Background: Prostate cancer (PC) can display very heterogeneous phenotypes ranging from indolent asymptomatic to aggressive lethal forms. Understanding how these PC subtypes vary in their striving for energy and anabolic molecules is of fundamental importance for developing more effective therapies and diagnostics. Here, we carried out an extensive analysis of prostate tissue samples to reveal metabolic alterations during PC development and disease progression and furthermore betweenTMPRSS2-ERGrearrangement-positive and -negative PC subclasses.Methods: Comprehensive metabolomics analysis of prostate tissue samples was performed by non-destructive high-resolution magic angle spinning nuclear magnetic resonance (¹H HR MAS NMR). Subsequently, samples underwent moderate extraction, leaving tissue morphology intact for histopathological characterization. Metabolites in tissue extracts were identified by¹H/³¹P NMR and liquid chromatography-mass spectrometry (LC-MS). These metabolomics profiles were analyzed by chemometric tools and the outcome was further validated using proteomic data from a separate sample cohort.Results: The obtained metabolite patterns significantly differed between PC and benign tissue and between samples with high and low Gleason score (GS). Five key metabolites (phosphocholine, glutamate, hypoxanthine, arginine and α-glucose) were identified, who were sufficient to differentiate between cancer and benign tissue and between high to low GS. In ERG-positive PC, the analysis revealed several acylcarnitines among the increased metabolites together with decreased levels of proteins involved in β-oxidation; indicating decreased acyl-CoAs oxidation in ERG-positive tumors. The ERG-positive group also showed increased levels of metabolites and proteins involved in purine catabolism; a potential sign of increased DNA damage and oxidative stress.Conclusions: Our comprehensive metabolomic analysis strongly indicates that ERG-positive PC and ERG-negative PC should be considered as different subtypes of PC; a fact requiring different, sub-type specific treatment strategies for affected patients.

High-resolution ³¹P magic angle spinning (MAS) NMR spectroscopy is presented as a direct and non-perturbing method for measuring changes in surface charge density occurring in mixed phospholipid membranes upon binding of charged surface-active peptides. ³¹P MAS NMR was used to investigate mixed lipid membranes of neutral phosphatidylcholine and negatively charged phosphatidylglycerol where the molar fraction of the charged lipid was varied from 0 to 1. The chemical shifts of the individual membrane lipids showed a simple variation in response to changes in the fraction of the negatively charged component phosphatidylglycerol. Addition of the positively charged amyloid-β_1-40 peptide, a key substance in Alzheimer's disease, resulted in changes in the isotropic chemical shifts of the membrane lipid phosphates in a way consistent with reduction in the negative surface charge of the mixed lipid bilayers. Binding of different amounts of the positively charged peptide pentalysine to L-α-dioleoylphosphatidylcholine/L-α-dioleoylphosphatidylglycerol (DOPC/DOPG) vesicles (2:1 molar ratio) also showed a systematic variation of both chemical shift values. These changes were described by a simple two-site model and indicate purely electrostatic binding of pentalysine.

Amyloid-β peptide (Aβ), a key substance in Alzheimer’s disease (AD), is characterized by its abnormal folding into neurotoxic aggregates. Since Aβ comprises an extracellular and transmembrane domain, some of its neurotoxic actions might be exerted via interactions with neuronal membranes. Wideline and magic angle spinning ¹⁴N and ³¹P NMR have been used in combination with differential scanning calorimetry and circular dichroism spectroscopy to investigate the association between Aβ_1–40 peptide and membranes with different electrostatic surface potentials. Calorimetric measurements showed that all membrane systems were in the liquid crystalline state at 308 K. Binding of Aβ_1–40 at a 30 ∶ 1 lipid/peptide ratio to membranes composed of neutral dimyristoyl-phosphatidylcholine (DMPC) and negatively charged dimyristoylphosphatidylglycerol (DMPG) at a 4 ∶ 1 molar ratio is mainly driven electrostatically, reflected in characteristic changes of the isotropic ³¹P chemical shift values for both lipids. In addition, the average orientation of the choline headgroup of DMPC, with its electric P⁻–N⁺(CH₃)₃ dipole, changed directly in response to the reduced negative membrane surface potential. The deviation in tilt angle of the PN vector relative to the membrane surface is manifested in the observed ¹⁴N NMR quadrupole splitting and can therefore be described semiquantitatively. Adding Aβ_1–40 to membranes with nominal neutral surface charge, but composed of a ternary mixture of DMPC with DMPG and the cationic amphiphile didodecyldimethyl–ammonium bromide (DDAB) at a 3 ∶ 1 ∶ 1 molar ratio revealed surprisingly electrostatic interactions visible in the NMR spectra. Since Aβ_1–40 does not bind to neutral DMPC bilayers a model is proposed, in which on a molecular level the charged residues of Aβ_1–40 peptide can interact independently with lipid headgroups of various charges in these microscopically heterogeneous systems.

Apotosis is an essential process tightly regulated by the Bcl-2 protein family where proapoptotic Bax triggers cell death by perforating the mitochondrial outer membrane. Although intensively studied, the molecular mechanism by which these proteins create apoptotic pores remains elusive. Here, we show that Bax creates pores by extracting lipids from outer mitochondrial membrane mimics by formation of Bax/lipid clusters that are deposited on the membrane surface. Time-resolved neutron reflectometry and Fourier transform infrared spectroscopy revealed two kinetically distinct phases in the pore formation process, both of which were critically dependent on cardiolipin levels. The initially fast adsorption of Bax on the mitochondrial membrane surface is followed by a slower formation of pores and Bax-lipid clusters on the membrane surface. Our findings provide a robust molecular understanding of mitochondrial membrane perforation by cell-killing Bax protein and illuminate the initial phases of programmed cellular death.