Search

The tumor microenvironment plays a critical role in cancer progression, but the precise mechanisms by which stromal cells influence the epithelium are poorly understood. Here we show that p62 levels were reduced in the stroma of several tumors and that its loss in the tumor microenvironment or stromal fibroblasts resulted in increased tumorigenesis of epithelial prostate cancer cells. The mechanism involves the regulation of cellular redox through an mTORC1/c-Myc pathway of stromal glucose and amino acid metabolism, resulting in increased stromal IL-6 production, which is required for tumor promotion in the epithelial compartment. Thus, p62 is an anti-inflammatory tumor suppressor that acts through the modulation of metabolism in the tumor stroma.

Prostate cancer is one of the most common neoplasias in men. The tumor suppressor Par-4 is an important negative regulator of the canonical NF-[kappa]B pathway and is highly expressed in prostate. Here we show that Par-4 expression is lost in a high percentage of human prostate carcinomas, and this occurs in association with phosphatase and tensin homolog deleted from chromosome 10 (PTEN) loss. Par-4 null mice, similar to PTEN-heterozygous mice, only develop benign prostate lesions, but, importantly, concomitant Par-4 ablation and PTEN-heterozygosity lead to invasive prostate carcinoma in mice. This strong tumorigenic cooperation is anticipated in the preneoplastic prostate epithelium by an additive increase in Akt activation and a synergistic stimulation of NF-[kappa]B. These results establish the cooperation between Par-4 and PTEN as relevant for the development of prostate cancer and implicate the NF-[kappa]B pathway as a critical event in prostate tumorigenesis. AKT | aPKC | IL-6 | inflammation | prostate cancer

Full-length pentameric WT-phospholamban (PLB) and its phosphorylated form (P-PLB) were employed to compare the structural changes in the acyl chains of 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) multilamellar vesicles (MLVs) upon the insertion of both proteins utilizing A [super 31]P, [super 2]H, and [super 13]C solid-state NMR spectroscopy. Results reveal that a direct interaction takes place between both proteins and the phospholipid head groups, but the interaction of P-PLB with POPC bilayers was less significant compared with that of PLB.

[sup.2.H] and [sup.15.N] solid-state NMR spectroscopic techniques are used to investigate the side chain and backbone dynamics of wild-type phospholamban (WT-PLB). The NMR data indicates the significant backbone motion for the cytoplasmic domain of WT-PLB when compared to the transmembrane section.

A transmembrane peptide containing the aminoproximate TM-A domain of the integral membrane acetylenase CREP-1 is used to illustrate the feasibility of using phospholipid bilayer nanotube arrays for solid-state NMR studies on integral membrane proteins. Studies on the integral membraneprotein TM-A indicate that the peptide is dominantly alpha-helical.

Studies showing reduced PKC... expression or enzymatic activity in different types of human cancers support the clinical relevance of PKC... as a tumor suppressor. However, the in vivo role of PKCC and its mechanisms of action in prostate cancer remain unclear. Here we demonstrate that the genetic inactivation of PKCC in mice results in invasive prostate carcinoma in vivo in the context of phosphatase and tensin homolog deficiency. Bioinformatic analysis of human prostate cancer gene-expression sets revealed increased c-Myc transcriptional activity in PKCC-inactive cells, which correlated with increased cell growth, invasion, and metastasis. Interestingly, PKC... knockdown or the overexpression of a kinase-inactive mutant resulted in enhanced cell proliferation and invasion in vitro through increased c-Myc mRNA and protein levels and decreased Ser-373 phosphorylation of c-Myc. Analysis of prostate cancer samples demonstrated increased expression and decreased phosphorylation of c-Myc at Ser-373 in PKC... knockout tumors. In vivo xenograft studies revealed that c-Myc phosphorylation by PKC... is a critical event in the control of metastasis. Collectively, these results establish PKC... as an important tumor suppressor and regulator of c-Myc function in prostate cancer. [ABSTRACT FROM AUTHOR]

Abstract: Wild-type phospholamban (WT-PLB) is a pentameric transmembrane protein that regulates the cardiac cycle (contraction and relaxation). From a physiological prospective, unphosphorylated WT-PLB inhibits sarcoplasmic reticulum ATPase activity; whereas, its phosphorylated form relieves the inhibition in a mechanism that is not completely understood. In this study, site-specifically 15N-Ala-11- and 15N-Leu-7-labeled WT-PLB and the corresponding phosphorylated forms (P-PLB) were incorporated into 1,2-dioleoyl-sn-glycero-3-phosphocholine/2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPC/DOPE) mechanically oriented lipid bilayers. The aligned 15N-labeled Ala-11 and Leu-7 WT-PLB samples show 15N resonance peaks at approximately 71ppm and 75ppm, respectively, while the corresponding phosphorylated forms P-PLB show 15N peaks at 92ppm and 99ppm, respectively. These 15N chemical shift changes upon phosphorylation are significant and in agreement with previous reports, which indicate that phosphorylation of WT-PLB at Ser-16 alters the structural properties of the cytoplasmic domain with respect to the lipid bilayers. [Copyright &y& Elsevier]

For the first time, 15N solid-state NMR experiments were conducted on wild-type phospholamban (WT-PLB) embedded inside mechanically oriented phospholipid bilayers to investigate the topology of its cytoplasmic and transmembrane domains. 15N solid-state NMR spectra of site-specific 15N-labeled WT-PLB indicate that the transmembrane domain has a tilt angle of 13° ± 6° with respect to the POPC (1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine) bilayer normal and that the cytoplasmic domain of WT-PLB lies on the surface of the phospholipid bilayers. Comparable results were obtained from site-specific 15N-labeled WT-PLB embedded inside DOPC/DOPE (1,2-dioleoyl- sn-glycero-3-phosphocholine/1,2-dioleoyl- sn-glycero-3-phosphoethanolamine) mechanically oriented phospholipids' bilayers. The new NMR data support a pinwheel geometry of WT-PLB, but disagree with a bellflower structure in micelles, and indicate that the orientation of the cytoplasmic domain of the WT-PLB is similar to that reported for the monomeric AFA-PLB mutant. [ABSTRACT FROM AUTHOR]

Phospholamban (PLB) is a 52-amino acid integral membrane protein that helps to regulate the flow of Ca2+ ions in cardiac muscle cells. Recent structural studies on the PLB pentamer and the functionally active monomer (AFA-PLB) debate whether its cytoplasmic domain, in either the phosphorylated or dephosphorylated states, is α-helical in structure as well as whether it associates with the lipid head groups (Oxenoid, K. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 10870–10875; Karim, C. B. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 14437–14442; Andronesi, C.A. (2005)J. Am. Chem. Soc. 127, 12965–12974; Li, J. (2003) Biochemistry 42, 10674–10682; Metcalfe, E. E. (2005) Biochemistry 44, 4386-4396: Clayton, J. C. (2005) Biochemistry 44, 17016–17026). Comparing the secondary structure of the PLB pentamer and its phosphorylated form (P-PLB) as well as their interaction with the lipid bilayer is crucial in order to understand its regulatory function. Therefore, in this study, the full-length wild-type (WT) PLB and P-PLB were incorporated into 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) phospholipid bilayers and studied utilizing solid-state NMR spectroscopy. The analysis of the ZH and 31p solid-state NMR data of PLB and P-PLB in POPC multilamellar vesicles (MLVs) indicates that a direct interaction takes place between both proteins and the phospholipid head groups. However, the interaction of P-PLB with POPC bilayers was less significant compared that with PLB. Moreover, the secondary structure using 13CO site-specific isotopically labeled Ala15-PLB and Ala15-P-PLB in POPC bilayers suggests that this residue, located in the cytoplasmic domain, is a part of an a-helical structure for both PLB and P-PLB. [ABSTRACT FROM AUTHOR]

Abstract: Saposin C (Sap C) is known to stimulate the catalytic activity of the lysosomal enzyme glucosylceramidase (GCase) that facilitates the hydrolysis of glucosylceramide to ceramide and glucose. Both Sap C and acidic phospholipids are required for full activity of GCase. In order to better understand this interaction, mixed bilayer samples prepared from dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylserine (DOPS) (5:3 ratio) and Sap C were investigated using 2H and 31P solid-state NMR spectroscopy at temperatures ranging from 25 to 50 °C at pH 4.7. The Sap C concentrations used to carry out these experiments were 0 mol%, 1 mol% and 3 mol% with respect to the phospholipids. The molecular order parameters (SCD) were calculated from the dePaked 2H solid-state NMR spectra of Distearoyl-d70-phosphatidylglycerol (DSPG-d70) incorporated with DOPG and DOPS binary mixed bilayers. The SCD profiles indicate that the addition of Sap C to the negatively charged phospholipids is concentration dependent. SCD profiles of 1 mol% of the Sap C protein show only a very slight decrease in the acyl chain order. However, the SCD profiles of the 3 mol% of Sap C protein indicate that the interaction is predominantly increasing the disorder in the first half of the acyl chain near the head group (C1–C8) indicating that the amino and the carboxyl termini of Sap C are not inserting deep into the DOPG and DOPS mixed bilayers. The 31P solid-state NMR spectra show that the chemical shift anisotropy (CSA) for both phospholipids decrease and the spectral broadening increases upon addition of Sap C to the mixed bilayers. The data indicate that Sap C interacts similarly with the head groups of both acidic phospholipids and that Sap C has no preference to DOPS over DOPG. Moreover, our solid-state NMR spectroscopic data agree with the structural model previously proposed in the literature [X. Qi, G.A. Grabowski, Differential membrane interactions of saposins A and C. Implication for the functional specificity, J. Biol. Chem. 276 (2001) 27010–27017] [1]. [Copyright &y& Elsevier]

Abstract: Solid-state NMR spectroscopic techniques were used to investigate the secondary structure of the transmembrane peptide phospholamban (TM-PLB), a sarcoplasmic Ca2+ regulator. 13C cross-polarization magic angle spinning spectra of 13C carbonyl-labeled Leu39 of TM-PLB exhibited two peaks in a pure 1-palmitoyl-2-oleoyl-phosphocholine (POPC) bilayer, each due to a different structural conformation of phospholamban as characterized by the corresponding 13C chemical shift. The addition of a negatively charged phospholipid (1-palmitoyl-2-oleoylphosphatidylglycerol (POPG)) to the POPC bilayer stabilized TM-PLB to an α-helical conformation as monitored by an enhancement of the α-helical carbonyl 13C resonance in the corresponding NMR spectrum. 13C–15N REDOR solid-state NMR spectroscopic experiments revealed the distance between the 13C carbonyl carbon of Leu39 and the 15N amide nitrogen of Leu42 to be 4.2±0.2Å indicating an α-helical conformation of TM-PLB with a slight deviation from an ideal 3.6 amino acid per turn helix. Finally, the quadrupolar splittings of three 2H labeled leucines (Leu28, Leu39, and Leu51) incorporated in mechanically aligned DOPE/DOPC bilayers yielded an 11°±5° tilt of TM-PLB with respect to the bilayer normal. In addition to elucidating valuable TM-PLB secondary structure information, the solid-state NMR spectroscopic data indicates that the type of phospholipids and the water content play a crucial role in the secondary structure and folding of TM-PLB in a phospholipid bilayer. [Copyright &y& Elsevier]

For the first time, 15N solid-state NMR experiments were conducted on wild-type phospholamban (WT-PLB) embedded inside mechanically oriented phospholipid bilayers to investigate the topology of its cytoplasmic and transmembrane domains. 15N solid-state NMR spectra of site-specific 15N-labeled WT-PLB indicate that the transmembrane domain has a tilt angle of 13 degrees+/-6 degrees with respect to the POPC (1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine) bilayer normal and that the cytoplasmic domain of WT-PLB lies on the surface of the phospholipid bilayers. Comparable results were obtained from site-specific 15N-labeled WT-PLB embedded inside DOPC/DOPE (1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) mechanically oriented phospholipids' bilayers. The new NMR data support a pinwheel geometry of WT-PLB, but disagree with a bellflower structure in micelles, and indicate that the orientation of the cytoplasmic domain of the WT-PLB is similar to that reported for the monomeric AFA-PLB mutant.