Synaptophysin I selectively specifies the exocytic pathway of synaptobrevin 2/VAMP2

Biogenesis and recycling of synaptic vesicles are accompanied by sorting processes that preserve the molecular composition of the compartments involved. In the present study, we have addressed the targeting of synaptobrevin 2/VAMP2 (vesicle-associated membrane protein 2), a critical component of the synaptic vesicle--fusion machinery, in a heterotypic context where its sorting is not confounded by the presence of other neuron-specific molecules. Ectopically expressed synaptophysin I interacts with VAMP2 and alters its default surface targeting to a prominent vesicular distribution, with no effect on the targeting of other membrane proteins. Protein-protein interaction is not sufficient for the control of VAMP2 sorting, which is mediated by the C-terminal domain of synaptophysin I. Synaptophysin I directs the sorting of VAMP2 to vesicles before surface delivery, without influencing VAMP2 endocytosis. Consistent with this, dynamin and alpha-SNAP (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein) mutants which block trafficking at the plasma membrane do not abrogate the effect of synaptophysin I on VAMP2 sorting. These results indicate that the sorting determinants of synaptic vesicle proteins can operate independently of a neuronal context and implicate the association of VAMP2 with synaptophysin I in the specification of the pathway of synaptic vesicle biogenesis.     

An experimental and theoretical study of the morphine binding capacity and kinetics of an engineered opioid receptor

Electrochemical real-time monitoring of ligand binding to an engineered opioid receptor specific for morphine is reported. In the particular systems studied, 90% of the binding was found to be completed after only 85-120 s. Thus, the binding kinetics has proven to be more rapid than previously believed. The observed association rate constant for the morphine binding reaction was calculated to be 215 M(-1)s(-1). A theoretical analysis of the experimental binding data suggested that the binding sites of the engineered opioid receptor could best be described by a model having two populations of binding sites: K(D)=40 microM (13 micromol/g) and K(D)=205 microM (29 micromol/g). Furthermore, a theoretical model was developed in order to explain the observed binding of the engineered opioid receptor. This model suggested that the binding sites on the polymer surface are up to 5.1A deep and they allow 100% of the ligand (morphine) to anchor itself into the site. The predicted theoretical maximum binding capacity for the reported receptor is calculated to be approximately 2 mmol/g polymer (based on an increase of cavity density).     

Time-resolved methods in biophysics. 4. Broadband pump-probe spectroscopy system with sub-20 fs temporal resolution for the study of energy transfer processes in photosynthesis.

In this paper we discuss how to push the temporal resolution limits of transient absorption spectroscopy in order to detect very fast processes (energy relaxation, energy or charge transfer, vibrational coherence) taking place in molecules of biological relevance. After reviewing the main principles of femtosecond pump-probe spectroscopy, we describe an experimental setup based on two synchronized non-collinear optical parametric amplifiers (NOPAs). Each NOPA can be independently configured to generate ultra-broadband sub-10 fs visible pulses, tunable 10-15 fs visible pulses, tunable 15-40 fs near-infrared pulses (900-1500 nm). This system enables to perform pump-probe experiments over nearly two octaves of spectrum with sub-20 fs temporal resolution. We then present an application example highlighting the capability of this instrument to track excited state dynamics in biomolecules on the sub-100 fs timescale: the study of carotenoid-bacteriochlorophyll energy transfer processes in peripheral light-harvesting complexes (LH2) from purple bacteria. We show that, by comparing excited-state dynamics of the carotenoids in organic solvents and inside the LH2 complexes, it is possible to visualize in the time domain the primary events in photosynthesis.     

Antibody-mediated delivery of IL-10 inhibits the progression of established collagen-induced arthritis

The antibody-mediated targeted delivery of cytokines to sites of disease is a promising avenue for cancer therapy, but it is largely unexplored for the treatment of chronic inflammatory conditions. Using both radioactive and fluorescent techniques, the human monoclonal antibodies L19 and G11 (specific to two markers of angiogenesis that are virtually undetectable in normal adult tissues) were found to selectively localize at arthritic sites in the murine collagen-induced model of rheumatoid arthritis following intravenous (i.v.) administration. The same animal model was used to study the therapeutic action of the L19 antibody fused to the cytokines IL-2, tumour necrosis factor (TNF) and IL-10. Whereas L19–IL-2 and L19–TNF treatment led to increased arthritic scores and paw swellings, the fusion protein L19–IL-10 displayed a therapeutic activity, which was superior to the activity of IL-10 fused to an antibody of irrelevant specificity in the mouse. The anti-inflammatory cytokine IL-10 has been investigated for the treatment of patients with rheumatoid arthritis, but clinical development plans have been discontinued because of a lack of efficacy. Because the antigen recognised by L19 is strongly expressed at sites of arthritis in humans and identical in both mice and humans, it suggests that the fusion protein L19–IL-10 might help overcome some of the clinical limitations of IL-10 and provide a therapeutic benefit to patients with chronic inflammatory disorders, including arthritis.     

RNASEH1 Mutations Impair mtDNA Replication and Cause Adult-Onset Mitochondrial Encephalomyopathy

Chronic progressive external ophthalmoplegia (CPEO) is common in mitochondrial disorders and is frequently associated with multiple mtDNA deletions. The onset is typically in adulthood, and affected subjects can also present with general muscle weakness. The underlying genetic defects comprise autosomal-dominant or recessive mutations in several nuclear genes, most of which play a role in mtDNA replication. Next-generation sequencing led to the identification of compound-heterozygous RNASEH1 mutations in two singleton subjects and a homozygous mutation in four siblings. RNASEH1, encoding ribonuclease H1 (RNase H1), is an endonuclease that is present in both the nucleus and mitochondria and digests the RNA component of RNA-DNA hybrids. Unlike mitochondria, the nucleus harbors a second ribonuclease (RNase H2). All affected individuals first presented with CPEO and exercise intolerance in their twenties, and these were followed by muscle weakness, dysphagia, and spino-cerebellar signs with impaired gait coordination, dysmetria, and dysarthria. Ragged-red and cytochrome c oxidase (COX)-negative fibers, together with impaired activity of various mitochondrial respiratory chain complexes, were observed in muscle biopsies of affected subjects. Western blot analysis showed the virtual absence of RNase H1 in total lysate from mutant fibroblasts. By an in vitro assay, we demonstrated that altered RNase H1 has a reduced capability to remove the RNA from RNA-DNA hybrids, confirming their pathogenic role. Given that an increasing amount of evidence indicates the presence of RNA primers during mtDNA replication, this result might also explain the accumulation of mtDNA deletions and underscores the importance of RNase H1 for mtDNA maintenance.     

Optimization of human dendritic cell sample preparation for mass spectrometry-based proteomic studies

Dendritic cells (DCs) are specialized leukocytes that orchestrate the adaptive immune response. Mass spectrometry (MS)-based proteomic study of these cells presents technical challenges, especially when the DCs are human in origin due to the paucity of available biological material. Here, to maximize MS coverage of the global human DC proteome, different cell disruption methods, lysis conditions, protein precipitation, and protein pellet solubilization and denaturation methods were compared. Mechanical disruption of DC cell pellets under cryogenic conditions, coupled with the use of RIPA (radioimmunoprecipitation assay) buffer, was shown to be the method of choice based on total protein extraction and on the solubilization and identification of nuclear proteins. Precipitation by acetone was found to be more efficient than that by 10% trichloroacetic acid (TCA)/acetone, allowing in excess of 28% more protein identifications. Although being an effective strategy to eliminate the detergent residue, the acetone wash step caused a loss of protein identifications. However, this potential drawback was overcome by adding 1% sodium deoxycholate into the dissolution buffer, which enhanced both solubility of the precipitated proteins and digestion efficiency. This in turn resulted in 6 to 11% more distinct peptides and 14 to 19% more total proteins identified than using 0.5 M triethylammonium bicarbonate alone, with the greatest increase (34%) for hydrophobic proteins.     

Elementary Energy Transfer Pathways in Allochromatium vinosum Photosynthetic Membranes

Allochromatium vinosum (formerly Chromatium vinosum) purple bacteria are known to adapt their light-harvesting strategy during growth according to environmental factors such as temperature and average light intensity. Under low light illumination or low ambient temperature conditions, most of the LH2 complexes in the photosynthetic membranes form a B820 exciton with reduced spectral overlap with LH1. To elucidate the reason for this light and temperature adaptation of the LH2 electronic structure, we performed broadband femtosecond transient absorption spectroscopy as a function of excitation wavelength in A. vinosum membranes. A target analysis of the acquired data yielded individual rate constants for all relevant elementary energy transfer (ET) processes. We found that the ET dynamics in high-light-grown membranes was well described by a homogeneous model, with forward and backward rate constants independent of the pump wavelength. Thus, the overall B800→B850→B890→ Reaction Center ET cascade is well described by simple triexponential kinetics. In the low-light-grown membranes, we found that the elementary backward transfer rate constant from B890 to B820 was strongly reduced compared with the corresponding constant from B890 to B850 in high-light-grown samples. The ET dynamics of low-light-grown membranes was strongly dependent on the pump wavelength, clearly showing that the excitation memory is not lost throughout the exciton lifetime. The observed pump energy dependence of the forward and backward ET rate constants suggests exciton diffusion via B850→ B850 transfer steps, making the overall ET dynamics nonexponential. Our results show that disorder plays a crucial role in our understanding of low-light adaptation in A. vinosum.     

The Voltage-dependent Anion Channel 1 Mediates Amyloid β Toxicity and Represents a Potential Target for Alzheimer Disease Therapy

The voltage-dependent anion channel 1 (VDAC1), found in the mitochondrial outer membrane, forms the main interface between mitochondrial and cellular metabolisms, mediates the passage of a variety of molecules across the mitochondrial outer membrane, and is central to mitochondria-mediated apoptosis. VDAC1 is overexpressed in post-mortem brains of Alzheimer disease (AD) patients. The development and progress of AD are associated with mitochondrial dysfunction resulting from the cytotoxic effects of accumulated amyloid β (Aβ). In this study we demonstrate the involvement of VDAC1 and a VDAC1 N-terminal peptide (VDAC1-N-Ter) in Aβ cell penetration and cell death induction. Aβ directly interacted with VDAC1 and VDAC1-N-Ter, as monitored by VDAC1 channel conductance, surface plasmon resonance, and microscale thermophoresis. Preincubated Aβ interacted with bilayer-reconstituted VDAC1 and increased its conductance ∼2-fold. Incubation of cells with Aβ resulted in mitochondria-mediated apoptotic cell death. However, the presence of non-cell-penetrating VDAC1-N-Ter peptide prevented Aβ cellular entry and Aβ-induced mitochondria-mediated apoptosis. Likewise, silencing VDAC1 expression by specific siRNA prevented Aβ entry into the cytosol as well as Aβ-induced toxicity. Finally, the mode of Aβ-mediated action involves detachment of mitochondria-bound hexokinase, induction of VDAC1 oligomerization, and cytochrome c release, a sequence of events leading to apoptosis. As such, we suggest that Aβ-mediated toxicity involves mitochondrial and plasma membrane VDAC1, leading to mitochondrial dysfunction and apoptosis induction. The VDAC1-N-Ter peptide targeting Aβ cytotoxicity is thus a potential new therapeutic strategy for AD treatment.