Hsp90 functions to balance the phosphorylation state of Akt during C2C12 myoblast differentiation

The function of the 90-kDa heat shock protein (Hsp90) is essential for the regulation of a myriad of signal transduction cascades that control all facets of a cell's physiology. Akt (PKB) is an Hsp90-dependent serine-threonine kinase that plays critical roles in the regulation of muscle cell physiology, including roles in the regulation of muscle differentiation and anti-apoptotic responses that modulate cell survival. In this report, we have examined the role of Hsp90 in regulating the activity of Akt in differentiating C2C12 myoblasts. While long-term treatment of differentiating C2C12 cells with the Hsp90 inhibitor geldanamycin led to the depletion of cellular Akt levels, pulse-chase analysis indicated that geldanamycin primarily enhanced the turnover rate of newly synthesized Akt. Hsp90 maintained an interaction with mature Akt, while Cdc37, Hsp90's kinase-specific co-chaperone, was lost from the chaperone complex upon Akt maturation. Geldanamycin partially disrupted the interaction of Cdc37 with Akt, but had a much less significant effect on the interaction of Hsp90 with Akt. Surprisingly, short-term treatment of differentiating C2C12 with geldanamycin increased the phosphorylation of Akt on Ser473, an effect mimicked by treatment of C2C12 cells with okadaic acid or the Hsp90 inhibitor novobiocin. Furthermore, Akt was found to interact directly with catalytic subunit of protein phosphatase 2A (PP2Ac) in C2C12 cells, and this interaction was not disrupted by geldanamycin. Thus, our findings indicate that Hsp90 functions to balance the phosphorylation state of Akt by modulating the ability of Akt to be dephosphorylated by PP2Ac during C2C12 myoblast differentiation.     

Protein mediated glycolipid transfer is inhibited FROM sphingomyelin membranes but enhanced TO sphingomyelin containing raft like membranes

The mammalian glycolipid transfer protein, GLTP, catalyzes the transfer in vitro of glycolipids between membranes. In this study we have examined on one hand the effect of the variations in the donor vesicle composition and on the other hand the effects of variations in the acceptor vesicle composition on the GLTP-catalyzed transfer kinetics of galactosylceramide between bilayer vesicles. For this purpose a resonance energy transfer assay was used, the energy donor being anthrylvinyl-galactosylceramide and the energy acceptor DiO-C16. First, we show that the transfer of anthrylvinyl-galactosylceramide from palmitoyl-oleoyl-phosphatidylcholine donor vesicles was faster than from dipalmitoyl-phosphatidylcholine vesicles, and that there is no transfer from palmitoyl-sphingomyelin vesicles regardless of the cholesterol amount. In this setup the acceptor vesicles were always 100% palmitoyl-oleoyl-phosphatidylcholine. We also showed that the transfer in general is faster from small highly curved vesicles compared to that from larger vesicles. Secondly, by varying the acceptor vesicle composition we showed that the transfer is faster to mixtures of sphingomyelin and cholesterol compared to mixtures of phosphatidylcholines and cholesterol. Based on these experiments we conclude that the GLTP mediated transfer of anthrylvinyl-galactosylceramide is sensitive to the matrix lipid composition and membrane bending. We postulate that a tightly packed membrane environment is most effective in preventing GLTP from accessing its substrates, and cholesterol is not required to protect the glycosphingolipid in the membrane from being transferred by GLTP. On the other hand GLTP can more easily transfer glycolipids to 'lipid raft' like membranes, suggesting that the protein could be involved in raft assembly.     

Identification of crucial histidines for heme binding in the N-terminal domain of the heme-regulated eIF2alpha kinase

The heme-regulated eukaryotic initiation factor-2alpha (eIF2alpha) kinase (HRI) regulates the initiation of protein synthesis in reticulocytes. The binding of NO to the N-terminal heme-binding domain (NTD) of HRI positively modulates its kinase activity. By utilizing UV-visible absorption, resonance Raman, EPR and CD spectroscopies, two histidine residues have been identified that are crucial for the binding of heme to the NTD. The UV-visible absorption and resonance Raman spectra of all the histidine to alanine mutants constructed were similar to those of the unmutated NTD. However, the change in the CD spectra of the NTD construct containing mutation of His78 to Ala (H78A) indicated loss of the specific binding of heme. The EPR spectrum for the ferric H78A mutant was also substantially perturbed. Thus, His78 is one of the axial ligands for the NTD of HRI. Significant changes in the EPR spectrum of the H123A mutant were also observed, and heme readily dissociated from both the H123A and the H78A NTD mutants, suggesting that His123 was also an axial heme ligand. However, the CD spectrum for the Soret region of the H123A mutant indicated that this mutant still bound heme specifically. Thus, while both His78 and His123 are crucial for stable heme binding, the effects of their mutations on the structure of the NTD differed. His78 appears to play the primary role in the specific binding of heme to the NTD, acting analogously to the "proximal histidine" ligand of globins, while His123 appears to act as the "distal" heme ligand.     

Definition of protein kinase sequence motifs that trigger high affinity binding of Hsp90 and Cdc37

Hsp90 cooperates with its co-chaperone Cdc37 to provide obligatory support to numerous protein kinases involved in the regulation of cellular signal transduction pathways. In this report, the crystal structure of the Src family tyrosine kinase Lck was used to guide the creation of kinase constructs to determine features recognized by Hsp90 and its "kinase-specific" co-chaperone Cdc37. Two parameters were assayed: the ability and extent to which the constructs bound to Hsp90 and Cdc37, and the ability of the constructs to trigger salt-resistant high affinity complexes with Hsp90 and Cdc37 independent of the presence of molybdate. Although Hsp90 interacted with both the N-terminal and C-terminal lobes (NL and CL, respectively) of the catalytic domains of the kinases, the lobes themselves were not sufficient to trigger the high affinity binding of Hsp90. Only constructs containing a complete N- or C-terminal lobe and part of the adjacent lobe bound to Hsp90 and Cdc37 in salt-stable complexes independent of molybdate. The two minimum constructs that bound Hsp90 and Cdc37 contained the alpha-C-helix and the beta4- and beta5-strands of the NL through to end of the CL and the NL through to the alpha-E-helix and the amino acids that cap the helix. Cdc37 interacted with only the NL and minimally required the alpha-C-helix and beta4- and beta5-strands of this lobe of Lck. The results indicate that the high affinity binding activity of Hsp90 is triggered through its interaction with adjacent subdomain structures of kinase catalytic domains. Furthermore, the alpha-C-helix and part of its adjoining loop connection to the beta4-strand appear to be the primary determinants recognized by Cdc37.     

Characterization of SCP-2 from Euphorbia lagascae reveals that a single Leu/Met exchange enhances sterol transfer activity

Sterol carrier protein-2 (SCP-2) is a small intracellular basic protein domain implicated in peroxisomal beta-oxidation. We extend our knowledge of plant SCP-2 by characterizing SCP-2 from Euphorbia lagascae. This protein consists of 122 amino acids including a PTS1 peroxisomal targeting signal. It has a molecular mass of 13.6 kDa and a pI of 9.5. It shares 67% identity and 84% similarity with SCP-2 from Arabidopsis thaliana. Proteomic analysis revealed that E. lagascae SCP-2 accumulates in the endosperm during seed germination. It showed in vitro transfer activity of BODIPY-phosphatidylcholine (BODIPY-PC). The transfer of BODIPY-PC was almost completely inhibited after addition of phosphatidylinositol, palmitic acid, stearoyl-CoA and vernolic acid, whereas sterols only had a very marginal inhibitory effect. We used protein modelling and site-directed mutagenesis to investigate why the BODIPY-PC transfer mediated by E. lagascae SCP-2 is not sensitive to sterols, whereas the transfer mediated by A. thaliana SCP-2 shows sterol sensitivity. Protein modelling suggested that the ligand-binding cavity of A. thaliana SCP-2 has four methionines (Met12, 14, 15 and 100), which are replaced by leucines (Leu11, 13, 14 and 99) in E. lagascae SCP-2. Changing Leu99 to Met99 was sufficient to convert E. lagascae SCP-2 into a sterol-sensitive BODIPY-PC-transfer protein, and correspondingly, changing Met100 to Leu100 abolished the sterol sensitivity of A. thaliana SCP-2.     

Molecular features of phospholipids that affect glycolipid transfer protein-mediated galactosylceramide transfer between vesicles

The glycolipid transfer protein (GLTP)-mediated movement of galactosylceramide from model membrane donor vesicles to acceptor vesicles is sensitive to the membrane environment surrounding the glycolipid. GLTP can catalyze the transfer of a fluorescently labeled GSL, anthrylvinyl-galactosylceramide (AV-GalCer), from vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and dipalmitoylphosphatidylcholine matrices, but not from vesicles prepared from N-palmitoylsphingomyelin, regardless of the cholesterol content of the vesicles. In this study, we have examined the structural features of sphingomyelin (SM) that are responsible for its inhibition of the rate of GLTP-catalyzed transfer of AV-GalCer. The rate of glycolipid transfer was enhanced when the N-palmitoyl chain of SM was replaced with an N-oleoyl chain. Analogs of N-palmitoyl-SM in which the 4,5-double bond of the long-chain base is reduced or the 3-hydroxy group is removed did not inhibit GLTP-catalyzed transfer of AV-GalCer. When the donor vesicles were prepared with phosphatidylcholines or ether-linked phosphatidylcholine analogs, the transfer rates of AV-GalCer increased with increasing degree of unsaturation. The rate of AV-GalCer transfer was strongly dependent on the unsaturation degree of the acyl and/or alkyl chains. For ester-linked PCs, the transfer rate increased in the order DPPC < POPC < DOPC, which have 0, 1, and 2 cis double bonds, respectively.     

A two-step strategy for identification of plasma protein biomarkers for endometrial and ovarian cancer

Background

Over 500,000 women worldwide are diagnosed with ovarian or endometrial cancer each year. We have used a two-step strategy to identify plasma proteins that could be used to improve the diagnosis of women with an indication of gynecologic tumor and in population screening.

Methods

In the discovery step we screened 441 proteins in plasma using the proximity extension assay (PEA) and five Olink Multiplex assays (CVD II, CVD III, INF I, ONC II, NEU I) in women with ovarian cancer (n?=?106), endometrial cancer (n?=?74), benign ovarian tumors (n?=?150) and healthy population controls (n?=?399). Based on the discovery analyses a set of 27 proteins were selected and two focused multiplex PEA assays were developed. In a replication step the focused assays were used to study an independent set of cases with ovarian cancer (n?=?280), endometrial cancer (n?=?228), women with benign ovarian tumors (n?=?76) and healthy controls (n?=?57).

Results

In the discovery step, 27 proteins that showed an association to cancer status were identified. In the replication analyses, the focused assays distinguished benign tumors from ovarian cancer stage III–IV with a sensitivity of 0.88 and specificity of 0.92 (AUC?=?0.92). The assays had a significantly higher AUC for distinguishing benign tumors from late stage ovarian cancer than using CA125 and HE4 (p?=?9.56e?22). Also, population controls could be distinguished from ovarian cancer stage III–IV with a sensitivity of 0.85 and a specificity of 0.92 (AUC?=?0.89).

Conclusion

The PEA assays represent useful tools for identification of new biomarkers for gynecologic cancers. The selected protein assays could be used to distinguish benign tumors from ovarian and endometrial cancer in women diagnosed with an unknown suspicious pelvic mass. The panels could also be used in population screening, for identification of women in need of specialized gynecologic transvaginal ultrasound examination.

Funding

The Swedish Cancer Foundation, Vinnova (SWELIFE), The Foundation for Strategic Research (SSF), Assar Gabrielsson Foundation.

     

p50(Cdc37) can buffer the temperature-sensitive properties of a mutant of Hck

Genetic studies have previously revealed that Cdc37p is required for the catalytic competence of v-Src in yeast. We have reasoned that temperature-sensitive mutants of Src family kinases might be more sensitive to the cellular level of p50(Cdc37), the mammalian homolog of Cdc37p, than their wild-type counterpart, thus potentially providing a unique opportunity to elucidate the involvement of p50(Cdc37) in the folding and stabilization of Src family kinases. A temperature-sensitive mutant of a constitutively active form of Hck (i.e., tsHck499F) was created by mutating two amino acids within the kinase domain of Hck499F. Significantly, overexpression of p50(Cdc37) rescues the catalytic activity of tsHck499F at 33 degrees C, while partially buffering it against inactivation at higher temperatures (e.g., 37 and 39 degrees C). Hsp90 function is required for tsHck499F activity and its stabilization by p50(Cdc37), but overexpression of Hsp90 is not sufficient to stabilize tsHck499F. Overexpression of p50(Cdc37) promotes the association of tsHck499F with Hsp90, suggesting that the cellular level of p50(Cdc37) might be the rate-limiting step in the association of tsHck499F with Hsp90. A truncation mutant of p50(Cdc37) that cannot bind Hsp90 still has a limited capacity to rescue the catalytic activity of tsHck499F and promote its association with Hsp90. This is a particularly important observation, since it argues that rather than solely acting as a passive adapter protein to tether tsHck499F to Hsp90, p50(Cdc37) may also act allosterically to enhance the association of tsHck499F with Hsp90. The findings presented here might also have implications for our understanding of the evolution of protein kinases and tumor development.     

Rapid suppression of mitochondrial permeability transition by methylglyoxal. Role of reversible arginine modification

Methylglyoxal (MG) (pyruvaldehyde) is a reactive carbonyl compound produced in glycolysis. MG can form covalent adducts on proteins resulting in advanced glycation end products that may alter protein function. Here we report that MG covalently modifies the mitochondrial permeability transition pore (PTP), a high conductance channel involved in the signal transduction of cell death processes. Incubation of isolated mitochondria with MG for a short period of time (5 min), followed by removal of excess free MG, prevented both ganglioside GD3- and Ca2+-induced PTP opening and the ensuing membrane depolarization, swelling, and cytochrome c release. Under these conditions MG did not significantly interfere with mitochondrial substrate transport, respiration, or oxidative phosphorylation. The suppression of permeability transition was reversible following extended incubation in MG-free medium. Of the 29 physiological carbonyl and dicarbonyl compounds tested only MG and its analogue glyoxal were able to specifically alter the behavior of the PTP. Using a set of arginine-containing peptides, we found that the major MG-derived arginine adduct formed, following a short time exposure to MG, was the 5-hydro-5-methylimidazol-4-one derivative. These findings demonstrate that MG rapidly modifies the PTP covalently and stabilizes the PTP in the closed conformation. This is probably due to the formation of an imidazolone adduct on an arginine residue involved in the control of PTP conformation (Linder, M. D., Morkunaite-Haimi, S., Kinnunen, P. J. K., Bernardi, P., and Eriksson, O. (2002) J. Biol. Chem. 277, 937-942). We deduce that the permeability transition constitutes a potentially important physiological target of MG.     

Stress–strain behavior of mitral valve leaflets in the beating ovine heart

Excised anterior mitral leaflets exhibit anisotropic, non-linear material behavior with pre-transitional stiffness ranging from 0.06 to 0.09 N/mm² and post-transitional stiffness from 2 to 9 N/mm². We used inverse finite element (FE) analysis to test, for the first time, whether the anterior mitral leaflet (AML), in vivo, exhibits similar non-linear behavior during isovolumic relaxation (IVR). Miniature radiopaque markers were sewn to the mitral annulus, AML, and papillary muscles in 8 sheep. Four-dimensional marker coordinates were obtained using biplane videofluoroscopic imaging during three consecutive cardiac cycles. A FE model of the AML was developed using marker coordinates at the end of isovolumic relaxation (when pressure difference across the valve is approximately zero), as the reference state. AML displacements were simulated during IVR using measured left ventricular and atrial pressures. AML elastic moduli in the radial and circumferential directions were obtained for each heartbeat by inverse FEA, minimizing the difference between simulated and measured displacements. Stress–strain curves for each beat were obtained from the FE model at incrementally increasing transmitral pressure intervals during IVR. Linear regression of 24 individual stress–strain curves (8 hearts, 3 beats each) yielded a mean (±SD) linear correlation coefficient (r²) of 0.994±0.003 for the circumferential direction and 0.995±0.003 for the radial direction. Thus, unlike isolated leaflets, the AML, in vivo, operates linearly over a physiologic range of pressures in the closed mitral valve.