Comparative analysis of the coordinated motion of Hsp70s from different organelles observed by single-molecule three-color FRET

Cellular function depends on the correct folding of proteins inside the cell. Heat-shock proteins 70 (Hsp70s), being among the first molecular chaperones binding to nascently translated proteins, aid in protein folding and transport. They undergo large, coordinated intra- and interdomain structural rearrangements mediated by allosteric interactions. Here, we applied a three-color single-molecule Förster resonance energy transfer (FRET) combined with three-color photon distribution analysis to compare the conformational cycle of the Hsp70 chaperones DnaK, Ssc1, and BiP. By capturing three distances simultaneously, we can identify coordinated structural changes during the functional cycle. Besides the known conformations of the Hsp70s with docked domains and open lid and undocked domains with closed lid, we observed additional intermediate conformations and distance broadening, suggesting flexibility of the Hsp70s in adopting the states in a coordinated fashion. Interestingly, the difference of this distance broadening varied between DnaK, Ssc1, and BiP. Study of their conformational cycle in the presence of substrate peptide and nucleotide exchange factors strengthened the observation of additional conformational intermediates, with BiP showing coordinated changes more clearly compared to DnaK and Ssc1. Additionally, DnaK and BiP were found to differ in their selectivity for nucleotide analogs, suggesting variability in the recognition mechanism of their nucleotide-binding domains for the different nucleotides. By using three-color FRET, we overcome the limitations of the usual single-distance approach in single-molecule FRET, allowing us to characterize the conformational space of proteins in higher detail.

Hsp70s, a family of chaperones that aids in protein folding, inhibits misfolding and aggregation, and transports proteins to their respective cellular destination, are among the most conserved proteins in the cell (1–4). They assist in the folding of nascent proteins co- or posttranslationally and prevent protein aggregation by specific interactions with protein sequences of five to seven hydrophobic amino acids (3, 4). Hsp70 chaperones are ubiquitously expressed in almost all organisms, including archaea and are found in many cellular compartments (5). Owing to their prokaryotic origin and uptake by endosymbiosis, they can be divided into cytosolic, endoplasmic reticulum (ER)-associated, mitochondrial, and plastid families. Hsp70s of these four families are more conserved throughout different phyla within one compartment than between the cellular organelles. They consist of a 44 kDa N-terminal nucleotide-binding domain (NBD) connected to a 28 kDa substrate-binding domain (SBD). The SBD itself is further divided into a β-sandwich domain consisting of two β-sheets with a hydrophobic cleft for substrate recognition and an α-helical lid. Despite their highly conserved sequence (48 to 58% sequence identity for the proteins investigated here), Hsp70s perform a multitude of additional functions in the different organelles with the aid of various cochaperones.To perform their functions, Hsp70 chaperones undergo a nucleotide-dependent conformational cycle. Multiple crystal structures, NMR, and single-pair Förster resonance energy transfer (FRET) experiments have elucidated the conformational states of the Hsp70s in their functional cycle (Fig. 1A) (6–12). When ATP is bound to the NBD, the domains contact each other and, for the SBD, the α-helical lid is found in an open conformation distant from the β-sheet (7–9, 11). Upon ATP hydrolysis, the NBD and SBD separate, and the lid closes upon the β-sheet of the SBD (6, 8–10). This conformational transition depends on the hydrolysis of ATP, which is intrinsically slow for Hsp70 chaperones (around 0.05 to 0.1/min at 30 °C) (13). The rate of ATP hydrolysis is increased by J-domain–containing heat-shock proteins 40 (Hsp40s, or J-proteins), which also confer specificity to Hsp70s for certain functionalities. J-domain–containing proteins play an important role in recruiting Hsp70s to their respective client proteins. In the ER, mitochondria, and plastids, Hsp70s are associated with the translocation of proteins across the respective organellar membrane. Mitochondria contain four different J-proteins conveying various functions to the mitochondrial Hsp70s. When interacting with membrane-anchored Pam16/18, they are involved in protein import (14–18), when binding to Jac1, they act as part of the iron–sulfur biogenesis (19), and, when interacting with Mdj1, they play a role in protein folding and prevent aggregation (20–23). The specialization by J-proteins is even more pronounced in the ER, where six J-proteins exist that modify the function of the Hsp70 (24). Protein folding occurs upon interaction of the Hsp70 BiP with ERdj3 and ERdj6 (25). SEC63 (or ERdj2) is important for protein translocation across the membrane (26), and ER-associated degradation is driven by binding to ERdj4 and ERdj5 (27–29).Open in a separate windowFig. 1.Conformational change of Hsp70 chaperones in the presence of different nucleotides observed by single-molecule 3C FRET using multiparameter fluorescence detection with pulsed interleaved excitation (MFD-PIE). (A) The conformational cycle of Hsp70s is shown schematically using the crystal structure of DnaK in its docked conformation with open lid (Protein Data Bank [PDB] accession code: 4B9Q) or with undocked domains and a closed lid (PDB accession code: 2KHO) (6, 7). The NBD is displayed in blue, the β-sheet of the SBD in green, and the α-helical lid in red. (B) A schematic representation of a single-molecule 3C-FRET experiment on DnaK (PDB accession code: 2KHO) showing the attachment sites of the fluorophores Atto488, Atto565, and Atto647N to the NBD and SBD of the molecule, respectively. (C–E) Histograms of the FRET efficiencies E_GR (Left), and E_BG (Right, dark shaded plots) and E_BR (Right, light shaded plots) of triple-labeled DnaK-PrK318-C425-C563 (Upper), Ssc1-PrK341-C448-C590 (Middle), and BiP-PrK167-C519-C638 (Lower) in their (C) apo form (gray, only measurable for BiP), (D) when bound to ADP (magenta), or (E) when bound to ATP (green).In prokaryotic cells, the major Hsp70 chaperone DnaK cooperates with trigger factor in the folding of nascent proteins upstream of the GroEL/GroES chaperonin complex (30, 31). Interestingly, DnaK only plays an essential role under stress conditions, such as elevated temperatures (3). Ssc1, the major Hsp70 of the mitochondrial matrix, on the other hand, has been shown to be essential for Saccharomyces cerevisiae growth, and its expression levels rise in response to stress through elevated temperatures (32, 33). It plays an important role in protein translocation into mitochondria, protein folding, and prevention of aggregation (32, 34). Unlike Ssc1 in mitochondria, BiP is the only Hsp70 member in the ER. It is involved in protein folding (25), protein translocation (26), ER-associated protein degradation (27–29), and the stress adaptability of the ER (35–37).Our comparative study focuses on intrinsic differences between Hsp70 chaperones regarding the allosteric interactions between the NBD and SBD throughout their functional cycle. As representative members of the different families, we chose the Hsp70s from the ER (BiP) and mitochondria (Ssc1) of eukaryotic cells and the well-characterized prokaryotic major Hsp70 DnaK (SI Appendix, Figs. S1 and S2 A–C). The selection of these three proteins allows us to compare Hsp70s from different cellular compartments and from different families of Hsp70s (11).To study the coordinated structural changes of these Hsp70s during their conformational cycle, we applied single-molecule three-color (3C) FRET. Single-molecule FRET with two fluorophores (also referred to as single-pair FRET) is a powerful tool to study conformational states of proteins in vitro but is limited to a single-distance readout. Three-color single-molecule FRET, on the other hand, gives access to three interdye distances simultaneously, allowing one to directly monitor the correlation of motions in different parts of the biomolecule. For solution-based, single-molecule FRET experiments, the recently developed 3C photon distribution analysis (3C-PDA) (38) enables a quantitative analysis of the underlying distance heterogeneity of the experimental system at hand. This methodology allows us to characterize the conformational landscape of the different Hsp70s with respect to the coordinated movement of the different domains.     

垂体催乳素瘤的诊断和治疗指南

首次测定确立高催乳血症必需避免过度的静脉穿刺压力,理想的情况是醒后或饭后致少1h来测试.     

Attenuated Salmonella typhimurium htrA mutants cause fatal infections in mice deficient in NADPH oxidase and destroy NADPH oxidase-deficient macrophage monolayers

Salmonella live vaccine strains harbouring mutations in htrA, a stress protein gene, display increased susceptibility to oxidative stress in vitro. This is believed to be connected to their reduced virulence, perhaps due to impaired survival inside phagocytes, although this has never been formally proven. We report that the in vitro phenotype of increased susceptibility to oxidative stress of Salmonella typhimurium htrA mutants newly prepared by transduction is rapidly lost on subculture, with the mutants becoming as resistant as the parent for reasons that remain unclear. However, despite this change, htrA mutants are still attenuated in normal mice. In contrast, they were found to be lethal for gene targeted gp91phox-/- mice deficient in NADPH oxidase, as was a S. typhimurium SPI-2 mutant known to be virulent in gp9lphox-/- mice. Infection with htrA mutants caused little damage to primary bone marrow macrophage cultures from normal mice; conversely, they caused extensive damage to macrophages from gp9lphox-/- mice, with more than 60% reduction in cell numbers 2.5h after being infected. The parental wild type strain similarly caused extensive damage to macrophages from both normal and gp9lphox-/- mice, whereas an aroA live vaccine strain had no effect on either normal or gp9lphox-/- macrophages. Taken collectively, the present results suggest that htrA is somehow involved in resistance to oxidative stress in vivo, with the avirulence of htrA mutants in mice being due to mechanisms which involve NADPH oxidase and suppression of bacterial growth within macrophages.     

Cell-specific expression of plasma membrane calcium ATPase isoforms in retinal neurons

Ca(2+) extrusion by high-affinity plasma membrane calcium ATPases (PMCAs) is a principal mechanism for the clearance of Ca(2+) from the cytosol. The PMCA family consists of four isoforms (PMCA1-4). Little is known about the selective expression of these isoforms in brain tissues or about the physiological function conferred upon neurons by any given isoform. We investigated the cellular and subcellular distribution of PMCA isoforms in a mammalian retina. Mouse photoreceptors, cone bipolar cells and horizontal cells, which respond to light with a graded polarization, express isoform 1 (PMCA1) of the PMCA family. PMCA2 is localized to rod bipolar cells, horizontal cells, amacrine cells, and ganglion cells, and PMCA3 is predominantly expressed in spiking neurons, including both amacrine and ganglion cells but is also found in horizontal cells. PMCA4 was found to be selectively expressed in both synaptic layers. Optical measurements of Ca(2+) clearance showed that PMCAs mediate Ca(2+) extrusion in both rod and cone bipolar cells. In addition, we found that rod bipolar cells, but not cone bipolar cells possess a prominent Na(+)/Ca(2+) exchange mechanism. We conclude that PMCA isoforms are selectively expressed in retinal neurons and that processes of Ca(2+) clearance are different in rod and cone bipolar cells.     

Specificity of mutagenesis by 4-aminobiphenyl: mutations at G residues in bacteriophage M13 DNA and G-->C transversions at a unique dG(8-ABP) lesion in single-stranded DNA

Mutagenesis by the human bladder carcinogen 4-aminobiphenyl (ABP) was studied in single-stranded DNA from a bacteriophage M13 cloning vector. In comparison to ABP lesions in double-stranded DNA, lesions in single- stranded DNA were approximately 70-fold more mutagenic and 50-fold more genotoxic. Sequencing analysis of ABP-induced mutations in the lacZ gene revealed exclusively base-pair substitutions, with over 80% of the mutations occurring at G sites; the G at position 6310 accounted for 25% of the observed mutations. Among the sequence changes at G sites, G- ->T transversions predominated, followed by G-->C transversions and G-- >A transitions. In order to further elucidate the mutagenic mechanism of ABP, an oligonucleotide containing the major DNA adduct, N- (deoxyguanosin-8-yl)-4-aminobiphenyl (dG(8-ABP)), was situated within the PstI site of a single-stranded M13 genome. After in vivo replication of the adduct containing ABP-modified and control (unadducted) genomes, the mutational frequency and mutational specificity of the dG(8-ABP) lesion were determined. The targeted mutational efficiency was approximately 0.01%, and the primary mutation observed was the G-->C transversion. Thus dG(8-ABP), albeit weakly mutagenic at the PstI site, can contribute to the mutational spectrum of ABP lesions.      

Metabolic activation of methyl-hydroxylated derivatives of 7,12- dimethylbenz[a]anthracene by human liver dehydroepiandrosterone-steroid sulfotransferase

Methyl-hydroxylated metabolites of the potent carcinogen, 7,12- dimethylbenz[a]anthracene (DMBA), namely, 7-hydroxymethyl-12- methylbenz[a]anthracene (7-OH-DMBA), 7-methyl-12- hydroxymethylbenz[a]anthracene (12-OH-DMBA) and 7,12- dihydroxymethylbenz[a]anthracene (7,12-diOH-DMBA), were examined as substrates for sulfotransferase bioactivation in different human tissue cytosols. Hepatic cytosols, which were able to catalyze the 3'- phosphoadenosine 5'-phosphosulfate (PAPS)-dependent DNA binding of 7-OH- DMBA, 12-OH-DMBA and 7,12-diOH-DMBA, were highly sensitive to inhibition by dehydroepiandrosterone (DHEA), a specific substrate for human DHEA-steroid sulfotransferase (IC50 = 5 microM). By comparison, 2,6-dichloro-4-nitrophenol, a potent inhibitor of the thermostable (TS)- phenol and estrogen sulfotransferases, did not have an appreciable inhibitory effect. Neither p-nitrophenol, a high affinity substrate for human TS-phenol and estrogen sulfotransferases, nor dopamine, a specific substrate for the thermolabile (TL)-phenol sulfotransferase, significantly inhibited the DNA binding of 12-OH-DMBA catalyzed by hepatic cytosols. Inter-subject variation (n = 12) of the PAPS- dependent DNA binding of 12-OH- and 7,12-diOH-DMBAs also correlated well with DHEA-sulfotransferase activity (r = 0.90; P < 0.00001 and r = 0.92; P < 0.00001, respectively). This sulfation-dependent metabolic activation was not detected in cytosols from human colon, pancreas, larynx or mammary gland. Both TS- and TL-phenol sulfotransferases were active in human liver and colon but only liver contained DHEA- sulfotransferase activity. These results indicate that the sulfotransferase-mediated activation of the methyl-hydroxylated DMBAs is predominantly catalyzed by DHEA-steroid sulfotransferase in human liver and that TS- and TL-phenol sulfotransferases and estrogen sulfotransferase are not involved in the catalysis.