 interacts with phosphorylated extracellular regulated protein kinase 2 (ERK2) and prevents its dephosphorylation by dual specificity phosphatase 6, leading to ERK2-mediated repression of autophagy and resulting in aggravated saturated fatty acid (SFA)-triggered hepatocyte death. Importantly, such adverse effects could be ameliorated by ERK inhibitor. Our data reveal a framework of how HSP20 increases susceptibility of SFA-induced liver injury through enhancing ERK2 phosphorylation, which represents a plausible therapeutic intervention to combat MASLD.
Asunto(s)
Autofagia , Proteínas del Choque Térmico HSP20 , Proteína Quinasa 1 Activada por Mitógenos , Animales , Humanos , Masculino , Ratones , Autofagia/efectos de los fármacos , Autofagia/fisiología , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , Proteínas del Choque Térmico HSP20/metabolismo , Proteínas del Choque Térmico HSP20/genética , Resistencia a la Insulina/fisiología , Hígado/metabolismo , Hígado/patología , Hígado/efectos de los fármacos , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , FosforilaciónRESUMEN
The tardigrade Ramazzottius varieornatus has remarkable resilience to a range of environmental stresses. In this study, we have characterised two members of the small heat shock protein (sHSP) family in R. varieornatus, HSP20-3 and HSP20-6. These are the most highly upregulated sHSPs in response to a 24 h heat shock at 35 0C of adult tardigrades with HSP20-3 being one of the most highly upregulated gene in the whole transcriptome. Both R. varieornatus sHSPs and the human sHSP, CRYAB (HSPB5), were produced recombinantly for comparative structure-function studies. HSP20-3 exhibited a superior chaperone activity than human CRYAB in a heat-induced protein aggregation assay. Both tardigrade sHSPs also formed larger oligomers than CRYAB as assessed by size exclusion chromatography and transmission electron microscopy of negatively stained samples. Whilst both HSP20-3 and HSP20-6 formed particles that were variable in size and larger than the particles formed by CRYAB, only HSP20-3 formed filament-like structures. The particles and filament-like structures formed by HSP20-3 appear inter-related as the filament-like structures often had particles located at their ends. Sequence analyses identified two unique features; an insertion in the middle region of the N-terminal domain (NTD) and preceding the critical-sequence identified in CRYAB, as well as a repeated QNTN-motif located in the C-terminal domain of HSP20-3. The NTD insertion is expected to affect protein-protein interactions and subunit oligomerisation. Removal of the repeated QNTN-motif abolished HSP20-3 chaperone activity and also affected the assembly of the filament-like structures. We discuss the potential contribution of HSP20-3 to protein condensate formation.
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Proteínas de Choque Térmico Pequeñas , Humanos , Proteínas de Choque Térmico Pequeñas/metabolismo , Secuencia de Aminoácidos , Proteínas del Choque Térmico HSP20/genética , Proteínas del Choque Térmico HSP20/metabolismo , Chaperonas Moleculares/metabolismo , Respuesta al Choque TérmicoRESUMEN
To better understand the pathogenesis of acute type A aortic dissection, high-sensitivity liquid chromatography-tandem mass spectrometry/mass spectrometry (LC-MS/MS)-based proteomics and phosphoproteomics approaches were used to identify differential proteins. Heat shock protein family B (small) member 6 (HSPB6) in aortic dissection was significantly reduced in human and mouse aortic dissection samples by real-time PCR, western blotting, and immunohistochemical staining techniques. Using an HSPB6-knockout mouse, we investigated the potential role of HSPB6 in ß-aminopropionitrile monofumarate-induced aortic dissection. We found increased mortality and increased probability of ascending aortic dissection after HSPB6 knockout compared with wild-type mice. Mechanistically, our data suggest that HSPB6 deletion promoted vascular smooth muscle cell apoptosis. More importantly, HSPB6 deletion attenuated cofilin activity, leading to excessive smooth muscle cell stiffness and eventually resulting in the development of aortic dissection and rupture. Our data suggest that excessive stiffness of vascular smooth muscle cells caused by HSPB6 deficiency is a new pathogenetic mechanism leading to aortic dissection.
Asunto(s)
Disección Aórtica , Espectrometría de Masas en Tándem , Ratones , Humanos , Animales , Cromatografía Liquida , Disección Aórtica/genética , Miocitos del Músculo Liso/metabolismo , Ratones Noqueados , Modelos Animales de Enfermedad , Proteínas del Choque Térmico HSP20/metabolismoRESUMEN
Heat shock proteins (HSP) are a large family of peptide proteins that are widely found in cells. Studies have shown that the expression and function of HSPs in cells are very complex, and they can participate in cellular physiological and pathological processes through multiple pathways. Multiple heat shock proteins are associated with cancer cell growth, proliferation, metastasis, and resistance to anticancer drugs, and they play a key role in cancer development by ensuring the correct folding or degradation of proteins in cancer cells. As research hotspots, HSP90, HSP70 and HSP27 have been extensively studied in cancer so far. However, HSP20, also referred to as HSPB6, as a member of the small heat shock protein family, has been shown to play an important role in the cardiovascular system, but little research has been conducted on HSP20 in cancer. This review summarizes the current cellular functions of HSP20 in different cancer types, as well as its effects on cancer proliferation, progression, prognosis, and its other functions in cancer, to illustrate the close association between HSP20 and cancer. We show that, unlike most HSPs, HSP20 mainly plays an active anticancer role in cancer development, which is expected to provide new ideas and help for cancer diagnosis and treatment and research.
Asunto(s)
Antineoplásicos , Neoplasias , Humanos , Proteínas de Choque Térmico/metabolismo , Neoplasias/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas del Choque Térmico HSP20/metabolismoRESUMEN
Heat shock protein B6 (HSPB6) plays a certain role in the formation of several cancers, whereas its effect on osteosarcoma remains unclear. In this study, the effect of HSPB6 on osteosarcoma was validated through numerous experiments. HSPB6 was down-regulated in osteosarcoma. As indicated by the result of CCK-8 and colony formation assays, HSPB6 overexpression was likely to inhibit the osteosarcoma cells proliferation, whereas the flow cytometry analysis suggested that apoptosis of osteosarcoma cells was increased after HSPB6 overexpression. Furthermore, transwell and wound healing assays suggested that when HSPB6 was overexpressed, osteosarcoma cells migration and invasion were declined. Moreover, the western blotting assay suggested that the protein level of p-ERK1/2 was down-regulated in osteosarcoma when HSPB6 was overexpressed. Besides, the effect of HSPB6 on osteosarcoma in vivo was examined. As indicated by the result, HSPB6 overexpression was likely to prevent osteosarcoma growth and lung metastasis in vivo. As revealed by the findings of this study, HSPB6 overexpression exerted anticancer effects in osteosarcoma through the ERK signaling pathway and HSPB6 may be suitable target for osteosarcoma molecular therapies.
Asunto(s)
Neoplasias Óseas , Osteosarcoma , Humanos , Apoptosis , Neoplasias Óseas/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Proteínas del Choque Térmico HSP20/genética , Proteínas del Choque Térmico HSP20/metabolismo , Sistema de Señalización de MAP Quinasas , Osteosarcoma/patología , Transducción de SeñalRESUMEN
To successfully complete malolactic fermentation (MLF), Oenococcus oeni must overcome wine stress conditions of low pH, high ethanol, and the presence of SO2. Failure to complete MLF may result in detrimental effects to the quality and stability of the resulting wines. Research efforts to date have focused on elucidating the mechanisms and genetic features that confer the ability to withstand low pH and high ethanol concentrations on O. oeni; however, the responses to SO2 stress are less well defined. This study focused on characterizing the transcriptional response of O. oeni to SO2 challenge during cultivation in a continuous system at wine-like pH (3.5). This experimental design allowed the precise discrimination of transcriptional changes linked to SO2 stress from responses associated with growth stage and cultivation parameters. Differential gene expression analysis revealed major transcriptional changes following SO2 exposure and suggested that this compound primarily interacts with intracellular proteins, DNA, and the cell envelope of O. oeni. The molecular chaperone hsp20, which has a demonstrated function in the heat, ethanol, and acid stress response, was highly upregulated, confirming its additional role in the response of this species to SO2 stress. This work also reports the first nanopore-based complete genome assemblies for O. oeni. IMPORTANCE Malolactic fermentation is an indispensable step in the elaboration of most wines and is generally performed by Oenococcus oeni, a Gram-positive heterofermentative lactic acid bacterium species. While O. oeni is tolerant to many of the wine stresses, including low pH and high ethanol concentrations, it has high sensitivity to SO2, an antiseptic and antioxidant compound regularly used in winemaking. Understanding the physiological changes induced in O. oeni by SO2 stress is essential for the development of more robust starter cultures and methods for their use. This study describes the main transcriptional changes induced by SO2 stress in the wine bacterium O. oeni and provides foundational understanding on how this compound interacts with the cellular components and the induced protective mechanisms of this species.
Asunto(s)
Regulación Bacteriana de la Expresión Génica/genética , Malatos/metabolismo , Oenococcus/genética , Oenococcus/metabolismo , Sulfitos/metabolismo , Membrana Celular/metabolismo , Daño del ADN/genética , Etanol/análisis , Fermentación , Genoma Bacteriano/genética , Proteínas del Choque Térmico HSP20/metabolismo , Concentración de Iones de Hidrógeno , Ácido Láctico/metabolismo , Estrés Fisiológico/fisiología , Transcripción Genética/genética , Transcriptoma/genética , Vino/microbiologíaRESUMEN
Inflammation and oxidative stress contribute to the progression of acute lung injury (ALI). MicroRNA-23a-5p (miR-23a-5p) has been reported to regulate inflammation and oxidative stress; however, its role in ALI is still poorly elucidated. Mice were intravenously treated with the miR-23a-5p antagomir, agomir, or the negative controls for 3 consecutive days and then received a single intratracheal injection of lipopolysaccharide (LPS, 5 mg/kg) to induce ALI. Pulmonary function, bronchoalveolar lavage fluids (BALFs), arterial blood gas, and molecular biomarkers associated with inflammation and oxidative stress were analyzed. In addition, murine peritoneal macrophages were isolated and treated with LPS to verify the role of miR-23a-5p in vitro. We detected an elevation of miR-23a-5p expression in the lungs from ALI mice. The miR-23a-5p antagomir was prevented, whereas the miR-23a-5p agomir aggravated inflammation, oxidative stress, lung tissue injury, and pulmonary dysfunction in LPS-treated mice. Besides, the miR-23a-5p antagomir also reduced the productions of proinflammatory cytokines and free radicals in LPS-treated primary macrophages, which were further augmented in cells following the miR-23a-5p agomir treatment. Additional findings demonstrated that the miR-23a-5p agomir exacerbated LPS-induced ALI via activating apoptosis signal-regulating kinase 1 (ASK1), and that pharmacological or genetic inhibition of ASK1 significantly repressed the deleterious effects of the miR-23a-5p agomir. Moreover, we proved that the miR-23a-5p agomir activated ASK1 via directly reducing heat shock protein 20 (HSP20) expression. miR-23a-5p is involved in the regulation of LPS-induced inflammation, oxidative stress, lung tissue injury, and pulmonary dysfunction by targeting HSP20/ASK1, and it is a valuable therapeutic candidate for the treatment of ALI.
Asunto(s)
Lesión Pulmonar Aguda/patología , Regulación de la Expresión Génica , Proteínas del Choque Térmico HSP20/metabolismo , Lipopolisacáridos/toxicidad , MAP Quinasa Quinasa Quinasa 5/metabolismo , MicroARNs/genética , Estrés Oxidativo , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/metabolismo , Animales , Apoptosis , Citocinas , Proteínas del Choque Térmico HSP20/genética , Inflamación/etiología , Inflamación/metabolismo , Inflamación/patología , MAP Quinasa Quinasa Quinasa 5/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
The angiotensin Converting Enzyme 2 (ACE2) receptor is a key component of the renin-angiotensin-aldesterone system (RAAS) that mediates numerous effects in the cardiovascular system. It is also the cellular point of contact for the coronavirus spike protein. Cleavage of the receptor is both important to its physiological function as well as being necessary for cell entry by the virus. Shedding of ACE2 by the metalloprotease ADAM17 releases a catalytically active soluble form of ACE2, but cleavage by the serine protease TMPRSS2 is necessary for virion internalization. Complicating the issue is the observation that circulating ACE2 can also bind to the virus effectively blocking attachment to the membrane-bound receptor. This work investigates the possibility that the inflammatory response to coronavirus infection can abrogate shedding by ADAM17, thereby favoring cleavage by TMPRSS2 and thus cell entry by the virion.
Asunto(s)
Proteína ADAM17/química , Proteína ADAM17/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Proteínas del Choque Térmico HSP20/metabolismo , Interacciones Huésped-Patógeno/fisiología , SARS-CoV-2/patogenicidad , Enzima Convertidora de Angiotensina 2/química , Sitios de Unión , Proteínas del Choque Térmico HSP20/química , Respuesta al Choque Térmico/fisiología , Humanos , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , Serina Endopeptidasas/metabolismo , Internalización del VirusRESUMEN
Progesterone (P4) and cyclic adenosine monophosphate (cAMP) are regarded as pro-quiescent factors that suppress uterine contractions during pregnancy. We previously used human primary cells in vitro and mice in vivo to demonstrate that simultaneously enhancing myometrial P4 and cAMP levels may reduce inflammation-associated preterm labor. Here, we assessed whether aminophylline (Ami; phosphodiesterase inhibitor) and P4 can reduce myometrial contractility and contraction-associated proteins (CAPs) better together than individually; both agents are clinically used drugs. Myometrial tissues from pregnant non-laboring women were treated ex vivo with Ami acutely (while spontaneous contracting) or throughout 24-h tissue culture (±P4); isometric tension measurements, PKA assays, and Western blotting were used to assess tissue contractility, cAMP action, and inflammation. Acute (1 h) treatment with 250 and 750 µM Ami reduced contractions by 50% and 84%, respectively, which was not associated with a directly proportional increase in whole tissue PKA activity. Sustained myometrial relaxation was observed during 24-h tissue culture with 750 µM Ami, which did not require P4 nor reduce CAPs. COX-2 protein can be reduced by 300 nM P4 but this did not equate to myometrial relaxation. Ami (250 µM) and P4 (100 and 300 nM) co-treatment did not prevent oxytocin-augmented contractions nor reduce CAPs during interleukin-1ß stimulation. Overall, Ami and P4 co-treatment did not suppress myometrial contractions more than either agent alone, which may be attributed to low specificity and efficacy of Ami; cAMP and P4 action at in utero neighboring reproductive tissues during pregnancy should also be considered.
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Aminofilina/farmacología , Miometrio/efectos de los fármacos , Progesterona/farmacología , Contracción Uterina/efectos de los fármacos , Conexina 43/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ciclooxigenasa 2/metabolismo , Interacciones Farmacológicas , Femenino , Proteínas del Choque Térmico HSP20/metabolismo , Humanos , Interleucina-1beta/farmacología , Miometrio/fisiología , Embarazo , Receptores de Progesterona/metabolismoRESUMEN
At dawn of a scorching summer day, land plants must anticipate upcoming extreme midday temperatures by timely establishing molecular defences that can keep heat-labile membranes and proteins functional. A gradual morning pre-exposure to increasing sub-damaging temperatures induces heat-shock proteins (HSPs) that are central to the onset of plant acquired thermotolerance (AT). To gain knowledge on the mechanisms of AT in the model land plant Physcomitrium patens, we used label-free LC-MS/MS proteomics to quantify the accumulated and depleted proteins before and following a mild heat-priming treatment. High protein crowding is thought to promote protein aggregation, whereas molecular chaperones prevent and actively revert aggregation. Yet, we found that heat priming (HP) did not accumulate HSP chaperones in chloroplasts, although protein crowding was six times higher than in the cytosol. In contrast, several HSP20s strongly accumulated in the cytosol, yet contributing merely 4% of the net mass increase of heat-accumulated proteins. This is in poor concordance with their presumed role at preventing the aggregation of heat-labile proteins. The data suggests that under mild HP unlikely to affect protein stability. Accumulating HSP20s leading to AT, regulate the activity of rare and specific signalling proteins, thereby preventing cell death under noxious heat stress.
Asunto(s)
Bryopsida/fisiología , Proteínas de Plantas/metabolismo , Termotolerancia/fisiología , Bryopsida/citología , Cromatografía Liquida , Citosol/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas del Choque Térmico HSP20/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Complejos Multiproteicos/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Proteínas de Plantas/análisis , Proteínas de Plantas/genética , Proteómica , Espectrometría de Masas en Tándem , Flujo de TrabajoRESUMEN
The cytoskeleton has a primary role in cardiomyocyte function, including the response to mechanical stimuli and injury. The small heat shock protein 20 (Hsp20) conveys protective effects in cardiac muscle that are linked to serine-16 (Ser16) Hsp20 phosphorylation by stress-induced PKA, but the link between Hsp20 and the cytoskeleton remains poorly understood. Herein, we demonstrate a physical and functional interaction of Hsp20 with the cytoskeletal protein 14-3-3. We show that, upon phosphorylation at Ser16, Hsp20 translocates from the cytosol to the cytoskeleton where it binds to 14-3-3. This leads to dissociation of 14-3-3 from the F-actin depolymerization regulator cofilin-2 (CFL2) and enhanced F-actin depolymerization. Importantly, we demonstrate that the P20L Hsp20 mutation associated with dilated cardiomyopathy exhibits reduced physical interaction with 14-3-3 due to diminished Ser16 phosphorylation, with subsequent failure to translocate to the cytoskeleton and inability to disassemble the 14-3-3/CFL2 complex. The topological sequestration of Hsp20 P20L ultimately results in impaired regulation of F-actin dynamics, an effect implicated in loss of cytoskeletal integrity and amelioration of the cardioprotective functions of Hsp20. These findings underscore the significance of Hsp20 phosphorylation in the regulation of actin cytoskeleton dynamics, with important implications in cardiac muscle physiology and pathophysiology.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citoesqueleto/metabolismo , Proteínas del Choque Térmico HSP20/metabolismo , Miocardio/metabolismo , Proteínas 14-3-3/metabolismo , Actinas/metabolismo , Animales , Cofilina 2/metabolismo , Células HEK293 , Proteínas del Choque Térmico HSP20/genética , Humanos , Ratones , Mutación , Fosforilación , Unión Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
Smoking increases the risk of cardiovascular diseases. The present study was designed to determine the effects of 2-month exposure to cigarette smoke (CS) on proteins in the left ventricles of spontaneously hypertensive rats (SHR) and to identify the molecular targets associated with the pathogenesis/progression of CS-induced cardiac hypertrophy. SHR and Wistar Kyoto rats (WKY) were exposed to CS at low (2 puffs/min for 40 min) or high dose (2 puffs/min for 120 min), 5 days a week for 2 months. Using the two-dimensional fluorescence difference gel electrophoresis combined with MALDI-TOF/TOF tandem mass spectrometry, we compared differences in the expression levels of proteins in the whole left ventricles induced by long-term smoking. High-dose CS mainly caused cardiac hypertrophy in SHR, but not WKY, but no change in blood pressure. Proteomic analysis identified 30 protein spots with significant alterations, with 14 up-regulated and 16 down-regulated proteins in the left ventricles of CS-exposed SHR, compared with control SHR. Among these proteins, two members of the heat shock proteins (HSP70 and HSP20) showed significant up-regulation in the left ventricles of CS high-dose SHR, and the results were confirmed by western blot analysis. Our findings suggested that HSPs play an important role in regulation of CS-induced cardiac hypertrophy.
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Cardiomegalia/etiología , Cardiomegalia/genética , Fumar Cigarrillos/efectos adversos , Fumar Cigarrillos/genética , Proteínas del Choque Térmico HSP20/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteómica/métodos , Animales , Cardiomegalia/metabolismo , Expresión Génica , Proteínas del Choque Térmico HSP20/genética , Proteínas HSP70 de Choque Térmico/genética , Ventrículos Cardíacos/metabolismo , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Riesgo , Regulación hacia ArribaRESUMEN
The small heat shock protein 20 (HSPB6) emerges as a potential upstream mediator of autophagy. Although autophagy is linked to several clinical disorders, how HSPB6 and autophagy are regulated in the setting of heart failure (HF) remains unknown. The goal of this study was to assess the activation of the HSPB6 and its association with other well-established autophagy markers in central and peripheral tissues from a preclinical Ossabaw swine model of cardiometabolic HF induced by Western diet and chronic cardiac pressure overload. We hypothesized HSPB6 would be activated in central and peripheral tissues, stimulating autophagy. We found that autophagy in the heart is interrupted at various stages of the process in a chamber-specific manner. Protein levels of HSPB6, Beclin 1, and p62 are increased in the right ventricle, whereas only HSPB6 was increased in the left ventricle. Unlike the heart, samples from the triceps brachii long head showed only an increase in the protein level of p62, highlighting interesting central versus peripheral differences in autophagy regulation. In the right coronary artery, total HSPB6 protein expression was decreased and associated with an increase in LC3B-II/LC3B-I ratio, demonstrating a different mechanism of autophagy dysregulation in the coronary vasculature. Thus, contrary to our hypothesis, activation of HSPB6 was differentially regulated in a tissue-specific manner and observed in parallel with variable states of autophagy markers assessed by protein levels of LC3B, p62, and Beclin 1. Our data provide insight into how the HSPB6/autophagy axis is regulated in a preclinical swine model with potential relevance to heart failure with preserved ejection fraction.NEW & NOTEWORTHY Our study shows that the activation of HSPB6 is tissue specific and associated with variable states of downstream markers of autophagy in a unique preclinical swine model of cardiometabolic HF with potential relevance to HFpEF. These findings suggest that targeted approaches could be an important consideration regarding the development of drugs aimed at this intracellular recycling process.
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Autofagia , Proteínas del Choque Térmico HSP20/metabolismo , Insuficiencia Cardíaca/metabolismo , Síndrome Metabólico/metabolismo , Animales , Beclina-1/genética , Beclina-1/metabolismo , Vasos Coronarios/metabolismo , Femenino , Proteínas del Choque Térmico HSP20/genética , Insuficiencia Cardíaca/etiología , Síndrome Metabólico/complicaciones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , PorcinosRESUMEN
Small heat shock proteins (sHSPs) increase stress tolerance in a wide variety of organisms and enable them to endure changes in their environment. However, the molecular mechanism by which sHSPs protect plants against cold stress is unknown. Here, the sHSP of tomato named SlHSP17.7 (Solyc06g076540.1.1) has the characteristic of low temperature induced expression in BL21(DE3) E. coli and a molecular chaperone function in vitro. Overexpression of SlHSP17.7 showed a tolerant response to cold stress treatment due to an induce intracellular sucrose and less accumulation of ROS. Yeast two-hybrid assays showed that SlHSP17.7 is a binding partner of the cation/Ca2+ exchanger (SlCCX1-like; Solyc07g006370.1.1). This interaction was confirmed by pull down and bimolecular fluorescence complementation (BiFC) assays. High SlHSP17.7 and low SlCCX1-like levels alleviated programed cell death (PCD) under cold stress. Thus, SlHSP17.7 might be a cofactor of SlCCX1-like targeting endoplasmic reticulum (ER) membrane proteins, retaining intracellular Ca2+ homeostasis, and decreasing cold stress sensitivity. These findings provide a sound basis for genetic engineering of cold stress tolerance in tomato.
Asunto(s)
Respuesta al Choque por Frío/genética , Proteínas del Choque Térmico HSP20/genética , Proteínas de Plantas/genética , Solanum lycopersicum/fisiología , Proteínas del Choque Térmico HSP20/metabolismo , Solanum lycopersicum/genética , Filogenia , Proteínas de Plantas/metabolismo , TermotoleranciaRESUMEN
Charcot-Marie-Tooth (CMT) disease is major hereditary neuropathy. CMT has been linked to mutations in a range of proteins, including the small heat shock protein HspB1. Here we review the properties of several HspB1 mutants associated with CMT. In vitro, mutations in the N-terminal domain lead to a formation of larger HspB1 oligomers when compared with the wild-type (WT) protein. These mutants are resistant to phosphorylation-induced dissociation and reveal lower chaperone-like activity than the WT on a range of model substrates. Mutations in the α-crystallin domain lead to the formation of yet larger HspB1 oligomers tending to dissociate at low protein concentration and having variable chaperone-like activity. Mutations in the conservative IPV motif within the C-terminal domain induce the formation of very large oligomers with low chaperone-like activity. Most mutants interact with a partner small heat shock protein, HspB6, in a manner different from that of the WT protein. The link between the altered physico-chemical properties and the pathological CMT phenotype is a subject of discussion. Certain HspB1 mutations appear to have an effect on cytoskeletal elements such as intermediate filaments and/or microtubules, and by this means damage the axonal transport. In addition, mutations of HspB1 can affect the metabolism in astroglia and indirectly modulate the viability of motor neurons. While the mechanisms of pathological mutations in HspB1 are likely to vary greatly across different mutations, further in vitro and in vivo studies are required for a better understanding of the CMT disease at molecular level.
Asunto(s)
Astrocitos/metabolismo , Enfermedad de Charcot-Marie-Tooth/metabolismo , Proteínas de Choque Térmico , Chaperonas Moleculares , Neuronas Motoras/metabolismo , Astrocitos/patología , Proteínas del Choque Térmico HSP20/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/fisiología , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/fisiología , Neuronas Motoras/patología , Mutación , Dominios Proteicos/genética , ProteostasisRESUMEN
Small heat shock proteins (shsps) are conserved across invertebrate species. They are implicated in the modulation of various biological processes, such as immune responses, abiotic stress tolerance metamorphosis, and embryonic development. Herein, we identified a heat shock protein 20 from the red swamp crayfish, Procambarus clarkii (named as Pc-Hsp20), and performed in vivo studies to elucidate its physiological functions in the innate immunity. The open reading frame of Pc-Hsp20 was 609 base pair, encoding a protein of 202 amino acid residues with a hsp20/alpha crystallin family domain. Pc-Hsp20 was ubiquitously expressed in various tissues; however, it was highest in the hepatopancreas. The challenge with immune elicitors remarkably enhanced the transcript level of Pc-Hsp20 in the hepatopancreas when compared with the control. Administration of double-stranded RNA could significantly reduce expression of the Pc-Hsp20 mRNAs, and most of the immune-related genes expression enhanced with a variable concentration in the hepatopancreas. Altogether, these results suggest that Pc-Hsp20 may participate in innate immunity against microbial pathogens.
Asunto(s)
Proteínas de Artrópodos/genética , Astacoidea/inmunología , Proteínas del Choque Térmico HSP20/genética , Hepatopáncreas/fisiología , Infecciones/inmunología , Animales , Proteínas de Artrópodos/metabolismo , Clonación Molecular , Proteínas del Choque Térmico HSP20/metabolismo , Inmunidad Innata , Filogenia , Dominios Proteicos/genética , ARN Bicatenario/inmunología , Alineación de Secuencia , Estrés Fisiológico , Transcriptoma , alfa-Cristalinas/genéticaRESUMEN
Adenylyl cyclases (ACs) convert ATP into the classical second messenger cyclic adenosine monophosphate (cAMP). Cardiac ACs, specifically AC5, AC6, and AC9, regulate cAMP signaling controlling functional outcomes such as heart rate, contractility and relaxation, gene regulation, stress responses, and glucose and lipid metabolism. With so many distinct functional outcomes for a single second messenger, the cell creates local domains of cAMP signaling to correctly relay signals. Targeting of ACs to A-kinase anchoring proteins (AKAPs) not only localizes ACs, but also places them within signaling nanodomains, where cAMP levels and effects can be highly regulated. Here we will discuss the recent work on the structure, regulation and physiological functions of AC9 in the heart, where it accounts for <3% of total AC activity. Despite the small contribution of AC9 to total cardiac cAMP production, AC9 binds and regulates local PKA phosphorylation of Yotiao-IKs and Hsp20, demonstrating a role for nanometric targeting of AC9.
Asunto(s)
Adenilil Ciclasas/metabolismo , Miocardio/enzimología , Nanoestructuras , Proteínas de Anclaje a la Quinasa A/metabolismo , Adenilil Ciclasas/química , Animales , Sitios de Unión , AMP Cíclico/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas del Choque Térmico HSP20/metabolismo , Humanos , Fosforilación , Conformación Proteica , Dominios ProteicosRESUMEN
Regulatory 14-3-3 proteins interact with a plethora of phosphorylated partner proteins, however 14-3-3 complexes feature intrinsically disordered regions and often a transient type of interactions making structural studies difficult. Here we engineer and examine a chimera of human 14-3-3 tethered to a nearly complete partner HSPB6 which is phosphorylated by protein kinase A (PKA). HSPB6 includes a long disordered N-terminal domain (NTD), a phosphorylation motif around Ser16, and a core α-crystallin domain (ACD) responsible for dimerisation. The chosen design enables an unstrained binding of pSer16 in each 1433 subunit and secures the correct 2:2 stoichiometry. Differential scanning calorimetry, limited proteolysis and small-angle X-ray scattering (SAXS) support the proper folding of both the 14-3-3 and ACD dimers within the chimera, and indicate that the chimera retains the overall architecture of the native complex of 14-3-3 and phosphorylated HSPB6 that has recently been resolved using crystallography. At the same time, the SAXS data highlight the weakness of the secondary interface between the ACD dimer and the C-terminal lobe of 14-3-3 observed in the crystal structure. Applied to other 14-3-3 complexes, the chimeric approach may help probe the stability and specificity of secondary interfaces for targeting them with small molecules in the future.