Pyrolysis of peptides and proteins. Analytical study and environmental applications

Analytical pyrolysis was used to investigate the formation of diketopiperazines (DKPs) which are cyclic dipeptides formed from the thermal degradation of proteins. A quali/quantitative procedure was developed combining microscale flash pyrolysis at 500 °C with gas chromatography-mass spectrometry (GC-MS) of DKPs trapped onto an adsorbent phase. Polar DKPs were silylated prior to GC-MS. Particular attention was paid to the identification of proline (Pro) containing DKPs due to their greater facility of formation. The GC-MS characteristics of more than 80 original and silylated DKPs were collected from the pyrolysis of sixteen linear dipeptides and four model proteins (e.g. bovine serum albumin, BSA). The structure of a novel DKP, cyclo(pyroglutamic-Pro) was established by NMR and ESI-MS analysis, while the structures of other novel DKPs remained tentative. DKPs resulted rather specific markers of amino acid sequence in proteins, even though the thermal degradation of DKPs should be taken into account. Structural information of DKPs gathered from the pyrolysis of model compounds was employed to the identification of these compounds in the pyrolysate of proteinaceous samples, including intrinsecally unfolded protein (IUP). Analysis of the liquid fraction (bio-oil) obtained from the pyrolysis of microalgae Nannochloropsis gaditana, Scenedesmus spp with a bench scale reactor showed that DKPs constituted an important pool of nitrogen-containing compounds. Conversely, the level of DKPs was rather low in the bio-oil of Botryococcus braunii. The developed micropyrolysis procedure was applied in combination with thermogravimetry (TGA) and infrared spectroscopy (FT-IR) to investigate surface interaction between BSA and synthetic chrysotile. The results showed that the thermal behavior of BSA (e.g. DKPs formation) was affected by the different form of doped synthetic chrysotile. The typical DKPs evolved from collagen were quantified in the pyrolysates of archaeological bones from Vicenne Necropolis in order to evaluate their conservation status in combination with TGA, FTIR and XRD analysis.

Melanocytes: a new potential tool to study and monitoring Duchenne muscular dystrophy

Dystrophin is a subsarcolemmal protein critical for the integrity of muscle fibers by linking the actin cytoskeleton to the extracellular matrix via the dystroglycan complex. It is reported that dystroglycans are also localized in the skin, at dermal-epidermal junction. Here we show that epidermal melanocytes express dystrophin at the interface with the basement membrane. The full-length muscle isoform mDp427 was clearly detectable in epidermis and in melanocyte cultures as assessed by RNA and western blot analysis. Dystrophin was absent in Duchenne Muscular Dystrophy (DMD) patients melanocytes, and the ultrastructural analysis revealed mitochondrial alterations, similar to those occurring in myoblasts from the same patients. Interestingly, mitochondrial dysfunction of DMD melanocytes reflected the alterations identified in dystrophin-deficient muscle cells. In fact, mitochondria of melanocytes from DMD patients accumulated tetramethylrhodamine methyl ester but, on the contrary of control donor, mitochondria of DMD patients readily depolarized upon the addition of oligomycin, suggesting either that they are maintaining the membrane potential at the expense of glycolytic ATP, or that they are affected by a latent dysfunction unmasked by inhibition of the ATP synthase. Melanocyte cultures can be easily obtained by conventional skin biopsies, less invasive procedure than muscular biopsy, so that they may represent an alternative cellular model to myoblast for studying and monitoring dystrophinopathies also in response to pharmacological treatments.