Formation of the hydrophobic core of ribonuclease A through sequential coordinated conformational transitions

With steady-state and time-resolved fluorescence energy-transfer measurements, we determined the distributions of intramolecular distances in nine mutants to study the conformations of wild-type ribonuclease A in the reduced state under folding conditions. Although far-UV-CD measurements show no evidence for a secondary-structure transition, temperature- and GdnHCl-induced changes in intramolecular distance distributions in the reduced state revealed evidence for long-range subdomain structures in the denatured protein. These poorly defined structures, reflected here by wide distributions corresponding to a wide range of energies, form during refolding in a complex sequence of multiple subdomain transitions. A more well-defined structure emerges only when this structural framework, which directs the successive steps in the folding process, matures and is reinforced by stronger interactions such as disulfide bonds.     

All-optical histology using ultrashort laser pulses

As a means to automate the three-dimensional histological analysis of brain tissue, we demonstrate the use of femtosecond laser pulses to iteratively cut and image fixed as well as fresh tissue. Cuts are accomplished with 1 to 10 microJ pulses to ablate tissue with micron precision. We show that the permeability, immunoreactivity, and optical clarity of the tissue is retained after pulsed laser cutting. Further, samples from transgenic mice that express fluorescent proteins retained their fluorescence to within microns of the cut surface. Imaging of exogenous or endogenous fluorescent labels down to 100 microm or more below the cut surface is accomplished with 0.1 to 1 nJ pulses and conventional two-photon laser scanning microscopy. In one example, labeled projection neurons within the full extent of a neocortical column were visualized with micron resolution. In a second example, the microvasculature within a block of neocortex was measured and reconstructed with micron resolution.     

Autosomal-Recessive Hypophosphatemic Rickets Is Associated with an Inactivation Mutation in the ENPP1 Gene

Human disorders of phosphate (Pi) handling and hypophosphatemic rickets have been shown to result from mutations in PHEX, FGF23, and DMP1, presenting as X-linked recessive, autosomal-dominant, and autosomal-recessive patterns, respectively. We present the identification of an inactivating mutation in the ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene causing autosomal-recessive hypophosphatemic rickets (ARHR) with phosphaturia by positional cloning. ENPP1 generates inorganic pyrophosphate (PPi), an essential physiologic inhibitor of calcification, and previously described inactivating mutations in this gene were shown to cause aberrant ectopic calcification disorders, whereas no aberrant calcifications were present in our patients. Our surprising result suggests a different pathway involved in the generation of ARHR and possible additional functions for ENPP1.     

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.     

A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms

Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce "miniSOG" (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.     

Effects of IKAP/hELP1 deficiency on gene expression in differentiating neuroblastoma cells: implications for familial dysautonomia

Familial dysautonomia (FD) is a developmental neuropathy of the sensory and autonomous nervous systems. The IKBKAP gene, encoding the IKAP/hELP1 subunit of the RNA polymerase II Elongator complex is mutated in FD patients, leading to a tissue-specific mis-splicing of the gene and to the absence of the protein in neuronal tissues. To elucidate the function of IKAP/hELP1 in the development of neuronal cells, we have downregulated IKBKAP expression in SHSY5Y cells, a neuroblastoma cell line of a neural crest origin. We have previously shown that these cells exhibit abnormal cell adhesion when allowed to differentiate under defined culture conditions on laminin substratum. Here, we report results of a microarray expression analysis of IKAP/hELP1 downregulated cells that were grown on laminin under differentiation or non-differentiation growth conditions. It is shown that under non-differentiation growth conditions, IKAP/hELP1 downregulation affects genes important for early developmental stages of the nervous system, including cell signaling, cell adhesion and neural crest migration. IKAP/hELP1 downregulation during differentiation affects the expression of genes that play a role in late neuronal development, in axonal projection and synapse formation and function. We also show that IKAP/hELP1 deficiency affects the expression of genes involved in calcium metabolism before and after differentiation of the neuroblastoma cells. Hence, our data support IKAP/hELP1 importance in the development and function of neuronal cells and contribute to the understanding of the FD phenotype.     

Structure-based analysis of VDAC1: N-terminus location, translocation, channel gating and association with anti-apoptotic proteins

Structural studies place the VDAC1 (voltage-dependent anion channel 1) N-terminal region within the channel pore. Biochemical and functional studies, however, reveal that the N-terminal domain is cytoplasmically exposed. In the present study, the location and translocation of the VDAC1 N-terminal domain, and its role in voltage-gating and as a target for anti-apoptotic proteins, were addressed. Site-directed mutagenesis and cysteine residue substitution, together with a thiol-specific cross-linker, served to show that the VDAC1 N-terminal region exists in a dynamic equilibrium, located within the pore or exposed outside the β-barrel. Using a single cysteine-residue-bearing VDAC1, we demonstrate that the N-terminal region lies inside the pore. However, the same region can be exposed outside the pore, where it dimerizes with the N-terminal domain of a second VDAC1 molecule. When the N-terminal region α-helix structure was perturbed, intra-molecular cross-linking was abolished and dimerization was enhanced. This mutant also displays reduced voltage-gating and reduced binding to hexokinase, but not to the anti-apoptotic proteins Bcl-2 and Bcl-xL. Replacing glycine residues in the N-terminal domain GRS (glycine-rich sequence) yielded less intra-molecular cross-linked product but more dimerization, suggesting that GRS provides the flexibility needed for N-terminal translocation from the internal pore to the channel face. N-terminal mobility may thus contribute to channel gating and interaction with anti-apoptotic proteins.     

The FSH-inhibin axis in Prader-Willi Syndrome: heterogeneity of gonadal dysfunction

ABSTRACT: BACKGROUND: We characterized the spectrum and etiology of hypogonadism in a cohort of Prader-Willi syndrome (PWS) adolescents and adults. METHODS: Reproductive hormonal profiles and physical examination were performed on 19 males and 16 females ages 16-34 years with PWS. Gonadotropins, sex-steroids, inhibin B (INB) and anti-Mullerian hormone (AMH) were measured. We defined 4 groups according to the relative contribution of central and gonadal dysfunction based on FSH and INB levels: Group A: primary hypogonadism (FSH >15 IU/l and undetectable INB (<10 pg/ml); Group B: central hypogonadism (FSH <0.5 IU/l, INB <10pg/ml); Group C: partial gonadal & central dysfunction (FSH 1.5-15 IU/l, INB >20 pg/ml); Group D: mild central and severe gonadal dysfunction (FSH 1.5-15 IU/l, INB < 10 pg/ml. RESULTS: There were 10, 8, 9 and 8 individuals in Groups A-D respectively; significantly more males in group A (9, 4, 4 and 2; P=0.04). Significant differences between the groups were found in mean testosterone (P=0.04), AMH (P=0.003) and pubic hair (P=0.04) in males and mean LH (P=0.003) and breast development (P=0.04) in females. Mean age, height, weight, BMI and the distribution of genetic subtypes were similar within the groups. CONCLUSIONS: Analysis of FSH and inhibin B revealed four distinct phenotypes ranging from primary gonadal to central hypogonadism. Primary gonadal dysfunction was common, while severe gonadotropin deficiency was rare. Longitudinal studies are needed to verify whether the individual phenotypes are consistent.