RESUMEN
The primary function of mitochondria is energy production, a task of particular importance especially for cells with a high energy demand like cardiomyocytes. The B-cell lymphoma (BCL-2) family member BCL-2 adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) is linked to mitochondrial targeting after homodimerization, where it functions in inner membrane depolarization and permeabilization of the mitochondrial outer membrane (MOM) mediating cell death. We investigated the basal distribution of cardiac BNIP3 in vivo and its physical interaction with the pro-death protein BCL2 associated X, apoptosis regulator (BAX) and with mitochondria using immunoblot analysis, co-immunoprecipitation, and continuous wave and pulsed electron paramagnetic resonance spectroscopy techniques. We found that BNIP3 is present as a dimer in the cytosol and in the outer membrane of cardiac mitochondria under basal conditions. It forms disulfide-bridged, but mainly non-covalent dimers in the cytosol. Heterodimers with BAX are formed exclusively in the MOM. Furthermore, our results suggest that BNIP3 interacts with the MOM directly via mitochondrial BAX. However, the physical interactions with BAX and the MOM did not affect the membrane potential and cell viability. These findings suggest that another stimulus other than the mere existence of the BNIP3/BAX dimer in the MOM is required to promote BNIP3 cell-death activity; this could be a potential disturbance of the BNIP3 distribution homeostasis, namely in the direction of the mitochondria.
Asunto(s)
Proteínas de la Membrana/metabolismo , Mitocondrias Cardíacas/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Apoptosis , Citoplasma/metabolismo , Potencial de la Membrana Mitocondrial , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Multimerización de ProteínaRESUMEN
Ras of complex proteins (Roc) is a Ras-like GTP-binding domain that always occurs in tandem with the C-terminal of Roc (COR) domain and is found in bacteria, plants and animals. Recently, it has been shown that Roco proteins belong to the family of G-proteins activated by nucleotide (nt)-dependent dimerization (GADs). We investigated the RocCOR tandem from the bacteria Chlorobium tepidum with site-directed spin labelling and pulse EPR distance measurements to follow conformational changes during the Roco G-protein cycle. Our results confirm that the COR domains are a stable dimerization device serving as a scaffold for the Roc domains that, in contrast, are structurally heterogeneous and dynamic entities. Contrary to other GAD proteins, we observed only minor structural alterations upon binding and hydrolysis of GTP, indicating significant mechanistic variations within this protein class. Mutations in the most prominent member of the Roco family of proteins, leucine-rich repeat (LRR) kinase 2 (LRRK2), are the most frequent cause of late-onset Parkinson's disease (PD). Using a stable recombinant LRRK2 Roc-COR-kinase fragment we obtained detailed kinetic data for the G-protein cycle. Our data confirmed that dimerization is essential for efficient GTP hydrolysis and PD mutations in the Roc domain result in decreased GTPase activity. Previous data have shown that these LRRK2 PD-mutations are located in the interface between Roc and COR. Importantly, analogous mutations in the conserved C. tepidum Roc/COR interface significantly influence the structure and nt-induced conformational changes of the Roc domains.
Asunto(s)
Proteínas Bacterianas/química , Chlorobium/química , Enfermedad de Parkinson/genética , Mutación Puntual , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Chlorobium/genética , Chlorobium/metabolismo , GTP Fosfohidrolasas/metabolismo , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Modelos Moleculares , Datos de Secuencia Molecular , Enfermedad de Parkinson/metabolismo , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de ProteínaRESUMEN
Rationally designed gold/silver nanoshells (Au/Ag-NS) with plasmon resonances optimized for red laser excitation in order to minimize autofluorescence from clinical samples exhibit scattering cross-sections, which are ca. one order of magnitude larger compared with solid quasi-spherical gold nanoparticles (Au-NPs) of the same size. Hydrophilic stabilization and sterical accessibility for subsequent bioconjugation of Au/Ag-NS is achieved by coating their surface with a self-assembled monolayer (SAM) of rationally designed Raman reporter molecules comprising terminal mono- and tri-ethylene glycol (EG) spacers, respectively. The stability of the hydrophilically stabilized metal colloid was tested under different conditions. In contrast to metal colloids coated with a SAM without terminal EG spacers, the hydrophilically stabilized SERS particles do not aggregate under physiologically relevant conditions, i.e., buffer solutions with high ionic strength. Using these rationally designed SERS particles in conjunction with a microspectroscopic acquisition scheme, a sandwich immunoassay for the sensitive detection of interleukin-6 (IL-6) was developed. Several control experiments demonstrate the high specificity of the assay towards IL-6, with a lowest detectable concentration of ca. 1 pg mL(-1). The signal strength of the Au/Ag-NS is at least one order of magnitude higher compared with hydrophilically stabilized, non-aggregated solid quasi-spherical Au-NPs of the same size.