Spinal muscular atrophy (SMA) is a motor-neuron disorder resulting from anterior-horn-cell death. The autosomal recessive form has a carrier frequency of 1 in 50 and is the most common genetic cause of infant death. SMA is categorized as types I-III, ranging from severe to mild, based upon age of onset and clinical course. Two closely flanking copies of the survival motor neuron (SMN) gene are on chromosome 5q13 (ref. 1). The telomeric SMN (SMN1) copy is homozygously deleted or converted in >95% of SMA patients, while a small number of SMA disease alleles contain missense mutations within the carboxy terminus. We have identified a modular oligomerization domain within exon 6 of SMN1. All previously identified missense mutations map within or immediately adjacent to this domain. Comparison of wild-type to mutant SMN proteins of type I, II and III SMA patients showed a direct correlation between oligomerization and clinical type. Moreover, the most abundant centromeric SMN product, which encodes exons 1-6 but not 7, demonstrated reduced self-association. These findings identify decreased SMN self-association as a biochemical defect in SMA, and imply that disease severity is proportional to the intracellular concentration of oligomerization-competent SMN proteins.

Age-related macular degeneration (AMD) is the major cause of blindness in developed nations. AMD is characterized by retinal pigmented epithelial (RPE) cell dysfunction and loss of photoreceptor cells. Epidemiologic studies indicate important contributions of dietary patterns to the risk for AMD, but the mechanisms relating diet to disease remain unclear. Here we investigate the effect on AMD of isocaloric diets that differ only in the type of dietary carbohydrate in a wild-type aged-mouse model. The consumption of a high-glycemia (HG) diet resulted in many AMD features (AMDf), including RPE hypopigmentation and atrophy, lipofuscin accumulation, and photoreceptor degeneration, whereas consumption of the lower-glycemia (LG) diet did not. Critically, switching from the HG to the LG diet late in life arrested or reversed AMDf.LG diets limited the accumulation of advanced glycation end products, long-chain polyunsaturated lipids, and their peroxidation end-products and increased C3-carnitine in retina, plasma, or urine. Untargeted metabolomics revealed microbial cometabolites, particularly serotonin, as protective against AMDf. Gut microbiota were responsive to diet, and we identified microbiota in the Clostridiales order as being associated with AMDf and the HG diet, whereas protection from AMDf was associated with the Bacteroidales order and the LG diet. Network analysis revealed a nexus of metabolites and microbiota that appear to act within a gut-retina axis to protect against diet-and age-induced AMDf. The findings indicate a functional interaction between dietary carbohydrates, the metabolome, including microbial cometabolites, and AMDf. Our studies suggest a simple dietary intervention that may be useful in patients to arrest AMD.age-related macular degeneration | glycemic index | advanced glycation end-product | gut microbiome | metabolomics A ge-related macular degeneration (AMD) is the leading cause of irremediable blindness in the industrialized world, with 200 million cases projected by 2020, at a cost of $300 billion (1, 2). Dry AMD accounts for the great majority of cases and is associated with photoreceptor cell loss, often preceded by compromise to the retina pigment epithelium (RPE) cells that nourish and remove waste from the photoreceptors. The etiology of AMD remains an enigma but is clearly multifactorial. Stresses associated with AMD include environment, age, and genetics (3). Frustratingly, there are no early biomarkers to anticipate AMD, and there are no therapies or cure.Recently, we and others observed in epidemiologic studies that, in addition to micronutrients (4-6), macronutrient quality [e.g., consuming a diet with a high glycemic index (GI)] is a significant risk factor for AMD onset and/or progress in nondiabetic humans (7)(8)(9). The GI appears to be an attractive dietary intervention target, because simple replacement of small amounts of high-index foods (such as white bread) with lower-index foods (such as wholegrain bread) can significantly reduce glycemic peaks without requiring ...

The blood coagulation and regulatory proteins that contain ␥-carboxyglutamic acid are a part of a unique class of membrane binding proteins that require calcium for their interaction with cell membranes. Following protein biosynthesis, glutamic acids on these proteins are converted to ␥-carboxyglutamic acid (Gla) in a reaction that requires vitamin K as a cofactor. The vitamin K-dependent proteins undergo a conformational transition upon metal ion binding, but only calcium ions mediate protein-phospholipid interaction. To identify the site on Factor IX that is required for phospholipid binding, we have determined the three-dimensional structure of the Factor IX Gla domain bound to magnesium ions by NMR spectroscopy. By comparison of this structure to that of the Gla domain bound to calcium ions, we localize the membrane binding site to a highly ordered structure including residues 1-11 of the Gla domain. In the presence of Ca 2؉ , Factor IX Gla domain peptides that contain the photoactivatable amino acid p-benzoyl-L-phenylalanine at positions 6 or 9 cross-link to phospholipid following irradiation, while peptides lacking this amino acid analog or with this analog at position 46 did not cross-link. These results indicate that the NH 2 terminus of the Gla domain, specifically including leucine 6 and phenylalanine 9 in the hydrophobic patch, is the contact surface on Factor IX that interacts with the phospholipid bilayer.

Using a combination of cysteine mutagenesis and covalent cross-linking, we have identified subunits in close proximity to specific sites within subunit B of the vacuolar (H(+))-ATPase (V-ATPase) of yeast. Unique cysteine residues were introduced into subunit B by site-directed mutagenesis, and the resultant V-ATPase complexes were reacted with the bifunctional, photoactivatable maleimide reagent 4-(N-maleimido)benzophenone (MBP) followed by irradiation. Cross-linked products were identified by Western blot using subunit-specific antibodies. Introduction of cysteine residues at positions Glu(106) and Asp(199) led to cross-linking of subunits B and E, at positions Asp(341) and Ala(424) to cross-linking of subunits B and D, and at positions Ala(15) and Lys(45) to cross-linking of subunits B and G. Using a molecular model of subunit B constructed on the basis of sequence homology between the V- and F-ATPases, the X-ray coordinates of the F(1)-ATPase, and energy minimization, Glu(106), Asp(199), Ala(15), and Lys(45) are all predicted to be located on the outer surface of the complex, with Ala(15) and Lys(45) located near the top of the complex furthest from the membrane. By contrast, Asp(341) and Ala(424) are predicted to face the interior of the A(3)B(3) hexamer. These results suggest that subunits E and G form part of a peripheral stalk connecting the V(1) and V(0) domains whereas subunit D forms part of a central stalk. Subunit D is thus the most likely homologue to the gamma subunit of F(1), which undergoes rotation during ATP hydrolysis and serves an essential function in rotary catalysis.

We have employed a combination of site-directed mutagenesis and covalent cross-linking to identify subunits in close proximity to subunit B in the vacuolar H ؉ -ATPase (V-ATPase) complex. Unique cysteine residues were introduced into a Cys-less form of subunit B, and the V-ATPase complex in isolated vacuolar membranes from each mutant strain was reacted with the bifunctional, photoactivable maleimide reagent 4-(N-maleimido)benzophenone. are predicted to reside near the bottom of V 1 , with all four residues predicted to be oriented toward the external surface of the complex. A model incorporating these and previous data is presented in which subunit E exists in an extended conformation on the outer surface of the A 3 B 3 hexamer that forms the core of the V 1 domain. This location for subunit E suggests that this subunit forms part of the peripheral stalk of the V-ATPase that links the V 1 and V 0 domains.The vacuolar H ϩ -ATPases (V-ATPases) 1 are a family of ATPdependent proton pumps that are responsible for acidification of intracellular compartments in eukaryotic cells. The VATPases are present in a variety of intracellular compartments, including clathrin-coated vesicles; endosomes; lysosomes; Golgi-derived vesicles; chromaffin granules; synaptic vesicles; and central vacuoles of yeast, Neurospora, and plants (1-8). Acidification of vacuolar compartments plays an important role in a variety of cellular processes, including receptormediated endocytosis, intracellular targeting, protein processing and degradation, and coupled transport. The V-ATPases are also present in the plasma membrane of various specialized cells, including osteoclasts (9), renal intercalated cells (10), and neutrophils (11), where they function in such processes as bone resorption, renal acidification, and pH homeostasis, respectively.The V-ATPases are composed of two functional domains, V 1 and V 0 (1-8). The V 1 domain is a 570-kDa peripheral complex containing eight different subunits with molecular masses of 70 to 14 kDa and is responsible for ATP hydrolysis. The nucleotide-binding sites are located on two subunits of the V 1 domain: the 69-kDa A subunit and the 57-kDa B subunit. The V 0 domain is a 260-kDa integral complex composed of five subunits with molecular masses of 100 to 17 kDa and is responsible for proton translocation.The V-ATPases are structurally and evolutionarily related to the ATP synthases (or F-ATPases) of mitochondria, chloroplasts, and bacteria (12-17). Thus, the nucleotide-binding subunits of the V-ATPase (A and B) are homologous to the corresponding ␤ and ␣ subunits of F 1 (18,19), and the proteolipid subunits of the two complexes are also homologous (20, 21). The structure of the peripheral F 1 domain of the mitochondrial F-ATPase has been determined by x-ray crystallography and shown to consist of a hexamer of alternating ␣ and ␤ subunits surrounding a central cavity containing the highly ␣-helical ␥ subunit (22-24). F 1 is attached to the F 0 domain via both a central stalk, which includes both the ␥ and ⑀...

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We recently identified a cellular protein named E6BP or ERC-55 that binds cancer-related papillomavirus E6 proteins (Chen, J. J., Reid, C. E., Band, V., and Androphy, E. J. (1995) Science 269, 529 -531). By construction of a series of deletion mutants, the region of E6BP that is necessary and sufficient for complex formation with human papillomavirus type 16 E6 has been mapped to a 25-amino acid domain. The corresponding peptide was synthesized and found by nuclear magnetic resonance spectroscopy to bind calcium and fold into a classical helix-loop-helix EF-hand conformation. Additional deletion mutagenesis showed that 13 amino acids that form the second ␣ helix mediated E6 association. Alanine replacement mutagenesis indicated that amino acids of this helix were most important for E6 binding. The transforming properties of HPVs reside in two genes, E6 and E7. The E6 and E7 genes are consistently expressed in HPV-positive cervical cancers and derived cell lines (5-7). They cooperate to immortalize primary human keratinocytes (8 -13). HPV-16 E6 also cooperates with activated Ras in the transformation and immortalization of baby mouse kidney cells and baby rat kidney cells (14, 15). Independently of E7 or ras, HPV-16 E6 can transform NIH 3T3 cells (16), immortalize human mammary epithelial cells (17), and induce keratinocyte resistance to calcium and serum-induced differentiation (18). The activity of E6 in different biological assays implies it may influence diverse cellular pathways.The ability of E6 protein to associate with the cellular tumor suppressor p53 has been suggested as the mechanism by which the viral protein promotes cell growth and proliferation (19). Although binding of high risk HPV E6s with p53 appears to be mediated by another cellular protein, E6AP (20), direct in vitro association of E6 with p53 has also been observed (21,22). The complex of E6 and E6AP functions as an ubiquitin-protein ligase that results in the specific ubiquitination and subsequent degradation of p53 (23). Accumulating evidence suggests that E6 has functions independent of inactivating p53 in cellular transformation (24 -36).We have recently identified a cDNA encoding a cellular protein that binds papillomavirus E6 (E6BP or ERC-55) (1). E6BP was identified as a calcium-binding protein of the endoplasmic reticulum (37). The localization of E6BP is consistent with the localization of E6 to nonnuclear membranes (38). In vitro binding experiments demonstrated that E6BP interacted specifically with E6 proteins from cancer-related HPV types and the bovine papillomavirus type 1 (BPV-1). The transforming activity of a set of previously characterized BPV-1 E6 mutants correlated well with their E6BP binding ability. These results suggest that the E6BP interaction plays an important role for BPV-1 E6-induced transformation. More recently, it was reported that BPV-1 E6 associated with paxillin (39) as well as the trans-Golgi network-specific clathrin adaptor complex AP-1 (40) and that E6 proteins from some high risk HPVs interacts wit...

Rab11-FIP2 is a member of a newly identified family of Rab11-binding proteins that have been implicated in the function of recycling endosomes. Here we show that Rab11-FIP2 may also be involved with the process of receptor-mediated endocytosis. First we demonstrate that Rab11-FIP2 contains an NPF motif that allows it to bind Reps1, a member of a family of EH domain proteins involved in endocytosis. We also show that Rab11-FIP2 associates with the ␣-adaptin subunit of AP-2 complexes, which are known to recruit receptors into clathrin-coated vesicles. Finally, we find that overexpression of Rab11-FIP2 suppresses the internalization of epidermal growth factor receptors, but not transferrin receptors, through binding sites that promote complex formation with Rab11, Reps1, and ␣-adaptin. These findings suggest that Rab11-FIP2 may participate in the coupling of receptor-mediated endocytosis to the subsequent sorting of receptor-containing vesicles in endosomes.