Activation of Archaeoglobus fulgidus Cu+-ATPase CopA by cysteine

AbstractCopA, a thermophilic ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu+ across the cell membrane. Millimolar concentration of Cys dramatically increases (≅800%) the activity of CopA and other PIB-type ATPases (Escherichia coli ZntA and Arabidopsis thaliana HMA2). The high affinity of CopA for metal (≅1 μM) together with the low Cu+–Cys KD (<10−10M) suggested a multifaceted interaction of Cys with CopA, perhaps acting as a substitute for the Cu+ chaperone protein present in vivo. To explain the activation by the amino acid and further understand the mechanism of metal delivery to transport ATPases, Cys effects on the turnover and partial reactions of CopA were studied. 2–20 mM Cys accelerates enzyme turnover with little effect on CopA affinity for Cu+, suggesting a metal independent activation. Furthermore, Cys activates the p-nitrophenyl phosphatase activity of CopA, even though this activity is metal independent. Cys accelerates enzyme phosphorylation and the forward dephosphorylation rates yielding higher steady state phosphoenzyme levels. The faster dephosphorylation would explain the higher enzyme turnover in the presence of Cys. The amino acid has no significant effect on low affinity ATP Km suggesting no changes in the E1↔E2 equilibrium. Characterization of Cu+ transport into sealed vesicles indicates that Cys acts on the cytoplasmic side of the enzyme. However, the Cys activation of truncated CopA lacking the N-terminal metal binding domain (N-MBD) indicates that activation by Cys is independent of the regulatory N-MBD. These results suggest that Cys is a non-essential activator of CopA, interacting with the cytoplasmic side of the enzyme while this is in an E1 form. Interestingly, these effects also point out that Cu+ can reach the cytoplasmic opening of the access path into the transmembrane transport sites either as a free metal or a Cu+–Cys complex

Cdc37 has distinct roles in protein kinase quality control that protect nascent chains from degradation and promote posttranslational maturation

Cdc37 is a molecular chaperone that functions with Hsp90 to promote protein kinase folding. Analysis of 65 Saccharomyces cerevisiae protein kinases (∼50% of the kinome) in a cdc37 mutant strain showed that 51 had decreased abundance compared with levels in the wild-type strain. Several lipid kinases also accumulated in reduced amounts in the cdc37 mutant strain. Results from our pulse-labeling studies showed that Cdc37 protects nascent kinase chains from rapid degradation shortly after synthesis. This degradation phenotype was suppressed when cdc37 mutant cells were grown at reduced temperatures, although this did not lead to a full restoration of kinase activity. We propose that Cdc37 functions at distinct steps in kinase biogenesis that involves protecting nascent chains from rapid degradation followed by its folding function in association with Hsp90. Our studies demonstrate that Cdc37 has a general role in kinome biogenesis

Ydj1 Protects Nascent Protein Kinases from Degradation and Controls the Rate of Their Maturation▿

Ydj1 is a Saccharomyces cerevisiae Hsp40 molecular chaperone that functions with Hsp70 to promote polypeptide folding. We identified Ydj1 as being important for maintaining steady-state levels of protein kinases after screening several chaperones and cochaperones in gene deletion mutant strains. Pulse-chase analyses revealed that a portion of Tpk2 kinase was degraded shortly after synthesis in a ydj1Δ mutant, while the remainder was capable of maturing but with reduced kinetics compared to the wild type. Cdc28 maturation was also delayed in the ydj1Δ mutant strain. Ydj1 protects nascent kinases in different contexts, such as when Hsp90 is inhibited with geldanamycin or when CDC37 is mutated. The protective function of Ydj1 is due partly to its intrinsic chaperone function, but this is minor compared to the protective effect resulting from its interaction with Hsp70. SIS1, a type II Hsp40, was unable to suppress defects in kinase accumulation in the ydj1Δ mutant, suggesting some specificity in Ydj1 chaperone action. However, analysis of chimeric proteins that contained the chaperone modules of Ydj1 or Sis1 indicated that Ydj1 promotes kinase accumulation independently of its client-binding specificity. Our results suggest that Ydj1 can both protect nascent chains against degradation and control the rate of kinase maturation

Hsp110 Chaperones Control Client Fate Determination in the Hsp70–Hsp90 Chaperone System

The Hsp110 family of protein chaperones was known to promote maturation of Hsp90 client proteins. The yeast Hsp110 ortholog Sse1 is now shown to influence the decision to fold or degrade substrates of the Hsp70–Hsp90 chaperone system when maturation is compromised

Self‐assembly and Neurotoxicity of Amyloid‐beta (21‐40) Peptide fragment: The regulatory Role of GxxxG Motifs

The three GxxxG repeating motifs from the C‐terminal region of β‐amyloid (Aβ) peptide play a significant role in regulating the aggregation kinetics of the peptide. Mutation of these glycine residues to leucine greatly accelerates the fibrillation process but generates a varied toxicity profile. Using an array of biophysical techniques, we demonstrated the uniqueness of the composite glycine residues in these structural repeats. We used solvent relaxation NMR spectroscopy to investigate the role played by the surrounding water molecules in determining the corresponding aggregation pathway. Notably, the conformational changes induced by Gly33 and Gly37 mutations result in significantly decreased toxicity in a neuronal cell line. Our results indicate that G33xxxG37 is the primary motif responsible for Aβ neurotoxicity, hence providing a direct structure–function correlation. Targeting this motif, therefore, can be a promising strategy to prevent neuronal cell death associated with Alzheimer's and other related diseases, such as type II diabetes and Parkinson's

Self‐assembly and Neurotoxicity of Amyloid‐beta (21‐40) Peptide fragment: The regulatory Role of GxxxG Motifs

The three GxxxG repeating motifs from the C‐terminal region of β‐amyloid (Aβ) peptide play a significant role in regulating the aggregation kinetics of the peptide. Mutation of these glycine residues to leucine greatly accelerates the fibrillation process but generates a varied toxicity profile. Using an array of biophysical techniques, we demonstrated the uniqueness of the composite glycine residues in these structural repeats. We used solvent relaxation NMR spectroscopy to investigate the role played by the surrounding water molecules in determining the corresponding aggregation pathway. Notably, the conformational changes induced by Gly33 and Gly37 mutations result in significantly decreased toxicity in a neuronal cell line. Our results indicate that G33xxxG37 is the primary motif responsible for Aβ neurotoxicity, hence providing a direct structure–function correlation. Targeting this motif, therefore, can be a promising strategy to prevent neuronal cell death associated with Alzheimer's and other related diseases, such as type II diabetes and Parkinson's

Biophysical Characterization of Essential Phosphorylation at the Flexible C-Terminal Region of C-Raf with 14-3-3ζ Protein

<div><p>Phosphorylation at the C-terminal flexible region of the C-Raf protein plays an important role in regulating its biological activity. Auto-phosphorylation at serine 621 (S621) in this region maintains C-Raf stability and activity. This phosphorylation mediates the interaction between C-Raf and scaffold protein 14-3-3ζ to activate the downstream MEK kinase pathway. In this study, we have defined the interaction of C-terminal peptide sequence of C-Raf with 14-3-3ζ protein and determined the possible structural adaptation of this region. Biophysical elucidation of the interaction was carried out using phosphopeptide (residue number 615–630) in the presence of 14-3-3ζ protein. Using isothermal titration calorimetry (ITC), a high binding affinity with micro-molar range was found to exist between the peptide and 14-3-3ζ protein, whereas the non-phosphorylated peptide did not show any appreciable binding affinity. Further interaction details were investigated using several biophysical techniques such as circular dichroism (CD), fluorescence, and nuclear magnetic resonance (NMR) spectroscopy, in addition to molecular modeling. This study provides the molecular basis for C-Raf C-terminal-derived phosphopeptide interaction with 14-3-3ζ protein as well as structural insights responsible for phosphorylated S621-mediated 14-3-3ζ binding at an atomic resolution.</p></div

Interaction of KH16p with 14-3-3ζ using ¹H NMR spectroscopy.

<p>¹H NMR spectra of the KH16p peptide with an increasing concentration of 14-3-3ζ demonstrating the linewidth broadening in the amide, aromatic, and aliphatic regions. The ¹H NMR experiments were carried out in aqueous solution on a Bruker AVANCE III 500MHz spectrometer at 288 K.</p

Relative changes in secondary structure of 14-3-3ζ protein upon interaction with KH16p peptide and <i>vice versa</i> by CD spectroscopy.

<p><b>(A)</b> Far-UV CD spectra of 14-3-3ζ protein in free and KH16p-peptide bound forms. (<b>B)</b> Far-UV CD spectra of KH16p peptide in absence and presence of 14-3-3ζ protein. Protein and peptides were in 10 mM Na₂HPO₄ buffer with 100 mM NaCl at pH 6.1.</p