Neurospora crassa FKBP22 is a novel ER chaperone and functionally cooperates with BiP

FK506 binding proteins (FKBPs) belong to the family of peptidyl prolyl cis-trans isomerases (PPIases) catalyzing the cis/trans isomerisation of Xaa-Pro bonds in oligopeptides and proteins. FKBPs are involved in folding, assembly and trafficking of proteins. However, only limited knowledge is available about the roles of FKBPs in the endoplasmic reticulum (ER) and their interaction with other proteins. Here we show the ER located Neurospora crassa FKBP22 to be a dimeric protein with PPIase and a novel chaperone activity. While the homodimerization of FKBP22 is mediated by its carboxy-terminal domain, the amino-terminal domain is a functional FKBP domain. The chaperone activity is mediated by the FKBP domain but is exhibited only by the full-length protein. We further demonstrate a direct interaction between FKBP22 and BiP, the major Hsp70 chaperone in the ER. The binding to BiP is mediated by the FKBP domain of FKBP22. Interestingly BiP enhances the chaperone activity of FKBP22. Both proteins form a stable complex with an unfolded substrate protein and thereby prevent its aggregation. These results suggest that BiP and FKBP22 form a folding helper complex with a high chaperoning capacity in the ER of Neurospora crassa.     

The chaperone function of cyclophilin 40 maps to a cleft between the prolyl isomerase and tetratricopeptide repeat domains

Cyclophilin 40 (CyP40), an immunophilin cochaperone present in steroid receptor-Hsp90 complexes, contains an N-terminal peptidylprolyl isomerase (PPIase) domain separated from a C-terminal Hsp90-binding tetratricopeptide repeat (TPR) domain by a 30-residue linker. To map CyP40 chaperone function, CyP40 deletion mutants were prepared and analysed for chaperone activity. CyP40 fragments containing the PPIase domain plus linker or the linker region and the adjoining TPR domain retained chaperone activity, whilst individually, the catalytic and TPR domains were devoid of chaperoning ability. CyP40 chaperone function then, is localized within the linker that forms a binding cleft with potential to accommodate non-native substrates.     

The role of the mitochondrial permeability transition pore in heart disease

Like Dr. Jeckyll and Mr. Hyde, mitochondria possess two distinct persona. Under normal physiological conditions they synthesise ATP to meet the energy needs of the beating heart. Here calcium acts as a signal to balance the rate of ATP production with ATP demand. However, when the heart is overloaded with calcium, especially when this is accompanied by oxidative stress, mitochondria embrace their darker side, and induce necrotic cell death of the myocytes. This happens acutely in reperfusion injury and chronically in congestive heart failure. Here calcium overload, adenine nucleotide depletion and oxidative stress combine forces to induce the opening of a non-specific pore in the mitochondrial membrane, known as the mitochondrial permeability transition pore (mPTP). The molecular nature of the mPTP remains controversial but current evidence implicates a matrix protein, cyclophilin-D (CyP-D) and two inner membrane proteins, the adenine nucleotide translocase (ANT) and the phosphate carrier (PiC). Inhibition of mPTP opening can be achieved with inhibitors of each component, but targeting CyP-D with cyclosporin A (CsA) and its non-immunosuppressive analogues is the best described. In animal models, inhibition of mPTP opening by either CsA or genetic ablation of CyP-D provides strong protection from both reperfusion injury and congestive heart failure. This confirms the mPTP as a promising drug target in human cardiovascular disease. Indeed, the first clinical trials have shown CsA treatment improves recovery after treatment of a coronary thrombosis with angioplasty.     

Identification of cyclophilin-40-interacting proteins reveals potential cellular function of cyclophilin-40

Cyclophilin-40 (CyP40) is part of the immunophilin family and is found in Hsp90-containing protein complexes. We were interested in identifying proteins that interact with CyP40. CyP40-interacting proteins in HeLa cells were identified using the tandem affinity purification approach. Adenovirus expressing human CyP40 protein (Ad–CyP40), fused with streptavidin and calmodulin binding peptides at the N terminus, was generated. Proteins were separated on a sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel after tandem affinity purification. Here 10 silver-stained protein bands that were enriched in the Ad–CyP40-infected lysate and the corresponding regions in the control lysate were excised, digested by trypsin, and identified by tandem mass spectrometric analysis. Of 11 interacting proteins that were identified, 4 (RACK1, Ku70, RPS3, and NF45) were expressed in rabbit reticulocyte lysate, bacteria, and MCF-7 cells. We confirmed that these proteins interact with CyP40. We observed that RACK1 suppressed the cobalt chloride-induced, hypoxia response element-dependent luciferase activity in MCF-7 cells but not in MCF-7 stable cells expressing approximately 10% of the cellular CyP40 content. In addition, RACK1 reduced the HIF-1α protein accumulation after cobalt chloride treatment, which was not observed when the CyP40 content was down-regulated. Collectively, we conclude that reduction of the HIF-1α protein by RACK1 is CyP40-mediated.     

The mitochondrial permeability transition from yeast to mammals

Regulated permeability changes have been detected in mitochondria across species. We review here their key features, with the goal of assessing whether a “permeability transition” similar to that observed in higher eukaryotes is present in other species. The recent discoveries (i) that treatment with cyclosporin A (CsA) unmasks an inhibitory site for inorganic phosphate (Pi) [Basso, E., Petronilli, V., Forte, M.A. and Bernardi, P. (2008) Phosphate is essential for inhibition of the mitochondrial permeability transition pore by cyclosporin A and by cyclophilin D ablation. J. Biol. Chem. 283, 26307-26311], the classical inhibitor of the permeability transition of yeast and (ii) that under proper experimental conditions a matrix Ca²⁺-dependence can be demonstrated in yeast as well [Yamada, A., Yamamoto, T., Yoshimura, Y., Gouda, S., Kawashima, S., Yamazaki, N., Yamashita, K., Kataoka, M., Nagata, T., Terada, H., Pfeiffer, D.R. and Shinohara Y. (2009) Ca²⁺-induced permeability transition can be observed even in yeast mitochondria under optimized experimental conditions. Biochim. Biophys. Acta 1787, 1486-1491] suggest that the mitochondrial permeability transition has been conserved during evolution.     

CyP40, but not Hsp70, in rabbit reticulocyte lysate causes the aryl hydrocarbon receptor-DNA complex formation

Upon ligand binding, the aryl hydrocarbon receptor (AhR) translocates into the nucleus and dimerizes with its partner aryl hydrocarbon receptor nuclear translocator (Arnt). The AhR-Arnt heterodimer binds to the dioxin response element (DRE) to regulate target gene expression. Using baculovirus expressed human AhR and Arnt, we showed that the formation of the ligand-dependent AhR-Arnt-DRE complex requires protein factors in vitro. Recently, we provided evidence that p23, an Hsp90-associated protein, is involved in the complex formation. The aim of this study was to determine whether two other Hsp90-associated proteins present in rabbit reticulocyte lysate (RRL), namely CyP40 and Hsp70, play any role in forming the AhR-Arnt-DRE complex. Fractionation and immunodepletion experiments revealed that Hsp70 is not necessary for the formation of this complex. In contrast, CyP40 is involved in forming the complex since (1) immunodepletion of CyP40 from a RRL fraction reduces the intensity of the AhR-Arnt-DRE complex by 48% and (2) recombinant human CyP40 alone causes the formation of this complex. In addition, CyP40-interacting proteins appear to be essential for the full CyP40 effect on the AhR gel shift complex.     

The role of mitochondria in protection of the heart by preconditioning

A prolonged period of ischaemia followed by reperfusion irreversibly damages the heart. Such reperfusion injury (RI) involves opening of the mitochondrial permeability transition pore (MPTP) under the conditions of calcium overload and oxidative stress that accompany reperfusion. Protection from MPTP opening and hence RI can be mediated by ischaemic preconditioning (IP) where the prolonged ischaemic period is preceded by one or more brief (2-5 min) cycles of ischaemia and reperfusion. Following a brief overview of the molecular characterisation and regulation of the MPTP, the proposed mechanisms by which IP reduces pore opening are reviewed including the potential roles for reactive oxygen species (ROS), protein kinase cascades, and mitochondrial potassium channels. It is proposed that IP-mediated inhibition of MPTP opening at reperfusion does not involve direct phosphorylation of mitochondrial proteins, but rather reflects diminished oxidative stress during prolonged ischaemia and reperfusion. This causes less oxidation of critical thiol groups on the MPTP that are known to sensitise pore opening to calcium. The mechanisms by which ROS levels are decreased in the IP hearts during prolonged ischaemia and reperfusion are not known, but appear to require activation of protein kinase Cε, either by receptor-mediated events or through transient increases in ROS during the IP protocol. Other signalling pathways may show cross-talk with this primary mechanism, but we suggest that a role for mitochondrial potassium channels is unlikely. The evidence for their activity in isolated mitochondria and cardiac myocytes is reviewed and the lack of specificity of the pharmacological agents used to implicate them in IP is noted. Some K⁺ channel openers uncouple mitochondria and others inhibit respiratory chain complexes, and their ability to produce ROS and precondition hearts is mimicked by bona fide uncouplers and respiratory chain inhibitors. IP may also provide continuing protection during reperfusion by preventing a cascade of MPTP-induced ROS production followed by further MPTP opening. This phase of protection may involve survival kinase pathways such as Akt and glycogen synthase kinase 3 (GSK3) either increasing ROS removal or reducing mitochondrial ROS production.     

The ADP and ATP transport in mitochondria and its carrier

Different from some more specialised short reviews, here a general although not encyclopaedic survey of the function, metabolic role, structure and mechanism of the ADP/ATP transport in mitochondria is presented. The obvious need for an “old fashioned” review comes from the gateway role in metabolism of the ATP transfer to the cytosol from mitochondria. Amidst the labours, 40 or more years ago, of unravelling the role of mitochondrial compartments and of the two membranes, the sequence of steps of how ATP arrives in the cytosol became a major issue. When the dust settled, a picture emerged where ATP is exported across the inner membrane in a 1:1 exchange against ADP and where the selection of ATP versus ADP is controlled by the high membrane potential at the inner membrane, thus uplifting the free energy of ATP in the cytosol over the mitochondrial matrix. Thus the disparate energy and redox states of the two major compartments are bridged by two membrane potential responsive carriers to enable their symbiosis in the eukaryotic cell. The advance to the molecular level by studying the binding of nucleotides and inhibitors was facilitated by the high level of carrier (AAC) binding sites in the mitochondrial membrane. A striking flexibility of nucleotide binding uncovered the reorientation of carrier sites between outer and inner face, assisted by the side specific high affinity inhibitors. The evidence of a single carrier site versus separate sites for substrate and inhibitors was expounded. In an ideal setting principles of transport catalysis were elucidated. The isolation of intact AAC as a first for any transporter enabled the reconstitution of transport for unravelling, independently of mitochondrial complications, the factors controlling the ADP/ATP exchange. Electrical currents measured with the reconstituted AAC demonstrated electrogenic translocation and charge shift of reorienting carrier sites. Aberrant or vital para-functions of AAC in basal uncoupling and in the mitochondrial pore transition were demonstrated in mitochondria and by patch clamp with reconstituted AAC. The first amino acid sequence of AAC and of any eukaryotic carrier furnished a 6-transmembrane helix folding model, and was the basis for mapping the structure by access studies with various probes, and for demonstrating the strong conformation changes demanded by the reorientation mechanism. Mutations served to elucidate the function of residues, including the particular sensitivity of ATP versus ADP transport to deletion of critical positive charge in AAC. After resisting for decades, at last the atomic crystal structure of the stabilised CAT-AAC complex emerged supporting the predicted principle fold of the AAC but showing unexpected features relevant to mechanism. Being a snapshot of an extreme abortive “c-state” the actual mechanism still remains a conjecture.     

家蚕CyPA基因的克隆、表达谱及进化分析

CyP蛋白家族在蛋白质折叠过程中起着重要作用。本研究克隆了家蚕Bombyx mori CyPA基因(BmCyPA),该基因由2个外显子和1个内含子组成,推导开放阅读框编码165个氨基酸,分子量为19.4 kD,等电点为8.79。序列分析表明BmCyPA在不同物种间具有高度的保守性,含有肽酰脯氨酸顺反异构酶活性位点及与CsA侧链结合的氨基酸,提示BmCyPA可能具有肽酰脯氨酸顺反异构酶活性和与CsA结合的特性。组织表达谱及EST数据分析显示,BmCyPA在丝腺中高丰度表达。通过对不同物种来源的CyP基因的进化分析,进一步预测了BmCyP基因的功能,BmCyP可能与丝蛋白的正确折叠相关。