RESUMO
Apolipoprotein B (ApoB) is the primary component of atherogenic lipoproteins, which transport serum fats and cholesterol. Therefore elevated levels of circulating ApoB are a primary risk factor for cardiovascular disease. During ApoB biosynthesis in the liver and small intestine under nutrient-rich conditions, ApoB cotranslationally translocates into the endoplasmic reticulum (ER) and is lipidated and ultimately secreted. Under lipid-poor conditions, ApoB is targeted for ER-associated degradation (ERAD). Although prior work identified select chaperones that regulate ApoB biogenesis, the contributions of cytoplasmic Hsp40s are undefined. To this end, we screened ApoB-expressing yeast and determined that a class A ER-associated Hsp40, Ydj1, associates with and facilitates the ERAD of ApoB. Consistent with these results, a homologous Hsp40, DNAJA1, functioned similarly in rat hepatoma cells. DNAJA1-deficient cells also secreted hyperlipidated lipoproteins in accordance with attenuated ERAD. In contrast to the role of DNAJA1 during ERAD, DNAJB1-a class B Hsp40-helped stabilize ApoB. Depletion of DNAJA1 and DNAJB1 also led to opposing effects on ApoB ubiquitination. These data represent the first example in which different Hsp40s exhibit disparate effects during regulated protein biogenesis in the ER and highlight distinct roles that chaperones can play on a single ERAD substrate.
Assuntos
Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Apolipoproteína B-100/metabolismo , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/fisiologia , Lipoproteínas/metabolismo , Chaperonas Moleculares/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , UbiquitinaçãoRESUMO
Hsp70s comprise a deeply conserved chaperone family that has a central role in maintaining protein homeostasis. In humans, Hsp70 client specificity is provided by 49 different co-factors known as J domain proteins (JDPs). However, the cellular function and client specificity of JDPs have largely remained elusive. We have combined affinity purification-mass spectrometry (AP-MS) and proximity-dependent biotinylation (BioID) to characterize the interactome of all human JDPs and Hsp70s. The resulting network suggests specific functions for many uncharacterized JDPs, and we establish a role of conserved JDPs DNAJC9 and DNAJC27 in histone chaperoning and ciliogenesis, respectively. Unexpectedly, we find that the J domain of DNAJC27 but not of other JDPs can fully replace the function of endogenous DNAJC27, suggesting a previously unappreciated role for J domains themselves in JDP specificity. More broadly, our work expands the role of the Hsp70-regulated proteostasis network and provides a platform for further discovery of JDP-dependent functions.
Assuntos
Proteínas de Choque Térmico HSP40/fisiologia , Proteínas de Choque Térmico HSP70/fisiologia , Domínios e Motivos de Interação entre Proteínas/fisiologia , Células HEK293 , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Células HeLa , Humanos , Chaperonas Moleculares/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
From biosynthesis to assembly into nucleosomes, histones are handed through a cascade of histone chaperones, which shield histones from non-specific interactions. Whether mechanisms exist to safeguard the histone fold during histone chaperone handover events or to release trapped intermediates is unclear. Using structure-guided and functional proteomics, we identify and characterize a histone chaperone function of DNAJC9, a heat shock co-chaperone that promotes HSP70-mediated catalysis. We elucidate the structure of DNAJC9, in a histone H3-H4 co-chaperone complex with MCM2, revealing how this dual histone and heat shock co-chaperone binds histone substrates. We show that DNAJC9 recruits HSP70-type enzymes via its J domain to fold histone H3-H4 substrates: upstream in the histone supply chain, during replication- and transcription-coupled nucleosome assembly, and to clean up spurious interactions. With its dual functionality, DNAJC9 integrates ATP-resourced protein folding into the histone supply pathway to resolve aberrant intermediates throughout the dynamic lives of histones.
Assuntos
Proteínas de Choque Térmico HSP40/metabolismo , Chaperonas de Histonas/metabolismo , Linhagem Celular Tumoral , Cromatina , Montagem e Desmontagem da Cromatina , Replicação do DNA , Proteínas de Choque Térmico HSP40/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Células HeLa , Chaperonas de Histonas/fisiologia , Histonas/metabolismo , Humanos , Componente 2 do Complexo de Manutenção de Minicromossomo/metabolismo , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Nucleossomos , Ligação Proteica , Proteômica/métodosRESUMO
The transmembrane Hsp40 DNAJB12 and cytosolic Hsp70 cooperate on the endoplasmic reticulum's (ER) cytoplasmic face to facilitate the triage of nascent polytopic membrane proteins for folding versus degradation. N1303K is a common mutation that causes misfolding of the ion channel CFTR, but unlike F508del-CFTR, biogenic and functional defects in N1303K-CFTR are resistant to correction by folding modulators. N1303K is reported to arrest CFTR folding at a late stage after partial assembly of its N-terminal domains. N1303K-CFTR intermediates are clients of JB12-Hsp70 complexes, maintained in a detergent-soluble state, and have a relatively long 3-h half-life. ER-associated degradation (ERAD)-resistant pools of N1303K-CFTR are concentrated in ER tubules that associate with autophagy initiation sites containing WIPI1, FlP200, and LC3. Destabilization of N1303K-CFTR or depletion of JB12 prevents entry of N1303K-CFTR into the membranes of ER-connected phagophores and traffic to autolysosomes. In contrast, the stabilization of intermediates with the modulator VX-809 promotes the association of N1303K-CFTR with autophagy initiation machinery. N1303K-CFTR is excluded from the ER-exit sites, and its passage from the ER to autolysosomes does not require ER-phagy receptors. DNAJB12 operates in biosynthetically active ER microdomains to triage membrane protein intermediates in a conformation-specific manner for secretion versus degradation via ERAD or selective-ER-associated autophagy.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Degradação Associada com o Retículo Endoplasmático/fisiologia , Proteínas de Choque Térmico HSP40/metabolismo , Animais , Autofagossomos , Autofagia/fisiologia , Células COS , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Retículo Endoplasmático/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Dobramento de ProteínaRESUMO
Mutations in the DNAJB6 gene have been identified as rare causes of myofibrillar myopathies. However, the underlying pathophysiologica mechanisms remain elusive. DNAJB6 has two known isoforms, including the nuclear isoform DNAJB6a and the cytoplasmic isoform DNAJB6b, which was thought to be the pathogenic isoform. Here, we report a novel recessive mutation c.695_699del (p. Val 232 Gly fs*7) in the DNAJB6 gene, associated with an apparently recessively inherited late onset distal myofibrillar myopathy in a Chinese family. Notably, the novel mutation localizes to exon 9 and uniquely encodes DNAJB6a. We further identified that this mutation decreases the mRNA and protein levels of DNAJB6a and results in an age-dependent recessive toxic effect on skeletal muscle in knock-in mice. Moreover, the mutant DNAJB6a showed a dose-dependent anti-aggregation effect on polyglutamine-containing proteins in vitro. Taking together, these findings reveal the pathogenic role of DNAJB6a insufficiency in myofibrillar myopathies and expand upon the molecular spectrum of DNAJB6 mutations.
Assuntos
Miopatias Distais/genética , Proteínas de Choque Térmico HSP40/genética , Chaperonas Moleculares/genética , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Mutação , Miopatias Congênitas Estruturais/genética , Proteínas do Tecido Nervoso/genética , Idoso , Animais , Povo Asiático , Miopatias Distais/diagnóstico por imagem , Miopatias Distais/patologia , Miopatias Distais/fisiopatologia , Técnicas de Introdução de Genes , Células HEK293 , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP40/fisiologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/fisiologia , Miopatias Congênitas Estruturais/diagnóstico por imagem , Miopatias Congênitas Estruturais/patologia , Miopatias Congênitas Estruturais/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/fisiologia , FenótipoRESUMO
Chloroplast-targeted proteins are actively imported into chloroplasts via the machinery spanning the double-layered membranes of chloroplasts. While the key translocons at the outer (TOC) and inner (TIC) membranes of chloroplasts are defined, proteins that interact with the core components to facilitate pre-protein import are continuously being discovered. A DnaJ-like chaperone ORANGE (OR) protein is known to regulate carotenoid biosynthesis as well as plastid biogenesis and development. In this study, we found that OR physically interacts with several Tic proteins including Tic20, Tic40, and Tic110 in the classic TIC core complex of the chloroplast import machinery. Knocking out or and its homolog or-like greatly affects the import efficiency of some photosynthetic and non-photosynthetic pre-proteins. Consistent with the direct interactions of OR with Tic proteins, the binding efficiency assay revealed that the effect of OR occurs at translocation at the inner envelope membrane (i.e. at the TIC complex). OR is able to reduce the Tic40 protein turnover rate through its chaperone activity. Moreover, OR was found to interfere with the interaction between Tic40 and Tic110, and reduces the binding of pre-proteins to Tic110 in aiding their release for translocation and processing. Our findings suggest that OR plays a new and regulatory role in stabilizing key translocons and in facilitating the late stage of plastid pre-protein translocation to regulate plastid pre-protein import.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis , Proteínas de Choque Térmico HSP40/fisiologia , Transporte Proteico , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismoRESUMO
Cysteine string protein (CSP) was discovered by use of a synapse-specific, monoclonal antibody to screen a cDNA expression library in Drosophila. A vertebrate CSP homolog was later identified and shown to co-purify with synaptic vesicles. CSP-α is now recognized as a membrane constituent of many regulated secretory organelles. Knockout of the csp gene in Drosophila produced temperature-sensitive paralysis reflecting a loss of evoked (but not spontaneous) transmitter release. However, CSP's role in regulated exocytosis remains ambiguous. Fruit flies lacking the csp gene also exhibited nerve terminal degeneration as did mice deficient in the csp-α gene. This phenotype has been ascribed to the depletion of a functional pool of the t-SNARE, SNAP-25. However, recent studies showing that an endosomal pool of CSP-α contributes to a novel, protein-export pathway argues that CSP's role in neurodegeneration is more complex. Clients of this later pathway include tau and α-synuclein, proteins linked to neurodegeneration. Additionally, mutations in the csp-α gene cause an adult-onset, neuronal ceroid lipofuscinosis and diminished CSP-α expression is an early event in Alzheimer's disease. Collectively, these findings indicate that much remains to be learned about the role of CSPs in cellular secretory pathways and human disease.
Assuntos
Proteínas de Choque Térmico HSP40/fisiologia , Proteínas de Membrana/fisiologia , Doenças Neurodegenerativas/metabolismo , Animais , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Doenças Neurodegenerativas/genéticaRESUMO
Maintenance of synaptic function across ageing is vital in sustaining cognitive function. Synaptic dysfunction is a key part of the pathophysiology of a number of neurodegenerative diseases. The synaptic co-chaperone, cysteine-string protein (CSP), is important for synaptic maintenance and function in Drosophila, mice and humans, and disruption of CSP results in synaptic degeneration. We sought to characterise synaptic ageing in Caenorhabditis elegans upon genetic disruption of CSP. To do this, we focused on the worms' neuromuscular junctions, which are the best characterised synapse. CSP mutant worms did not display reduced lifespan or any neuromuscular-dependent behavioural deficits across ageing. Pharmacological interrogation of the neuromuscular synapse of CSP mutant animals showed no sign of synaptic dysfunction even at advanced age. Lastly, patch clamp analysis of neuromuscular transmission across ageing in wild-type and CSP mutant animals revealed no obvious CSP-dependent deficits. Electrophysiological spontaneous postsynaptic current analysis reinforced pharmacological observations that the C. elegans neuromuscular synapse increases in strength during early ageing and remains relatively intact in old, immotile worms. Taken together, this study shows that surprisingly, despite disruption of CSP in other animals having severe synaptic phenotypes, CSP does not seem to be important for maintenance of the neuromuscular junction across ageing in C. elegans.
Assuntos
Envelhecimento , Proteínas de Choque Térmico HSP40/fisiologia , Proteínas de Membrana/fisiologia , Junção Neuromuscular/fisiologia , Animais , Caenorhabditis elegans/genética , Proteínas de Choque Térmico HSP40/genética , Longevidade , Proteínas de Membrana/genética , Mutação , Proteínas do Tecido Nervoso/metabolismo , Junção Neuromuscular/metabolismo , Técnicas de Patch-Clamp , Sinapses/fisiologia , Transmissão Sináptica/fisiologiaRESUMO
Nuclear factor kappa B (NF-κB) controls the transcription of various genes in response to immune stimuli. Our previous study revealed that the Droj2/DNAJA3 cochaperone contributes to the NF-κB pathway in Drosophila and humans. In general, the cochaperone is associated with the 70-kDa heat shock protein (HSP70) chaperone and the complex supports the folding of diverse target proteins. The cochaperone/chaperone functions in the NF-κB pathway, however, are not clearly understood. Here, we report that HSP70 proteins are involved in activating canonical NF-κB signaling during immune responses. In human cultured cells, HSP70 inhibitor destabilized the IKKß/IκBα/NF-κB p65 complex and dampened the phosphorylation of NF-κB p65 in response to flagellin stimulation. We identified HSPA1A and HSPA8 as the HSP70 family proteins that physically interact with DNAJA3, and established their requirement for the phosphorylation of NF-κB p65. Furthermore, as in flies with knockdown of Droj2, flies with knockdown of Hsc70-4, a Drosophila homolog of HSPA8, were more susceptible to infection. Our results suggest that the chaperone/cochaperone complex regulates NF-κB immune signaling in an evolutionarily conserved manner.
Assuntos
Proteínas de Choque Térmico HSP40/fisiologia , Proteínas de Choque Térmico HSP70/fisiologia , Imunidade , Chaperonas Moleculares/fisiologia , NF-kappa B/metabolismo , Animais , Células Cultivadas , Drosophila , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Chaperonas Moleculares/imunologia , NF-kappa B/imunologia , FosforilaçãoRESUMO
The human ether-a-go-go-related gene (hERG) encodes a voltage-gated potassium channel that controls repolarization of cardiac action potentials. Accumulating evidence suggests that most disease-related hERG mutations reduce the function of the channel by disrupting protein biogenesis of the channel in the endoplasmic reticulum (ER). However, the molecular mechanism underlying the biogenesis of ERG K+ channels is largely unknown. By forward genetic screening, we identified an ER-located chaperone CNX-1, the worm homologue of mammalian chaperone Calnexin, as a critical regulator for the protein biogenesis of UNC-103, the ERG-type K+ channel in Caenorhabditis elegans Loss-of-function mutations of cnx-1 decreased the protein level and current density of the UNC-103 K+ channel and suppressed the behavioral defects caused by a gain-of-function mutation in unc-103 Moreover, CNX-1 facilitated tetrameric assembly of UNC-103 channel subunits in a liposome-assisted cell-free translation system. Further studies showed that CNX-1 act in parallel to DNJ-1, another ER-located chaperone known to regulate maturation of UNC-103 channels, on controlling the protein biogenesis of UNC-103. Importantly, Calnexin interacted with hERG proteins in the ER in HEK293T cells. Deletion of calnexin reduced the expression and current densities of endogenous hERG K+ channels in SH-SY5Y cells. Collectively, we reveal an evolutionarily conserved chaperone CNX-1/Calnexin controlling the biogenesis of ERG-type K+ channels.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Proteínas de Ligação ao Cálcio/fisiologia , Retículo Endoplasmático/metabolismo , Canais de Potássio Éter-A-Go-Go/biossíntese , Proteínas de Choque Térmico HSP40/fisiologia , Canais de Potássio/biossíntese , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ligação ao Cálcio/genética , Calnexina/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP40/genética , Humanos , CamundongosRESUMO
Hepatitis C is a disease caused by the hepatitis C virus (HCV), and an estimated 3% of the world population is infected with the virus. During replication, HCV interacts with several cellular proteins. Studies have shown that several heat shock proteins (HSPs) have an altered expression profile in the presence of the virus, and some HSPs interact directly with HCV proteins. In the present study, we evaluated the expression levels of heat shock proteins in vitro in the presence and absence of HCV. The differential expression of 84 HSPs and chaperones was observed using a qPCR array, comparing HCV uninfected and infected Huh7.5 cells. To validate qPCR array, the differentially expressed genes were tested by real-time PCR in three different HCV models: subgenomic HCV replicon cells (SGR-JFH-1), JFH-1 infected cells (both genotype 2a) and subgenomic S52 cells (genotype 3). The HSPB8 gene showed increased expression in all three viral models. We silenced HSPB8 expression and observed an increase in viral replication. In contrast, when we increased the expression of HSPB8, a decrease in the HCV replication rate was observed. The same procedure was adopted for DNAJC5B, and HCV showed a similar replication pattern as that observed for HSPB8. These results suggest that HSPB8 may act as an intracellular factor against hepatitis C virus replication and that DNAJC5B has the same function, with more relevant results for genotype 3. We also evaluated the direct interactions between HCV and HSP proteins, and the IP experiments showed that the HCV NS4B protein interacts with HSPB8. These results contribute to a better understanding of the mechanisms involved in HCV replication.
Assuntos
Proteínas de Choque Térmico HSP40/fisiologia , Proteínas de Choque Térmico/fisiologia , Hepacivirus/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Linhagem Celular Tumoral , Humanos , Chaperonas Moleculares , Reação em Cadeia da Polimerase em Tempo Real , Replicação ViralRESUMO
Tsetse flies (Glossina spp.) are the sole vectors of the protozoan parasites of the genus Trypanosoma, the causative agents of African Trypanosomiasis. Species of Glossina differ in vector competence and Glossina morsitans morsitans is associated with transmission of Trypanosoma brucei rhodesiense, which causes an acute and often fatal form of African Trypanosomiasis. Heat shock proteins are evolutionarily conserved proteins that play critical roles in proteostasis. The activity of heat shock protein 70 (Hsp70) is regulated by interactions with its J-protein (Hsp40) co-chaperones. Inhibition of these interactions are emerging as potential therapeutic targets. The assembly and annotation of the G. m. morsitans genome provided a platform to identify and characterize the Hsp70s and J-proteins, and carry out an evolutionary comparison to its well-studied eukaryotic counterparts, Drosophila melanogaster and Homo sapiens, as well as Stomoxys calcitrans, a comparator species. In our study, we identified 9 putative Hsp70 proteins and 37 putative J-proteins in G. m. morsitans. Phylogenetic analyses revealed three evolutionarily distinct groups of Hsp70s, with a closer relationship to orthologues from its blood-feeding dipteran relative Stomoxys calcitrans. G. m. morsitans also lacked the high number of heat inducible Hsp70s found in D. melanogaster. The potential localisations, functions, domain organisations and Hsp70/J-protein partnerships were also identified. A greater understanding of the heat shock 70 (Hsp70) and J-protein (Hsp40) families in G. m. morsitans could enhance our understanding of the cell biology of the tsetse fly.
Assuntos
Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP70/genética , Insetos Vetores/genética , Moscas Tsé-Tsé/genética , Animais , Drosophila melanogaster/genética , Redes Reguladoras de Genes , Genoma de Inseto , Proteínas de Choque Térmico HSP40/fisiologia , Proteínas de Choque Térmico HSP70/fisiologia , Interações Hospedeiro-Parasita/genética , Insetos Vetores/metabolismo , Família Multigênica , Filogenia , Transdução de Sinais/genética , Tripanossomíase Africana/transmissão , Moscas Tsé-Tsé/parasitologiaRESUMO
OBJECTIVE: To investigate the presence of interactions between DNAJB13 and HK1. METHODS: The open reading frame of Dnajb13 gene was amplified from mouse testis cDNA by PCR. The PCR products were then inserted into pGEX-4T-1 vector after double digestion and identified by sequencing. The recombinant plasmids were transformated into competent DH5a cells, and the fusion protein was expressed with IPTG induction. SDS-PAGE Coomassie brilliant blue staining and Western blot analysis were used to detect the fusion protein expression. The protein precipitated by GST-DNAJB13 in GST pull down assay was detected by Western blotting. RESULTS: The recombinant plasmid pGEX-4T-1-Dnajb13 was successfully constructed and verified. E.coli transformed with the recombinant plasmid expressed abundant fusion protein. GST pull down assay showed interactions between DNAJB13 and HK1. CONCLUSION: DNAJB13 interacts with HK1 in mouse testis and probably participates in spermatogenesis and the regulation of sperm motility.
Assuntos
Proteínas de Choque Térmico HSP40/fisiologia , Hexoquinase/fisiologia , Plasmídeos , Proteínas Recombinantes , Espermatogênese/fisiologia , Animais , Proteínas Reguladoras de Apoptose , Western Blotting , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Camundongos , Chaperonas Moleculares , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão , Motilidade dos EspermatozoidesRESUMO
Detergent-resistant, ubiquitinated and hyperphosphorylated Tar DNA binding protein 43 (TDP-43, encoded by TARDBP) neuronal cytoplasmic inclusions are the pathological hallmark in â¼95% of amyotrophic lateral sclerosis and â¼60% of frontotemporal lobar degeneration cases. We sought to explore the role for the heat shock response in the clearance of insoluble TDP-43 in a cellular model of disease and to validate our findings in transgenic mice and human amyotrophic lateral sclerosis tissues. The heat shock response is a stress-responsive protective mechanism regulated by the transcription factor heat shock factor 1 (HSF1), which increases the expression of chaperones that refold damaged misfolded proteins or facilitate their degradation. Here we show that manipulation of the heat shock response by expression of dominant active HSF1 results in a dramatic reduction of insoluble and hyperphosphorylated TDP-43 that enhances cell survival, whereas expression of dominant negative HSF1 leads to enhanced TDP-43 aggregation and hyperphosphorylation. To determine which chaperones were mediating TDP-43 clearance we over-expressed a range of heat shock proteins (HSPs) and identified DNAJB2a (encoded by DNAJB2, and also known as HSJ1a) as a potent anti-aggregation chaperone for TDP-43. DNAJB2a has a J domain, allowing it to interact with HSP70, and ubiquitin interacting motifs, which enable it to engage the degradation of its client proteins. Using functionally deleted DNAJB2a constructs we demonstrated that TDP-43 clearance was J domain-dependent and was not affected by ubiquitin interacting motif deletion or proteasome inhibition. This indicates that TDP-43 is maintained in a soluble state by DNAJB2a, leaving the total levels of TDP-43 unchanged. Additionally, we have demonstrated that the levels of HSF1 and heat shock proteins are significantly reduced in affected neuronal tissues from a TDP-43 transgenic mouse model of amyotrophic lateral sclerosis and patients with sporadic amyotrophic lateral sclerosis. This implies that the HSF1-mediated DNAJB2a/HSP70 heat shock response pathway is compromised in amyotrophic lateral sclerosis. Defective refolding of TDP-43 is predicted to aggravate the TDP-43 proteinopathy. The finding that the pathological accumulation of insoluble TDP-43 can be reduced by the activation of HSF1/HSP pathways presents an exciting opportunity for the development of novel therapeutics.
Assuntos
Esclerose Lateral Amiotrófica/fisiopatologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteínas de Choque Térmico/metabolismo , Fatores de Transcrição/fisiologia , Esclerose Lateral Amiotrófica/metabolismo , Animais , Sobrevivência Celular/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP40/fisiologia , Fatores de Transcrição de Choque Térmico , Humanos , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Fosforilação , Fatores de Transcrição/biossíntese , Ubiquitina/metabolismoRESUMO
Chaperones, nucleosome remodeling complexes, and histone acetyltransferases have been implicated in nucleosome disassembly at promoters of particular yeast genes, but whether these cofactors function ubiquitously, as well as the impact of nucleosome eviction on transcription genome-wide, is poorly understood. We used chromatin immunoprecipitation of histone H3 and RNA polymerase II (Pol II) in mutants lacking single or multiple cofactors to address these issues for about 200 genes belonging to the Gcn4 transcriptome, of which about 70 exhibit marked reductions in H3 promoter occupancy on induction by amino acid starvation. Examining four target genes in a panel of mutants indicated that SWI/SNF, Gcn5, the Hsp70 cochaperone Ydj1, and chromatin-associated factor Yta7 are required downstream from Gcn4 binding, whereas Asf1/Rtt109, Nap1, RSC, and H2AZ are dispensable for robust H3 eviction in otherwise wild-type cells. Using ChIP-seq to interrogate all 70 exemplar genes in single, double, and triple mutants implicated Gcn5, Snf2, and Ydj1 in H3 eviction at most, but not all, Gcn4 target promoters, with Gcn5 generally playing the greatest role and Ydj1 the least. Remarkably, these three cofactors cooperate similarly in H3 eviction at virtually all yeast promoters. Defective H3 eviction in cofactor mutants was coupled with reduced Pol II occupancies for the Gcn4 transcriptome and the most highly expressed uninduced genes, but the relative Pol II levels at most genes were unaffected or even elevated. These findings indicate that nucleosome eviction is crucial for robust transcription of highly expressed genes but that other steps in gene activation are more rate-limiting for most other yeast genes.
Assuntos
Adenosina Trifosfatases/fisiologia , Proteínas de Choque Térmico HSP40/fisiologia , Histona Acetiltransferases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Fatores de Transcrição/fisiologia , Ativação Transcricional , Epigênese Genética , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Nucleossomos/genética , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Saccharomyces cerevisiae/enzimologia , TranscriptomaRESUMO
BACKGROUND: Wound healing depends on a well-balanced regulation of inflammation and angiogenesis. In chronic wounds the healing process is disturbed and inflammation persists. Regulation of wound closure is controlled by transmembrane and extracellular proteins, the folding and maturation of which occur in the endoplasmic reticulum (ER) by ER-resident chaperone machinery. OBJECTIVES: To study the role of the ER-resident chaperones BiP/Grp78, its cochaperone Mdg1/ERdJ4, and Grp94 in chronic, nonhealing wounds. METHODS: Immunohistochemical staining of these chaperones in individual human biopsies and investigation of the possible role of BiP and Mdg1 in endothelial cells, focusing on their inflammatory response and angiogenic potential. RESULTS: In all chronic wounds investigated, the levels of these ER-resident chaperones were elevated in endothelial cells and leucocytes. The proangiogenic role of BiP has been shown in tumour growth studies before and was confirmed in this study. Proangiogenic activity of the cochaperone Mdg1 has been postulated before but could not be confirmed in this study. The chemokine tumour necrosis factor (TNF)-α was shown to trigger the presentation of proinflammatory adhesion molecules and the release of proinflammatory cytokines. Here we show that TNF-α does not affect endogenous chaperone levels, but that the ER-resident chaperones BiP and Mdg1 modulate the cellular TNF-α-induced proinflammatory response. CONCLUSIONS: According to the presented data we assume that in chronic wounds upregulated levels of ER-resident chaperones might contribute to persistent inflammation in chronic wounds. Therapies to downregulate chaperone levels might provide a tool that switches the imbalanced chronic wound microenvironment from inflammation to healing.
Assuntos
Chaperonas Moleculares/fisiologia , Cicatrização/fisiologia , Células Cultivadas , Doença Crônica , Regulação para Baixo/fisiologia , Retículo Endoplasmático/fisiologia , Chaperona BiP do Retículo Endoplasmático , Células Endoteliais/fisiologia , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP40/fisiologia , Proteínas de Choque Térmico/metabolismo , Humanos , Inflamação/fisiopatologia , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Chaperonas Moleculares/metabolismo , Neovascularização Fisiológica/fisiologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Viruses hijack host machineries for replicating themselves efficiently. Host protein quality control machineries (QC) not only assist protein folding to form bona fide proteins with active functions but also get rid of un/misfolded proteins via degradation to maintain the protein homeostasis. Previous studies have reported that viruses utilize QC at various steps for their lifecycles. Recently we defined Hsp70s and their cochaperones, DnaJs functions on Dengue lifecycle. Here we summarize the significance of QC on Dengue virus.
Assuntos
Vírus da Dengue/crescimento & desenvolvimento , Vírus da Dengue/metabolismo , Vírus da Dengue/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP70/fisiologia , Estágios do Ciclo de Vida/genética , Estágios do Ciclo de Vida/fisiologia , Replicação Viral/genética , Replicação Viral/fisiologia , Animais , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP40/fisiologia , Homeostase , Humanos , Camundongos , Chaperonas Moleculares , Dobramento de Proteína , Modificação Traducional de ProteínasRESUMO
Epithelial-mesenchymal transition (EMT) is a developmental program, which is associated with hepatocellular carcinoma (HCC) development and progression. DNAJC6 (DNA/HSP40 homolog subfamily C member 6) encodes auxilin, which is responsible for juvenile Parkinsonism with phenotypic variability. However, the role of DNAJC6 in HCC development and progression is limited. Here, we report that DNAJC6 is up-regulated in HCC tissues and up-regulation of DNAJC6 expression predicts poor outcome in patients with HCC. Furthermore, overexpression of DNAJC6 enhances the ability for acquisition of mesenchymal traits, enhanced cell proliferation and invasion. DNAJC6 positively regulated expression of EMT-related transcription factor, also activating transforming growth factor ß (TGF-ß) pathway to contribute to EMT. Our findings demonstrated an important function of DNAJC6 in the progression of HCC by induction of EMT, and they implicate DNAJC6 as a marker of poor outcome in HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP40/fisiologia , Neoplasias Hepáticas/metabolismo , Idoso , Animais , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Proteínas de Choque Térmico HSP40/genética , Humanos , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Fator de Crescimento Transformador beta/metabolismo , Resultado do Tratamento , Regulação para CimaRESUMO
Microsporidia are obligate intracellular parasites, and a derivative of fungi, which harbor a rigid spore wall to resist adverse environmental pressures. The spore wall protein, which is thought to be the first and direct protein interacting with the host cell, may play a key role in the process of microsporidia infection. In this study, we report a protein, NbHSWP11, with a dnaJ domain. The protein also has 6 heparin-binding motifs which are known to interact with extracellular glycosaminoglycans. Syntenic analysis indicated that gene loci of Nbhswp11 are conserved and syntenic between Nosema bombycis and Nosema ceranae. Phylogenetic tree analysis showed that Nbhswp11 clusters with fungal dnaJ proteins and has 98% identity with an N. bombycis dnaJ protein. Nbhswp11 was transcribed throughout the entire life stages, and gradually increased during 1-7 days, in a silkworm that was infected by N. bombycis, as determined by reverse-transcription PCR (RT-PCR). The recombinant protein NbHSWP11 (rSWP11-HIS) was obtained and purified using gene cloning and prokaryotic expression. Western blotting analysis displayed NbHSWP11 expressed in the total mature spore proteins and spore coat proteins. Indirect immunofluorescence assay revealed NbHSWP11 located at the spore wall of mature spores and the spore coats. Furthermore, immune electron microscopy showed that NbHSWP11 localized in the cytoplasm of the sporont. Within the developmental process of N. bombycis, a portion of NbHSWP11 is targeted to the spore wall of sporoblasts and mature spores. However, most of NbHSWP11 distributes on the membraneous structures of the sporoblast and mature spore. In addition, using a host cell binding assay, native protein NbHSWP11 in the supernatant of total soluble mature spore proteins is shown to bind to the host cell BmE surface. Finally, an antibody blocking assay showed that purified rabbit antibody of NbHSWP11 inhibits spore adherence and decreases the adherence rate of spores by 20% compared to untreated spores. Collectively, the present results suggest that NbHSWP11 is involved in host cell adherence in vitro. Therefore NbHSWP11, which has a dnaJ domain, may modulate protein assembly, disassembly, and translocation in N. bombycis.
Assuntos
Proteínas Fúngicas/fisiologia , Proteínas de Membrana/fisiologia , Nosema/química , Sequência de Aminoácidos , Animais , Anticorpos Bloqueadores/imunologia , Bombyx/citologia , Bombyx/embriologia , Bombyx/microbiologia , Adesão Celular , Linhagem Celular , Parede Celular/química , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/imunologia , Proteínas de Choque Térmico HSP40/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Nosema/genética , Nosema/imunologia , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esporos Fúngicos/químicaRESUMO
TDP-43 aggregation in the cytoplasm or nucleus is a key feature of the pathology of amyotrophic lateral sclerosis and frontotemporal dementia and is observed in numerous other neurodegenerative diseases, including Alzheimer's disease. Despite this fact, the inciting events leading to TDP-43 aggregation remain unclear. We observed that endogenous TDP-43 undergoes reversible aggregation in the nucleus after the heat shock and that this behavior is mediated by the C-terminal prion domain. Substitution of the prion domain from TIA-1 or an authentic yeast prion domain from RNQ1 into TDP-43 can completely recapitulate heat shock-induced aggregation. TDP-43 is constitutively bound to members of the Hsp40/Hsp70 family, and we found that heat shock-induced TDP-43 aggregation is mediated by the availability of these chaperones interacting with the inherently disordered C-terminal prion domain. Finally, we observed that the aggregation of TDP-43 during heat shock led to decreased binding to hnRNPA1, and a change in TDP-43 RNA-binding partners suggesting that TDP-43 aggregation alters its function in response to misfolded protein stress. These findings indicate that TDP-43 shares properties with physiologic prions from yeast, in that self-aggregation is mediated by a Q/N-rich disordered domain, is modulated by chaperone proteins and leads to altered function of the protein. Furthermore, they indicate that TDP-43 aggregation is regulated by chaperone availability, explaining the recurrent observation of TDP-43 aggregates in degenerative diseases of both the brain and muscle where protein homeostasis is disrupted.