Hsc66 Substrate Specificity Is Directed toward a Discrete Region of the Iron-Sulfur Cluster Template Protein IscU

Hoff, Kevin G.; Ta, Dennis T.; Tapley, Tim L.; Silberg, Jonathan J.; Vickery, Larry E.

doi:10.1074/jbc.m202814200

Cited by 104 publications

(131 citation statements)

References 44 publications

(79 reference statements)

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“…RecA and RecN have important roles in the repair of DNA damage which have been extensively characterized in UV damage experiments in many organisms, including E. coli and H. influenzae (43,50,51,77,82). Further repair mechanisms are potentially afforded by the gene products of NTHI0493 and NTHI0495, which encode homologues of the cochaperone proteins HscA and HscB, respectively, and that have roles in iron-sulfur cluster assembly (2,31,78). NTHI1588 is a homologue of a gene that encodes ImpA, an error-prone DNA polymerase originally identified in the incompatibility group plasmid TP110.…”

Section: Resultsmentioning

confidence: 99%

The OxyR Regulon in Nontypeable Haemophilus influenzae

et al. 2007

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Nontypeable Haemophilus influenzae (NTHi) is a gram-negative bacterium and a common commensal organism of the upper respiratory tract in humans. NTHi causes a number of diseases, including otitis media, sinusitis, conjunctivitis, exacerbations of chronic obstructive pulmonary disease, and bronchitis. During the course of colonization and infection, NTHi must withstand oxidative stress generated by insult due to multiple reactive oxygen species produced endogenously by other copathogens and by host cells. Using an NTHi-specific microarray containing oligonucleotides representing the 1821 open reading frames of the recently sequenced NTHi isolate 86-028NP, we have identified 40 genes in strain 86-028NP that are upregulated after induction of oxidative stress due to hydrogen peroxide. Further comparisons between the parent and an isogenic oxyR mutant identified a subset of 11 genes that were transcriptionally regulated by OxyR, a global regulator of oxidative stress. Interestingly, hydrogen peroxide induced the OxyR-independent upregulation of expression of the genes encoding components of multiple iron utilization systems. This finding suggested that careful balancing of levels of intracellular iron was important for minimizing the effects of oxidative stress during NTHi colonization and infection and that there are additional regulatory pathways involved in iron utilization.In the evolution of life, a delicate physiological balance has arisen to compensate for both an organism's need for oxygen and the attendant lethal consequences of oxygen's toxicity. For example, in the electron transport chain, electrons can be inadvertently transferred from redox-active proteins to oxygen, producing reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H 2 O 2 ). The effects of ROS on cells can then be exacerbated by the presence of free iron which, via the Fenton reaction, can react with H 2 O 2 to produce more highly reactive hydroxyl radicals (34, 40, 52). Within a host, this problem is compounded by the release of extracellular ROS by phagocytes, which again, can have deleterious effects on invading pathogens (13). Thus, bacteria have evolved an array of increasingly well-defined mechanisms to abrogate the effects of oxidative stress. Proteins involved in such processes include catalase, which decomposes H 2 O 2 , and members of both the AhpCF/TsaA family of alkylhydroperoxidases and the organic hydroperoxide resistance proteins (Ohr) that decompose organic peroxides. Also, DNA-binding ferritin-like proteins (Dps) sequester free iron and bind to DNA, thus protecting DNA from damage (27,33,45,53,59,60,67). In addition, many bacterial species possess genes encoding OxyR, a global regulator of antioxidant defenses (24, 66).Protection against oxidative stress is especially important to nontypeable Haemophilus influenzae (NTHi). NTHi is one of three bacterial commensal organisms, the others being Streptococcus pneumoniae and Moraxella catarrhalis, which can ascend a virus-compromised eustachian tube and caus...

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Section: Resultsmentioning

confidence: 99%

The OxyR Regulon in Nontypeable Haemophilus influenzae

et al. 2007

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“…HscA and HscB function together as a nucleotide-dependent molecular chaperone system that is specific for IscU (15)(16)(17). HscA is a specialized member of the ubiquitous 70-kDa heat shock protein (Hsp70 or DnaK) family of molecular chaperones that facilitate protein folding, (dis)assembly and transport via nucleotide-dependent binding to unfolded, misfolded or unstable polypeptides in order to prevent non-specific aggregation processes (18).…”

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confidence: 99%

HscA and HscB Stimulate [2Fe-2S] Cluster Transfer from IscU to Apoferredoxin in an ATP-Dependent Reaction

2006

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The role of the Azotobacter vinelandii HscA/HscB co-chaperone system in ISC-mediated iron-sulfur cluster biogenesis has been investigated in vitro by using CD and EPR spectrometry to monitor the effect of HscA, HscB, MgATP, and MgADP on the time course of cluster transfer from [2Fe-2S] IscU to apo-Isc ferredoxin. CD spectra indicate that both HscB and HscA interact with [2Fe-2S] IscU and the rate of cluster transfer was stimulated more than 20-fold in the presence stoichiometric HscA and HscB and excess MgATP. No stimulation was observed in the absence of either HscB or MgATP and cluster transfer was found to be an ATP-dependent reaction based on concomitant phosphate production and the enhanced rates of cluster transfer in the presence of KCl which is known to stimulated HscA ATPase activity. The results demonstrate a role of the ISC HscA/HscB cochaperone system in facilitating efficient [2Fe-2S] cluster transfer from the IscU scaffold protein to acceptor proteins and that [2Fe-2S] cluster transfer from IscU is an ATP-dependent process. The data are consistent with the proposed regulation of the HscA ATPase cycle by HscB and IscU (Silberg, J. J., Tapley, T. L., Hoff, K. G., and Vickery, L. E. (2004) J. Biol. Chem. 279,[53924][53925][53926][53927][53928][53929][53930][53931], and mechanistic proposals for coupling of the HscA ATPase cycle with cluster transfer from [2Fe-2S] IscU to apo-IscFdx are discussed.Iron-sulfur cluster biogenesis proteins in bacteria are commonly encoded by a highly conserved isc (iron-sulfur cluster) gene cluster, iscRSUA-hscBA-fdx (1). Moreover, with the exception of the regulatory protein IscR, homologs of each of these proteins have been shown to be involved in Fe-S cluster biogenesis in eukaryotic organisms (2). Extensive biochemical and genetic studies have established well-defined roles for IscS and IscU, but specific roles for the IscA, HscA, HscB and Fdx proteins remain elusive. IscS is a cysteine desulfurase that catalyzes the conversion of cysteine to alanine (3;4) and thereby provides the inorganic sulfur for assembly or [4Fe-4S] clusters on the IscU scaffold protein (5). In vitro studies have shown thatIscA has the ability to function as either an alternative scaffold for IscS-mediated cluster assembly of or [4Fe-4S] clusters (6;7) or as a specific Fe donor for cluster assembly on IscU (8), but the physiologically relevant role has yet to be determined. Likewise, the specific redox role of the Isc [2Fe-2S] 2+,+ Fdx (ferredoxin) in IscS-mediated cluster assembly has yet to be identified, with reduction of S 0 to S 2− , oxidation of Fe 2+ to Fe 3+ in the assembly of [2Fe-2S] 2+ clusters, and reductive coupling of two [2Fe-2S] 2+ clusters on IscU to yield one [4Fe-4S] 2+ cluster as the most likely candidates (5;9).In vivo gene knockout studies have demonstrated that both HscA (heat shock cognate 66-kDa; Hsc66) and HscB (heat shock cognate 20-kDa; Hsc20), and their eukaryotic homologs, Ssq1 and Jac1, have crucial roles in the maturation of Fe-S proteins in both prokary...

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“…Recently, we found that Hsc66 interacts with cellulose-bound peptides containing a conserved motif of IscU (residues 99 -103, LPPVK), suggesting that Hsc66 binds a single discrete region of the protein (14). In addition, a synthetic peptide corresponding to IscU residues 98 -106 (ELPPVKIHC) was capable of eliciting IscU-like effects on Hsc66.…”

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confidence: 99%

“…In addition, a synthetic peptide corresponding to IscU residues 98 -106 (ELPPVKIHC) was capable of eliciting IscU-like effects on Hsc66. The Glu 98 -Cys 106 peptide stimulates Hsc66 ATPase activity with affinity similar to that of full-length IscU (14), whereas denatured proteins that bind Hsc66 but lack the LPPVK motif fail to stimulate Hsc66 ATPase activity (3). These results indicate that residues in the LPPVK motif play a key role in regulation of Hsc66 by IscU.…”

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confidence: 99%