Structural, thermodynamic and functional studies of human 71 kDa heat shock cognate protein (HSPA8/hHsc70)

Silva, Noeli Soares Melo; Rodrigues, Luiz Fernando de Camargo; Dores-Silva, Paulo R.; Montanari, Carlos Alberto; Ramos, Carlos Henrique Inácio; Barbosa, Leandro R.S.; Borges, Júlio César

doi:10.1016/j.bbapap.2021.140719

Cited by 11 publications

(11 citation statements)

References 92 publications

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“…6a ; one-sample t -test, p = 0.14 and p = 0.68), which is consistent with the NBD being denatured and hence making Cys17 amenable to reaction irrespective of other ligands. This conclusion is supported by a recent study reporting a urea denaturation curve of HSPA8 that showed it displayed two-domain folding behavior 33 . One domain, likely the NBD, had a C m around 1.5 M urea and the other domain a C m around 3.6 M urea.…”

Section: Resultssupporting

confidence: 70%

Hidden information on protein function in censuses of proteome foldedness

Cox

Nillegoda

et al. 2022

Nat Commun

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Methods that assay protein foldedness with proteomics have generated censuses of apparent protein folding stabilities in biological milieu. However, different censuses poorly correlate with each other. Here, we show that the reason for this is that methods targeting foldedness through monitoring amino acid sidechain reactivity also detect changes in conformation and ligand binding, which can be a substantial fraction of the data. We show that the reactivity of only one quarter of cysteine or methionine sidechains in proteins in a urea denaturation curve of mammalian cell lysate can be confidently explained by a two-state unfolding isotherm. Contrary to that expected from unfolding, up to one third of the cysteines decreased reactivity. These cysteines were enriched in proteins with functions relating to unfolded protein stress. One protein, chaperone HSPA8, displayed changes arising from ligand and cofactor binding. Unmasking this hidden information using the approaches outlined here should improve efforts to understand both folding and the remodeling of protein function directly in complex biological settings.

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Section: Resultssupporting

confidence: 70%

Hidden information on protein function in censuses of proteome foldedness

Cox

Nillegoda

et al. 2022

Nat Commun

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“…HSPA8 was the most downregulated DE gene [log2(FC) = -1.12] and also one core hub in PPI networks in this study. Located on chromosome 11q24.1, HSPA8 encodes the human soluble constitutive 71-kDa heat shock cognate protein, also known as Hsc70 ( 35 ). HSPA8 had been identified as the uncoating ATPase of clathrin-coated vesicles, which would disassemble the clathrin cages to modulate the clathrin-coated vesicle cycle and endocytic trafficking, acting as the essential late step in clathrin-mediated endocytosis ( 36 ).…”

Section: Discussionmentioning

confidence: 99%

Transcriptomic signatures responding to PKM2 activator TEPP-46 in the hyperglycemic human renal proximal epithelial tubular cells

Wang

Hao

et al. 2022

Front. Endocrinol.

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Pyruvate kinase M2 (PKM2), as the terminal and last rate-limiting enzyme of the glycolytic pathway, is an ideal enzyme for regulating metabolic phenotype. PKM2 tetramer activation has shown a protective role against diabetic kidney disease (DKD). However, the molecular mechanisms involved in diabetic tubular have not been investigated so far. In this study, we performed transcriptome gene expression profiling in human renal proximal tubular epithelial cell line (HK-2 cells) treated with 25 mM high D-glucose (HG) for 7 days before the addition of 10 μM TEPP-46, an activator of PKM2 tetramerization, for a further 1 day in the presence of HG. Afterwards, we analyzed the differentially expressed (DE) genes and investigated gene relationships based on weighted gene co-expression network analysis. The results showed that 2,902 DE genes were identified (adjusted P-value ≤ 0.05), where 2,509 DE genes (86.46%) were co-expressed in the key module. Four extremely downregulated DE genes (HSPA8, HSPA2, HSPA1B, and ARRB1) and three extremely upregulated DE genes (GADD45A, IGFBP3, and SIAH1) enriched in the downregulated endocytosis (hsa04144) and upregulated p53 signaling pathway (hsa04115), respectively, were validated by qRT-PCR experiments. The qRT-PCR results showed that the relative expression levels of HSPA8 [adjusted P-value = 4.45 × 10-34 and log2(FC) = -1.12], HSPA2 [adjusted P-value = 6.09 × 10-14 and log2(FC) = -1.27], HSPA1B [adjusted P-value = 1.14 × 10-11 and log2(FC) = -1.02], and ARRB1 [adjusted P-value = 2.60 × 10-5 and log2(FC) = -1.13] were significantly different (P-value < 0.05) from the case group to the control group. Furthermore, the interactions and predicted microRNAs of the key genes (HSPA8, HSPA2, HSPA1B, and ARRB1) were visualized in networks. This study identified the key candidate transcriptomic biomarkers and biological pathways in hyperglycemic HK-2 cells responding to the PKM2 activator TEPP-46 that can highlight a possibility of PKM2 tetramerization reshaping the interplay among endocytic trafficking through the versatile networks of Hsp70s and rewiring the crosstalk between EGFR signal transduction circuits and metabolic stress to promote resilience, which will be valuable for further research on PKM2 in DKD.

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“…O supracitado kit colorimétrico baseia-se na alteração da absorbância em 590 nm do corante verde malaquita na presença de moléculas de Pi, resultante da formação de fosfomolibdatos, de forma que o Pi liberado é proporcional à taxa de hidrólise de nucleotídeos ATP pelas proteínas (SILVA et al, 2021).…”

Section: Atividade Atpásicaunclassified

Section: Expressão E Purificação Do Nbd E Pbd Da Hspa1aunclassified

“…C., RAMOS, C. H. I., 2006;DORES-SILVA et al, 2015;KIRALY et al, 2020;SILVA et al, 2021), estas apresentam conteúdo de estrutura secundária majoritário A proteína HspA1A_NBD apresentou 30% de conteúdo estrutural hélices-α em sua forma apo e, quando ligada aos nucleotídeos adenosina (ATP e ADP) apresentou 33% e 35%, respectivamente. Os dados observados sugerem que na presença de nucleotídeos ADP e íons Mg 2+ , a proteína apresenta um leve aumento estrutural correspondente a hélices-α, como foi I., 2006;DORES-SILVA et al, 2015;KIRALY et al, 2020;SILVA et al, 2021). Quando a proteína está enovelada e detém estrutura terciária local, parte dos resíduos de W se encontram no ambiente hidrofóbico, entretanto, em condições desnaturantes, esses são expostos à água, de forma que, a polaridade do solvente implica diretamente no deslocamento do espectro de emissão para λ menos energéticos (LAKOWICS, J. R., 2006).…”

Section: Expressão E Purificação Do Nbd E Pbd Da Hspa1aunclassified

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Estudo estrutural-funcional do NBD (>i<Nucleotide Binding Domain>/i<) e do PBD (>i<Peptide Binding Domain>/i<) da proteína HspA1A

Oliveira¹

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AGRADECIMENTOSA Deus e Nossa Senhora Aparecida, por terem me dado à permissão de chegar até aqui, por toda a força concedida na concretização dessa etapa, força para não desistir e proteção para me amparar.Aos meus pais, Cleuza e Benedito, minhas bases, por tanto amor, por tudo o que sou, por terem me proporcionado educação e amor pelos estudos, por sempre acreditarem em minha capacidade e, apesar das inúmeras dificuldades, por sempre me estimularem a continuar.Aos meus tios, tias, primos e primas pelo apoio, pela "propaganda" positiva a meu respeito e pela força.Ao meu orientador, Prof. Dr. Júlio César Borges, agradeço, primeiramente, por ter me aceitado, sem, ao menos, me conhecer, e por ter acreditado em mim desde sempre. Só tenho a agradecer aos seus ensinamentos (pessoais e acadêmicos), orientações, palavras de incentivo, puxões de orelha, paciência e dedicação. Tenho orgulho em dizer que fui seu orientando.

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Structural, thermodynamic and functional studies of human 71 kDa heat shock cognate protein (HSPA8/hHsc70)

Cited by 11 publications

References 92 publications

Hidden information on protein function in censuses of proteome foldedness

Hidden information on protein function in censuses of proteome foldedness

Transcriptomic signatures responding to PKM2 activator TEPP-46 in the hyperglycemic human renal proximal epithelial tubular cells

Estudo estrutural-funcional do NBD (>i<Nucleotide Binding Domain>/i<) e do PBD (>i<Peptide Binding Domain>/i<) da proteína HspA1A

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