The Nutrigenetics of Hyperhomocysteinemia: QUANTITATIVE PROTEOMICS REVEALS DIFFERENCES IN THE METHIONINE CYCLE ENZYMES OF GENE-INDUCED VERSUS DIET-INDUCED HYPERHOMOCYSTEINEMIA*

Hyperhomocysteinemia has long been associated with atherosclerosis and thrombosis and is an independent risk factor for cardiovascular disease. Its causes include both genetic and environmental factors. Although homocysteine is produced in every cell as an intermediate of the methionine cycle, the liver contributes the major portion found in circulation, and fatty liver is a common finding in homocystinuric patients. To understand the spectrum of proteins and associated pathways affected by hyperhomocysteinemia, we analyzed the mouse liver proteome of gene-induced (cystathionine β-synthase (CBS)) and diet-induced (high methionine) hyperhomocysteinemic mice using two-dimensional difference gel electrophoresis and Ingenuity Pathway Analysis. Nine proteins were identified whose expression was significantly changed by 2-fold (p ≤ 0.05) as a result of genotype, 27 proteins were changed as a result of diet, and 14 proteins were changed in response to genotype and diet. Importantly, three enzymes of the methionine cycle were up-regulated. S-Adenosylhomocysteine hydrolase increased in response to genotype and/or diet, whereas glycine N-methyltransferase and betaine-homocysteine methyltransferase only increased in response to diet. The antioxidant proteins peroxiredoxins 1 and 2 increased in wild-type mice fed the high methionine diet but not in the CBS mutants, suggesting a dysregulation in the antioxidant capacity of those animals. Furthermore, thioredoxin 1 decreased in both wild-type and CBS mutants on the diet but not in the mutants fed a control diet. Several urea cycle proteins increased in both diet groups; however, arginase 1 decreased in the CBS^+/− mice fed the control diet. Pathway analysis identified the retinoid X receptor signaling pathway as the top ranked network associated with the CBS^+/− genotype, whereas xenobiotic metabolism and the NRF2-mediated oxidative stress response were associated with the high methionine diet. Our results show that hyperhomocysteinemia, whether caused by a genetic mutation or diet, alters the abundance of several liver proteins involved in homocysteine/methionine metabolism, the urea cycle, and antioxidant defense.Homocysteine (Hcy)1 is a thiol-containing amino acid that is produced in every cell of the body as an intermediate of the methionine cycle (Fig. 1, Reactions 1–5) (1). Once formed, the catabolism of homocysteine occurs via three enzymatic pathways. 1) Hcy is remethylated back to methionine using vitamin B₁₂-dependent methionine synthase (Fig. 1, Reaction 4) and/or 2) betaine-homocysteine methyltransferase (BHMT) (Fig. 1, Reaction 5), and 3) Hcy is converted to cysteine via the transsulfuration pathway using CBS and γ-cystathionase (Fig. 1, Reactions 6 and 7). Under normal conditions ∼40–50% of the Hcy that is produced in the liver is remethylated, ∼40–50% is converted to cysteine, and a small amount is exported (1–3). However, when Hcy production is increased (i.e. increased dietary methionine/protein intake) or when Hcy catabolism is decreased (i.e. CBS deficiency or B vitamin deficiencies), excess Hcy is exported into the extracellular space, resulting in hyperhomocysteinemia (1–5).Open in a separate windowFig. 1.Homocysteine metabolism in liver and kidney. In classical homocystinuria, the initial enzyme of the transsulfuration pathway, CBS (Reaction 6), is deficient. MTHF, methylenetetrahydrofolate; THF, tetrahydrofolate; DHF, dihydrofolate; MeCbl, methylcobalamin; DMG, dimethylglycine; PLP, pyridoxal 5′-phosphate.Homocystinuria was first described in the 1960s by Carson et al. (6): they observed 10 pediatric patients with severely elevated levels of Hcy in the urine and hypermethioninemia. Normal concentrations of plasma total homocysteine (tHcy) range from 5 to 12 μm (7); however, in homocystinuria, tHcy levels can exceed 100 μm. Homocystinuric patients present with mental retardation, abnormal bone growth, fine hair, malar flush, and dislocation of the lens of the eye, and most die from premature cardiovascular disease (6, 8). Autopsy findings indicate widespread thromboembolism, arteriosclerosis, and fatty livers (6, 8). Mudd et al. (9, 10) identified the cause of homocystinuria as a defect in the enzyme cystathionine β-synthase. A recent study of newborn infants in Denmark estimated the birth prevalence for CBS heterozygosity to be about 1:20,000 (11).Plasma tHcy concentrations are also directly correlated with dietary methionine/protein intake (12, 13). Guttormsen et al. (13) demonstrated that a protein-rich meal affected tHcy for at least 8–24 h. When normal subjects were fed a low protein-containing breakfast (12–15 g), plasma methionine levels increased slightly after 2 h (22.5–27.5 μm), but tHcy levels did not change significantly. However, when these same subjects were fed a high protein meal (52 g), plasma methionine levels peaked after 4 h (38 μm), and tHcy rose steadily until a maximum level was reached 8 h postmeal (7.6 versus 8.5 μm) (13). Thus, the following questions can be raised. How does the hepatic proteome respond to a hyperhomocysteinemic diet, and are the changes that accompany such a diet the same as or different from those that may be observed in gene-induced hyperhomocysteinemia?Because hyperhomocysteinemia is a strong independent risk factor for cardiovascular, cerebrovascular, and peripheral vascular disease, most of the current research has focused on the mechanisms involved in Hcy-induced endothelial dysfunction (14–24). The results of those studies have concluded that Hcy induces intracellular oxidative stress by generating ROS, which in turn lead to decreased bioavailable nitric oxide (NO), altered gene expression, increased endoplasmic reticulum stress, and activation of cholesterol biosynthesis. Also, several studies have examined the association between hyperhomocysteinemia and alcoholic liver disease, but few have looked at the effect of Hcy on the non-alcoholic liver even though fatty liver is a constant finding in homocystinuria (6, 8), and the liver is the major source of circulating Hcy (4, 5, 10). We hypothesize that 1) the liver proteome will respond to hyperhomocysteinemia by altering the expression of proteins involved in methionine/homocysteine metabolism and antioxidant defense and that 2) the set of proteins that are expressed when hyperhomocysteinemia is induced by CBS deficiency will differ from those expressed as a result of a high methionine diet. In the present study, we use a well established mouse model of CBS deficiency to study the early changes in the liver proteome that accompany hyperhomocysteinemia (25).     

FANCI Protein Binds to DNA and Interacts with FANCD2 to Recognize Branched Structures

In this study, we report that the purified wild-type FANCI (Fanconi anemia complementation group I) protein directly binds to a variety of DNA substrates. The DNA binding domain roughly encompasses residues 200–1000, as suggested by the truncation study. When co-expressed in insect cells, a small fraction of FANCI forms a stable complex with FANCD2 (Fanconi anemia complementation group D2). Intriguingly, the purified FANCI-FANCD2 complex preferentially binds to the branched DNA structures when compared with either FANCI or FANCD2 alone. Co-immunoprecipitation with purified proteins indicates that FANCI interacts with FANCD2 through its C-terminal amino acid 1001–1328 fragment. Although the C terminus of FANCI is dispensable for direct DNA binding, it seems to be involved in the regulation of DNA binding activity. This notion is further enhanced by two C-terminal point mutations, R1285Q and D1301A, which showed differentiated DNA binding activity. We also demonstrate that FANCI forms discrete nuclear foci in HeLa cells in the absence or presence of exogenous DNA damage. The FANCI foci are colocalized perfectly with FANCD2 and partially with proliferating cell nuclear antigen irrespective of mitomycin C treatment. An increased number of FANCI foci form and become resistant to Triton X extraction in response to mitomycin C treatment. Our data suggest that the FANCI-FANCD2 complex may participate in repair of damaged replication forks through its preferential recognition of branched structures.Fanconi anemia (FA)³ is a genetic disorder characterized by chromosome instability, predisposition to cancer, hypersensitivity to DNA cross-linking agents, developmental abnormalities, and bone marrow failure (1–9). There are at least 13 distinct FA complementation groups, each of which is associated with an identified gene (2, 9, 10). Eight of them are components of the FA core complex (FANC A, B, C, E, F, G, L, and M) that is epistatic to the monoubiquitination of both FANCI and FANCD2, a key event to initiate interstrand cross-link (ICL) repair (2, 9, 11). Downstream of or parallel to the FANCI and FANCD2 monoubiquitination are the proteins involved in double strand break repair and breast cancer susceptibility (i.e. FANCD1/BRCA2, FANCJ/BRIP1, and FANCN/PALB2) (2, 9).FANCI is the most recently identified FA gene (11–13). FANCI protein is believed to form a FANCI-FANCD2 (ID) complex with FANCD2, because they co-immunoprecipitate with each other from cell lysates and their stabilities are interdependent of each other (9, 11, 13). FANCI and FANCD2 are paralogs to each other, since they share sequence homology and co-evolve in the same species (11). Both FANCI and FANCD2 can be phosphorylated by ATR/ATM (ataxia telangiectasia and Rad3-related/ataxia telangiectasia-mutated) kinases under genotoxic stress (11, 14, 15). The phosphorylation of FANCI seems to function as a molecular switch to turn on the FA repair pathway (16). The monoubiquitination of FANCD2 at lysine 561 plays a critical role in cellular resistance to DNA cross-linking agents and is required for FANCD2 to form damage-induced foci with BRCA1, BRCA2, RAD51, FANCJ, FANCN, and γ-H2AX on chromatin during S phase of the cell cycle (17–25). In response to DNA damage or replication stress, FANCI is also monoubiquitinated at lysine 523 and recruited to the DNA repair nuclear foci (11, 13). The monoubiquitination of both FANCI and FANCD2 depends on the FA core complex (11, 13, 26), and the ubiquitination of FANCI relies on the FANCD2 monoubiquitination (2, 11). In an in vitro minimally reconstituted system, FANCI enhances FANCD2 monoubiquitination and increases its specificity toward the in vivo ubiquitination site (27).FANCI is a leucine-rich peptide (14.8% of leucine residues) with limited sequence information to indicate which processes it might be involved in. Besides the monoubiquitination site Lys⁵²³ and the putative nuclear localization signals (Fig. 1A), FANCI contains both ARM (armadillo) repeats and a conserved C-terminal EDGE motif as FANCD2 does (11, 28). The EDGE sequence in FANCD2 is not required for monoubiquitination but is required for mitomycin C (MMC) sensitivity (28). The ARM repeats form α-α superhelix folds and are involved in mediating protein-protein interactions (11, 29). In addition, FANCI, at its N terminus, contains a leucine zipper domain (aa 130–151) that could be involved in mediating protein-protein or protein-DNA interactions (Fig. 1A) (30–33). FANCD2, the paralog of FANCI, was reported to bind to double strand DNA ends and Holliday junctions (34).Open in a separate windowFIGURE 1.Purified human FANCI binds to DNA promiscuously. A, schematic diagram of predicted FANCI motifs and mutagenesis strategy to define the DNA binding domain. The ranges of numbers indicate how FANCI was truncated (e.g. 801–1328 represents FANCI-(801–1328)). NLS, predicted nuclear localization signal (aa 779–795 and 1323–1328); K523, lysine 523, the monoubiquitination site. The leucine zipper (orange bars, aa 130–151), ARM repeats (green bars), and EDGE motif (blue bars) are indicated. Red bars with a slash indicate the point mutations shown on the left. B, SDS-PAGE of the purified proteins stained with Coomassie Brilliant Blue R-250. R1285Q and D1301A are two point mutants of FANCI. All FANCI variants are tagged by hexahistidine. FANCD2 is in its native form. Protein markers in kilodaltons are indicated. C, titration of WT-FANCI for the DNA binding activity. Diagrams of the DNA substrates are shown at the top of each set of reactions. *, ³²P-labeled 5′-end. HJ, Holliday junction. Concentrations of FANCI were 0, 20, 40, 60, and 80 nm (ascending triangles). The substrate concentration was 1 nm. Protein-DNA complex is indicated by an arrow. D, supershift assay. 1 nm of ssDNA was incubated with PBS (lane 1), 80 nm FANCI alone (lane 2), and 80 nm FANCI preincubated with a specific FANCI antibody (lane 3) in the condition described under “Experimental Procedures.”In order to delineate the function of FANCI protein, we purified the recombinant FANCI from the baculovirus expression system. In this study, we report the DNA binding activity of FANCI. Unlike FANCD2, FANCI binds to different DNA structures, including single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), 5′-tailed, 3′-tailed, splayed arm, 5′-flap, 3′-flap, static fork, and Holliday junction with preference toward branched structures in the presence of FANCD2. Our data suggest that the dynamic DNA binding activity of FANCI and the preferential recognition of branched structures by the ID complex are likely to be the mechanisms to initiate downstream repair events.     

Direct Fe2+ Sensing by Iron-responsive Messenger RNA��Repressor Complexes Weakens Binding

Fe²⁺ is now shown to weaken binding between ferritin and mitochondrial aconitase messenger RNA noncoding regulatory structures ((iron-responsive element) (IRE)-RNAs) and the regulatory proteins (IRPs), which adds a direct role of iron to regulation that can complement the well known regulatory protein modification and degradative pathways related to iron-induced mRNA translation. We observe that the K_d value increases 17-fold in 5′-untranslated region IRE-RNA·repressor complexes; Fe²⁺, is studied in the absence of O₂. Other metal ions, Mn²⁺ and Mg²⁺ have similar effects to Fe²⁺ but the required Mg²⁺ concentration is 100 times greater than for Fe²⁺ or Mn²⁺. Metal ions also weaken ethidium bromide binding to IRE-RNA with no effect on IRP fluorescence, using Mn²⁺ as an O₂-resistant surrogate for Fe²⁺, indicating that metal ions bound IRE-RNA but not IRP. Fe²⁺ decreases IRP repressor complex stability of ferritin IRE-RNA 5–10 times compared with 2–5 times for mitochondrial aconitase IRE-RNA, over the same concentration range, suggesting that differences among IRE-RNA structures contribute to the differences in the iron responses observed in vivo. The results show the IRE-RNA·repressor complex literally responds to Fe²⁺, selectively for each IRE-mRNA.Iron (e.g. ferrous sulfate, ferric citrate, and hemin) added to animal cells changes translation rates of messenger RNAs encoding proteins of iron traffic and oxidative metabolism (1–4). To cross cell membranes, iron ions are transported by membrane proteins such as DMT1 or carried on proteins such as transferrin. Inside the cells, iron is mainly in heme, FeS clusters, non-heme iron cofactors of proteins, and iron oxide minerals coated by protein nanocages (ferritins). Iron in transit is thought to be Fe²⁺ in labile “pools” accessible to small molecular weight chelators, and/or bound loosely by chaperones.When iron concentrations in the cells increase, a group of mRNAs with three-dimensional, noncoding structures in the 5′-untranslated region (UTR)³ are derepressed (Fig. 1A), i.e. the fraction of the mRNAs in mRNA·repressor protein complexes, which inhibit ribosome binding, decreases and the fraction of the mRNAs in polyribosomes increases (5–7). The three-dimensional, noncoding mRNA structure, representing a family of related structures, is called the iron-responsive element, or IRE, and the repressors are called iron regulatory proteins (IRPs). Together they are one of the most extensively studied eukaryotic messenger RNA regulatory systems (1–4). In addition to large numbers of cell studies, structures of IRE-RNAs are known from solution NMR (8–12), and the RNA·protein complex from x-ray crystallography (13). Recent data indicate that demetallation of IRP1 and disruption of the [4Fe-4S] cluster that inhibits IRP1 binding to RNA will be enhanced by phosphorylation and low iron concentrations (1, 2, 14–16). Such results can explain the increased IRP1 binding to IRE-mRNAs and increased translational repression when iron concentrations are abnormally low. However, the mechanism to explain dissociation of IRE-RNA·IRP complexes, thereby allowing ribosome assembly and increased proteosomal degradation of IRPs (1, 2, 14, 15) (Fig. 1A), when high iron concentrations are abnormally high, is currently unknown.Open in a separate windowFIGURE 1.IRE-RNA·IRP complexes and a model for depression by excess iron. A, a representative model of iron-induced translation of 5′-UTR IRE-RNAs. This figure is modified from Ref. 7. B, IRE-RNA sites influenced by metal binding related to the crystal structure of the ferritin-IRE-RNA·IRP complex from Ref. 13. The figure was created by T. Tosha using Discovery Studio 1.6 and Protein Data Bank file 2IPY. ■, hydrated Mg²⁺, determined by solution NMR; ▴, Cu¹⁺-1.10-phenanthroline, determined by RNA cleavage in O₂.Metal ion binding changes conformation and function of most RNA classes, e.g. rRNA (17), tRNA (18, 19), ribozymes (20–23), riboswitches (24, 25), possibly hammerhead mRNAs in mammals (26), and proteins. Although the effects of metal ion binding on eukaryotic mRNAs have not been extensively studied, Mg²⁺ is known to cause changes in conformation, shown by changes in radical cleavage sites of IRE-RNA with 1,10-phenanthrolene-iron and proton shifts in the one-dimensional NMR spectrum (12, 27). The Mg²⁺ effects are observed at low magnesium concentrations (0.1–0.5 mm) and low molar stoichiometries (1:1 and 2:1 = Mg:RNA).We hypothesized that Fe²⁺ could directly change the binding of the IRE-mRNA to the iron regulatory protein for several reasons. First, other metal ions influence the IRE-RNA structure (12, 27). Second, in IRE-RNA/IRP cocrystals there are exposed RNA sites in the IRE-RNA/IRP complex that are accessible for interactions (13) (Fig. 1B). Third, regions in the IRE-RNA are hypersensitive to Fe²⁺-EDTA/ascorbate/H₂O₂, suggesting selective interactions with metals and/or solvent (28). We now report that Fe²⁺ weakens IRE-RNA/IRP binding, whereas Mg²⁺ requires 100 times the concentration and Mn²⁺ is comparable with Fe²⁺; the Fe²⁺ effect was diminished in mutant IRE-RNA and IRE-RNA selective in wild type sequences: ferritin IRE-RNA > mt-aconitase IRE-RNA.     

Dissecting an Allosteric Switch in Caspase-7 Using Chemical and Mutational Probes

Apoptotic caspases, such as caspase-7, are stored as inactive protease zymogens, and when activated, lead to a fate-determining switch to induce cell death. We previously discovered small molecule thiol-containing inhibitors that when tethered revealed an allosteric site and trapped a conformation similar to the zymogen form of the enzyme. We noted three structural transitions that the compounds induced: (i) breaking of an interaction between Tyr-223 and Arg-187 in the allosteric site, which prevents proper ordering of the catalytic cysteine; (ii) pinning the L2′ loop over the allosteric site, which blocks critical interactions for proper ordering of the substrate-binding groove; and (iii) a hinge-like rotation at Gly-188 positioned after the catalytic Cys-186 and Arg-187. Here we report a systematic mutational analysis of these regions to dissect their functional importance to mediate the allosteric transition induced by these compounds. Mutating the hinge Gly-188 to the restrictive proline causes a massive ∼6000-fold reduction in catalytic efficiency. Mutations in the Arg-187–Tyr-223 couple have a far less dramatic effect (3–20-fold reductions). Interestingly, although the allosteric couple mutants still allow binding and allosteric inhibition, they partially relieve the mutual exclusivity of binding between inhibitors at the active and allosteric sites. These data highlight a small set of residues critical for mediating the transition from active to inactive zymogen-like states.Caspases are a family of dimeric cysteine proteases whose members control the ultimate steps for apoptosis (programmed cell death) or innate inflammation among others (for reviews, see Refs. 1 and 2). During apoptosis, the upstream initiator caspases (caspase-8 and -9) activate the downstream executioner caspases (caspase-3, -6, and-7) via zymogen maturation (3). The activated executioner caspases then cleave upwards of 500 key proteins (4–6) and DNA, leading to cell death. Due to their pivotal role in apoptosis, the caspases are involved both in embryonic development and in dysfunction in diseases including cancer and stroke (7). The 11 human caspases share a common active site cysteine-histidine dyad (8), and derive their name, cysteine aspartate proteases, from their exquisite specificity for cleaving substrate proteins after specific aspartate residues (9–13). Thus, it has been difficult to develop active site-directed inhibitors with significant specificity for one caspase over the others (14). Despite difficulties in obtaining specificity, there has been a long-standing correlation between efficacy of caspase inhibitors in vitro and their ability to inhibit caspases and apoptosis in vivo (for review, see Ref. 31). Thus, a clear understanding of in vitro inhibitor function is central to the ability control caspase function in vivo.Caspase-7 has been a paradigm for understanding the structure and dynamics of the executioner caspases (15–21). The substrate-binding site is composed of four loops; L2, L3, and L4 are contributed from one-half of the caspase dimer, and L2′ is contributed from the other half of the caspase dimer (Fig. 1). These loops appear highly dynamic as they are only observed in x-ray structures when bound to substrate or substrate analogs in the catalytically competent conformation (17–19, 22) (Fig. 1B).Open in a separate windowFIGURE 1.Allosteric site and dimeric structure in caspase-7. A, the surface of active site-bound caspase-7 shows a large open allosteric (yellow) site at the dimer interface. This cavity is distinct from the active sites, which are bound with the active site inhibitor DEVD (green sticks). B, large subunits of caspase-7 dimers (dark green and dark purple) contain the active site cysteine-histidine dyad. The small subunits (light green and light purple) contain the allosteric site cysteine 290. The conformation of the substrate-binding loops (L2, L2′, L3, and L4) in active caspase-7 (Protein Data Bank (PDB) number 1f1j) is depicted. The L2′ loop (spheres) from one-half of the dimer interacts with the L2 loop from the other half of the dimer. C, binding of allosteric inhibitors influences the conformation of the L2′ loop (spheres), which folds over the allosteric cavity (PDB number 1shj). Subunit rendering is as in panel A. Panels A, B, and C are in the same orientation.A potential alternative to active site inhibitors are allosteric inhibitors that have been seeded by the discovery of selective cysteine-tethered allosteric inhibitors for either apoptotic executioner caspase-3 or apoptotic executioner caspase-7 (23) as well as the inflammatory caspase-1 (24). These thiol-containing compounds bind to a putative allosteric site through disulfide bond formation with a thiol in the cavity at the dimer interface (Fig. 1A) (23, 24). X-ray structures of caspase-7 bound to allosteric inhibitors FICA³ and DICA (Fig. 2) show that these compounds trigger conformational rearrangements that stabilize the inactive zymogen-like conformation over the substrate-bound, active conformation. The ability of small molecules to hold mature caspase-7 in a conformation that mimics the naturally occurring, inactive zymogen state underscores the utility and biological relevance of the allosteric mechanism of inhibition. Several structural changes are evident between these allosterically inhibited and active states. (i) The allosteric inhibitors directly disrupt an interaction between Arg-187 (next to the catalytic Cys-186) and Tyr-223 that springs the Arg-187 into the active site (Fig. 3), (ii) this conformational change appears to be facilitated by a hinge-like movement about Gly-188, and (iii) the L2′ loop folds down to cover the allosteric inhibitor and assumes a zymogen-like conformation (Fig. 1C) (23).Open in a separate windowFIGURE 2.Structure of allosteric inhibitors DICA and FICA. DICA and FICA are hydrophobic small molecules that bind to an allosteric site at the dimer interface of caspase-7. Binding of DICA/FICA is mediated by a disulfide between the compound thiol and Cys-290 in caspase-7.Open in a separate windowFIGURE 3.Movement of L2′ blocking arm. The region of caspase-7 encompassing the allosteric couple Arg-187 and Tyr-223 is boxed. The inset shows the down orientation of Arg-187 and Tyr-223 in the active conformation with DEVD substrate mimic (orange spheres) in the active site. In the allosteric/zymogen conformation, Arg-187 and Tyr-223 are pushed up by DICA (blue spheres).Here, using mutational analysis and small molecule inhibitors, we assess the importance of these three structural units to modulate both the inhibition of the enzyme and the coupling between allosteric and active site labeling. Our data suggest that the hinge movement and pinning of the L2-L2′ are most critical for transitioning between the active and inactive forms of the enzyme.     

Ceramide Synthesis Is Modulated by the Sphingosine Analog FTY720 via a Mixture of Uncompetitive and Noncompetitive Inhibition in an Acyl-CoA Chain Length-de pend ent Manner

FTY720, a sphingosine analog, is in clinical trials as an immunomodulator. The biological effects of FTY720 are believed to occur after its metabolism to FTY720 phosphate. However, very little is known about whether FTY720 can interact with and modulate the activity of other enzymes of sphingolipid metabolism. We examined the ability of FTY720 to modulate de novo ceramide synthesis. In mammals, ceramide is synthesized by a family of six ceramide synthases, each of which utilizes a restricted subset of acyl-CoAs. We show that FTY720 inhibits ceramide synthase activity in vitro by noncompetitive inhibition toward acyl-CoA and uncompetitive inhibition toward sphinganine; surprisingly, the efficacy of inhibition depends on the acyl-CoA chain length. In cultured cells, FTY720 has a more complex effect, with ceramide synthesis inhibited at high (500 nm to 5 μm) but not low (<200 nm) sphinganine concentrations, consistent with FTY720 acting as an uncompetitive inhibitor toward sphinganine. Finally, electrospray ionization-tandem mass spectrometry demonstrated, unexpectedly, elevated levels of ceramide, sphingomyelin, and hexosylceramides after incubation with FTY720. Our data suggest a novel mechanism by which FTY720 might mediate some of its biological effects, which may be of mechanistic significance for understanding its mode of action.FTY720 (2-amino-(2-2-[4-octylphenyl]ethyl)propane 1,3-diol hydrochloride), also known as Fingolimod, is an immunosuppressant drug currently being tested in clinical trials for organ transplantation and autoimmune diseases such as multiple sclerosis (1). FTY720 is a structural analog of sphingosine, a key biosynthetic intermediate in sphingolipid (SL)² metabolism (see Fig. 1). In vivo, FTY720 is rapidly phosphorylated by sphingosine kinase 2 (2, 3) to form FTY720 phosphate (FTY720-P), an analog of sphingosine 1-phosphate (S1P) (see Fig. 1A). FTY720-P binds to S1P receptors (S1PRs) (4, 5) and thereby induces a variety of phenomena such as T-lymphocyte migration from lymphoid organs (6–9); accordingly, FTY720 treatment results in lymphopenia as lymphocytes (especially T-cells) become sequestered inside lymphoid organs (10–12). The ability of FTY720 to sequester lymphocytes has stimulated its use in treatment of allograft rejection and autoimmune diseases (13), and FTY720 is currently under phase III clinical trials for treatment of relapsing-remitting multiple sclerosis (14).Open in a separate windowFIGURE 1.SL structure and metabolism. A, structures of SLs and SL analogs used in this study. B, metabolic inter-relationships between SLs and the metabolism of FTY720. The enzymes are denoted in italics. LPP3, lipid phosphate phosphatase 3; LPP1α, lipid phosphate phosphatase 1α.Apart from the binding of FTY720-P to S1PRs, the ability of FTY720 to inhibit S1P lyase (15) (see Fig. 1B), and its inhibitory effect on cytosolic phospholipase A2 (16), whose activity can be modulated by ceramide 1-phosphate (17), little is known about whether FTY720 or FTY720-P can modulate the activity of other enzymes of SL metabolism. Because FTY720 is an analog of sphingosine, one of the two substrates of ceramide synthase (CerS) (see Fig. 1), we now examine whether FTY720 can modulate CerS activity. CerS utilizes fatty acyl-CoAs to N-acylate sphingoid long chain bases. Six CerS exist in mammals, each of which uses a restricted subset of acyl-CoAs (18–23). We demonstrate that FTY720 inhibits CerS activity and that the extent of inhibition varies according to the acyl chain length of the acyl-CoA substrate. Surprisingly, FTY720 inhibits CerS activity toward acyl-CoA via noncompetitive inhibition and toward sphinganine via uncompetitive inhibition. Finally, the mode of interaction of FTY720 with CerS in cultured cells depends on the amount of available sphinganine. Together, we show that FTY720 modulates ceramide synthesis, which may be of relevance for understanding its biological effects in vivo and its role in immunomodulation.     

Novel Lipofuscin Bisretinoids Prominent in Human Retina and in a Model of Recessive Stargardt Disease

Bisretinoid adducts accumulate as lipofuscin in retinal pigment epithelial (RPE) cells of the eye and are implicated in the pathology of inherited and age-related macular degeneration. Characterization of the bisretinoids A2E and the all-trans-retinal dimer series has shown that these pigments form from reactions in photoreceptor cell outer segments that involve all-trans-retinal, the product of photoisomerization of the visual chromophore 11-cis-retinal. Here we have identified two related but previously unknown RPE lipofuscin compounds. By high performance liquid chromatography-elec tro spray ionization-tandem mass spectrometry, we determined that the first of these compounds is a phosphatidyl-dihydropyridine bisretinoid; to indicate this structure and its formation from two vitamin A-aldehyde (A2), we will refer to it as A2-dihydropyridine-phosphatidyleth a nol amine (A2-DHP-PE). The second pigment, A2-dihydropyridine-eth a nol amine, forms from phosphate hydrolysis of A2-DHP-PE. The structure of A2-DHP-PE was corroborated by Fourier transform infrared spectroscopy, and density functional theory confirmed the presence of a dihydropyridine ring. This lipofuscin pigment is a fluorescent compound with absorbance maxima at ∼490 and 330 nm, and it was identified in human, mouse, and bovine eyes. We found that A2-DHP-PE forms in reaction mixtures of all-trans-retinal and phosphatidyleth a nol amine, and in mouse eyecups we observed an age-related accumulation. As compared with wild-type mice, A2-DHP-PE is more abundant in mice with a null mutation in Abca4 (ATP-binding cassette transporter 4), the gene causative for recessive Stargardt macular degeneration. Efforts to clarify the composition of RPE lipofuscin are important because these compounds are targets of gene-based and drug therapies that aim to alleviate ABCA4-related retinal disease.Throughout the life of an individual, retinal pigment epithelial (RPE)² cells of the eye accumulate bisretinoid adducts that comprise the lipofuscin of these cells (1–3). The compounds form as a byproduct of light-mediated isomerization of the visual chromophore 11-cis-retinal. Accordingly, conditions that reduce the production of all-trans-retinal (atRAL) from 11-cis-retinal photoisomerization, such as reduced serum vitamin A (4–6), variants, or mutations in the visual cycle protein RPE65 (7–9) and inhibitors of RPE65 and 11-cis retinol dehydrogenase (10–13), substantially reduce the formation of this material.Up to the present time, at least 17 constituents of RPE lipofuscin have been identified chromatographically and characterized structurally; added to these are biosynthetic intermediates such as N-retinylidene-phosphatidylethanolamine (NRPE), A2PE and dihydropyridinium-A2PE (see Fig. 1, A and B) (14–19). The first RPE lipofuscin constituent to be described was A2E (see Fig. 1A, inset). The pyridinium bisretinoid (14–16, 20, 21) structure of A2E (C₄₂H₅₈NO; molecular weight, 592) was confirmed by extensive nuclear magnetic resonance studies (14) and by total synthesis (22). A2E formation begins in photoreceptor outer segments when atRAL, instead of being reduced to all-trans-retinol, reacts with phosphatidylethanolamine (PE) in a 2:1 ratio. Although the double bonds along the side arms of A2E are all in the trans (E) position, Z-isomers of A2E have double bonds at the C-13/14 (isoA2E), C-9/9′-10/10′, and C-11/11′-12/12′ positions, and all are detectable in human and mouse RPE (16). These pigments exhibit absorbances in both the UV and visible regions of the spectrum (A2E: λ_max, 439 and 338 nm; iso-A2E: λ_max, 428 and 337 nm).Open in a separate windowFIGURE 1.Some bisretinoid compounds associated with RPE lipofuscin formation. Structures, molecular weight (Mw), UV-visible absorbance (nm), and electronic transition assignments (↔). Phosphate hydrolysis (dashed lines) of A2PE (A) and A2-DHP-PE (C) generates A2E and A2-DHP-E (insets in A and C). A2PE (A), dihydropyridinium-A2PE (B), A2-DHP-PE (C). The molecular weights are based on dipalmitic acid as the fatty acid constituent.Another bisretinoid compound of RPE lipofuscin also absorbs in the short wavelength region of the visible spectrum (17, 18, 23). This pigment, all-trans-retinal dimer (atRAL dimer; λ_max, 432 and 290 nm) forms from the condensation of two atRAL and is present in RPE lipofuscin as Schiff base conjugates with either PE or ethanolamine (atRAL dimer-PE and atRAL dimer-E, respectively) or as unconjugated atRAL dimer. The pigments atRAL dimer-PE and atRAL dimer-E absorb in the visible range at about 510 nm, a “red” shift relative to atRAL dimer that is attributable to protonation of the Schiff base linkage. Although A2E is a pyridinium salt containing a quaternary amine nitrogen that does not deprotonate or reprotonate (24), the protonation state of the Schiff base linkage in atRAL dimer-PE and atRAL dimer-E is pH-dependent (18).Other known constituents of RPE lipofuscin are generated by photooxidation. By mass spectrometry, the photooxidation products of A2E and atRAL dimer present as a series of peaks differing by increments of mass 16 beginning with M+ 592 (A2E) or M+ 552 (atRAL dimer) (18, 25). The moieties generated by the addition of oxygens at CC bonds of these bisretinoid compounds include endoperoxides, furanoid oxides, and epoxides (25–27). These oxidized products are more polar than the parent compound, and mono- and bis-oxidized forms of A2E and atRAL dimer have been detected in RPE from human eyes and in eyecups from mice with null mutations in Abca4^−/− (18, 25), the gene responsible for recessive Stargardt macular degeneration. It is also notable that unconjugated atRAL dimer is a more efficient generator of singlet oxygen than is A2E and is also a more efficient quencher of singlet oxygen (18).Insight into the composition of RPE lipofuscin and the biosynthetic pathways by which these compounds form aids in an understanding of retinal diseases characterized by lipofuscin overload, particularly those associated with mutations in ABCA4 (ATP-binding cassette transporter 4) of photoreceptor cells (1–3). We report that a previously unrecognized bisretinoid molecule absorbing with maxima at 490 and 331 nm is detected at elevated levels in Abca4^−/− mice, a model of recessive Stargardt macular degeneration. This compound is also present in human RPE. By high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS), with corroboration by Fourier transform infrared spectroscopy (FTIR), we determined that this molecule is a bisretinoid presenting with a noncharged dihydropyridine core (Fig. 1C). We propose a biosynthetic pathway by which this pigment may form and demonstrate that enzymatic removal of the phosphatidic acid portion of the molecule generates a second novel component of RPE lipofuscin.     

Hyaluronan Molecular Weight Is Controlled by UDP-N-acetylglucosamine Concentration in Streptococcus zooepidemicus

The molecular weight of hyaluronan is important for its rheological and biological function. The molecular mechanisms underlying chain termination and hence molecular weight control remain poorly understood, not only for hyaluronan synthases but also for other β-polysaccharide synthases, e.g. cellulose, chitin, and 1,3-betaglucan synthases. In this work, we manipulated metabolite concentrations in the hyaluronan pathway by overexpressing the five genes of the hyaluronan synthesis operon in Streptococcus equi subsp. zooepidemicus. Overexpression of genes involved in UDP-glucuronic acid biosynthesis decreased molecular weight, whereas overexpression of genes involved in UDP-N-acetylglucosamine biosynthesis increased molecular weight. The highest molecular mass observed was at 3.4 ± 0.1 MDa twice that observed in the wild-type strain, 1.8 ± 0.1 MDa. The data indicate that (a) high molecular weight is achieved when an appropriate balance of UDP-N-acetylglucosamine and UDP-glucuronic acid is achieved, (b) UDP-N-acetylglucosamine exerts the dominant effect on molecular weight, and (c) the wild-type strain has suboptimal levels of UDP-N-acetylglucosamine. Consistent herewith molecular weight correlated strongly (ρ = 0.84, p = 3 × 10⁻⁵) with the concentration of UDP-N-acetylglucosamine. Data presented in this paper represent the first model for hyaluronan molecular weight control based on the concentration of activated sugar precursors. These results can be used to engineer strains producing high molecular weight hyaluronan and may provide insight into similar polymerization mechanisms in other polysaccharides.Hyaluronan (HA)³ is a linear polymer of a repeating disaccharide, β1–3 d-N-acetylglucosamine (GlcNAc) β1–4 d-glucuronic acid (GlcUA) (1) (see Fig. 1). Ubiquitous in the extracellular matrix in vertebrates, HA is particularly abundant in cartilage, synovial fluid, dermis, and the vitreous humor of the eye, where it serves specialized functions. HA also plays a critical role during fertilization and embryogenesis. In many group A and C streptococci, HA forms a capsule that helps these microbes evade the host immune system (2). HA molecular weight is important for the physiochemical as well as biological properties of HA. High molecular weight is important for HA to exert its unique rheological properties (3), for mucoadherence (4, 5), and anti-inflammatory effects (6, 7), whereas low molecular weight is a potent signaling molecule (8).Open in a separate windowFIGURE 1.Biosynthetic pathway of HA in S. zooepidemicus.HA is produced by a processive synthase (9, 10) from the activated sugar precursors, UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc) (see Fig. 1). In addition to the HA synthase (hasA), streptococcal has operons encode for one or more enzymes involved in biosynthesis of the activated sugars (11). The Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) operon encodes for five genes: HA synthase (EC 2.4.1.212; hasA), UDP-glucose dehydrogenase (EC 1.1.1.22; hasB), UDP-glucose pyrophosphorylase (EC 2.7.7.9; hasC), a glmU paralog encoding for a dual function enzyme acetyltransferase and pyrophosphorylase activity (EC 2.3.1.4/EC 2.7.7.23; hasD), and a pgi paralog encoding for phosphoglucoisomerase (EC 5.3.1.9; hasE).Although the biosynthetic mechanism is well established, little is known about what controls HA molecular weight. This is true not only for HA, but also for the highly abundant β-polysaccharides: cellulose, chitin, and 1,3-betaglucan. Molecular weight is partly an intrinsic parameter of the HA synthase. Weigel and colleagues have demonstrated that, at least in vitro, mutation of conserved cysteine or polar residues in streptococcal HA synthases results in reduced molecular weight with limited effect on biosynthetic rate (12–14). In a vertebrate HA synthase from Xenopus, the mutation of a serine or a cysteine residue yielded HA of higher, lower, or similar molecular weight depending on the amino acid substitution (15).We and others have demonstrated that in vivo molecular weight is also affected by culture parameters, e.g. temperature and aeration (16–20). Although changed culture conditions affect the physiochemical environment of the HA synthase, a more likely explanation is that molecular weight is affected by the availability of activated sugar substrates (UDP-GlcUA and UDP-GlcNAc) as well as the concentration of possible effector molecules, such as free UDP (21). Although such a mechanism has been suggested for several processive synthases (22, 23), there has never been any direct evidence linking molecular weight to the concentration of a substrate.Experimental support for the hypothesis has been obtained for the type 3 polysaccharide of Streptococcus pneumoniae (24). Like HA, the type 3 polysaccharide in S. pneumoniae is synthesized by a processive synthase from alternating addition of activated sugars, in this case UDP-glucose (UDP-Glc) and UDP-GlcUA. Mutants with reduced UDP-glucose dehydrogenase (“hasB”) activity not only produce less polysaccharide, but also polysaccharide with lower molecular weight (24). Although the levels of UDP-GlcUA were below detection in all strains, this supports the idea that UDP-GlcUA concentration controls molecular weight. Moreover, it is consistent with previous in vitro studies showing that low levels of UDP-GlcUA cause chain termination and hence low molecular weight (25). It was proposed that the concentration of UDP-GlcUA is critical for the successful transition from oligosaccharide lipid to highly processive polysaccharide synthesis (26). A similar mechanism is not likely for HA biosynthesis, because there is no indication that the HA synthase needs a primer (27).In the present study, we manipulated metabolite concentrations in the HA pathway by overexpressing the five genes in the has operon of S. zooepidemicus (Fig. 1). Overexpression of these genes had a profound effect on HA molecular weight, which correlated with the levels of UDP-sugars and in particular, UDP-GlcNAc.     

Motogenic Sites in Human Fibronectin Are Masked by Long Range Interactions

Fibronectin (FN) is a large extracellular matrix glycoprotein important for development and wound healing in vertebrates. Recent work has focused on the ability of FN fragments and embryonic or tumorigenic splicing variants to stimulate fibroblast migration into collagen gels. This activity has been localized to specific sites and is not exhibited by full-length FN. Here we show that an N-terminal FN fragment, spanning the migration stimulation sites and including the first three type III FN domains, also lacks this activity. A screen for interdomain interactions by solution-state NMR spectroscopy revealed specific contacts between the Fn N terminus and two of the type III domains. A single amino acid substitution, R222A, disrupts the strongest interaction, between domains ^4–5FnI and ³FnIII, and restores motogenic activity to the FN N-terminal fragment. Anastellin, which promotes fibril formation, destabilizes ³FnIII and disrupts the observed ^4–5FnI-³FnIII interaction. We discuss these findings in the context of the control of cellular activity through exposure of masked sites.Fibronectin (FN),4 a large multidomain glycoprotein found in all vertebrates, plays a vital role in cell adhesion, tissue development, and wound healing (1). It exists in soluble form in plasma and tissue fluids but is also present in fibrillar networks as part of the extracellular matrix. The structures of many FN domains of all three types, FnI, FnII, and FnIII, are known, for example (2–4). Although interactions between domains that are close in primary sequence have been demonstrated (3, 5), studies of multidomain fragments generally assume a beads-on-string model (2). There is, however, much evidence for the presence of long range order in soluble FN as a number of functional sites, termed cryptic, are not active in the native molecule, until exposed through conformational change. These include self-association sites (5–8), sites of protein interactions (9), and sites that control cellular activity (10, 11). Low resolution studies of the FN dimer suggest a compact conformation under physiological conditions (12–14); however, attempts to define large scale structure in FN by small angle scattering or electric birefringence (15–17) have yielded contradictory results. Interpretation of domain stability changes in terms of interaction sites (18) has also not been straightforward (2), possibly because of domain stabilization through nearest-neighbor effects (19, 20).A FN splicing variant produced in fetal and cancer patient fibroblasts, termed migration stimulation factor (MSF), stimulates migration of adult skin fibroblasts into type I collagen gels (10, 21) and breast carcinoma cells using the Boyden chamber (22). MSF comprises FN domains ¹FnI to ⁹FnI, a truncated ¹FnIII, and a small C-terminal extension; a recombinant FN fragment corresponding to ¹FnI-⁹FnI (Fn70kDa) displays the same activity (10). An overview of FN domain structure and nomenclature is presented in Fig. 1a. Further experiments sub-localized full motogenic activity to the gelatin binding domain of FN (GBD, domains ⁶FnI-⁹FnI) (23) and partial activity to a shorter fragment spanning domains ^7–9FnI (24). Two IGD tripeptides of domains ⁷FnI and ⁹FnI were shown to be essential through residue substitutions and reconstitution of partial motogenic activity in synthetic peptides (10, 24, 25); however, similar IGD tripeptides outside the GBD, on domains ³FnI and ⁵FnI, appear to have little effect (10, 23). Full-length adult FN does not affect cell migration in similar assays (10, 23); thus motogenic activity sites are presumed to be masked in the conformation adopted by soluble FN, although they could be exposed by molecular rearrangement.Open in a separate windowFIGURE 1.Motogenic activity of FN fragments. a, schematic representation of the FN domain structure (top) and enlargement of the FN N terminus (bottom). Type I domains are shown as pentagons; type II domains as hexagons; and type III domains as ovals. b, comparison of motogenic activity versus protein concentration of wild-type Fn70kDa and Fn100kDa fragments. Error bars are derived from duplicate experiments, and a gray band denotes migration activity of media without additives. c, similar comparisons for mutant Fn100kDa fragments. d, analytical size exclusion chromatography of large FN fragments. The trace of UV absorbance at 280 nm versus elution volume shown here indicates a larger hydrodynamic radius for Fn100kDa R222A compared with the wild type, consistent with our model (Fig. 6a).Here we show that a recombinant fragment, closely matching a truncated form of FN identified in zebrafish (26), as well as amphibians, birds, and mammals (27), does not stimulate cell migration. This fragment is similar to MSF but includes the first three FnIII domains (^1–3FnIII), suggesting that these domains are responsible for a conformational transition that masks the activity sites in this construct and probably in full-length FN. To identify the mechanism behind this transition, we performed structural studies by solution NMR spectroscopy and identified a specific long range interaction between domains ^4–5FnI and ³FnIII as essential for this masking effect. Interestingly, this interaction does not involve direct contacts with the GBD but possibly represses motogenic activity through chain compaction, evident in analytical size exclusion assays. Intramolecular interactions thus provide a mechanism by which conformational rearrangement induced, for example, by tension or splicing variation can result in cellular activity differences.     

Metal Deficiency Increases Aberrant Hydrophobicity of Mutant Superoxide Dismutases That Cause Amyotrophic Lateral Sclerosis

The mechanisms by which mutant variants of Cu/Zn-superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis are not clearly understood. Evidence to date suggests that altered conformations of amyotrophic lateral sclerosis mutant SOD1s trigger perturbations of cellular homeostasis that ultimately cause motor neuron degeneration. In this study we correlated the metal contents and disulfide bond status of purified wild-type (WT) and mutant SOD1 proteins to changes in electrophoretic mobility and surface hydrophobicity as detected by 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence. As-isolated WT and mutant SOD1s were copper-deficient and exhibited mobilities that correlated with their expected negative charge. However, upon disulfide reduction and demetallation at physiological pH, both WT and mutant SOD1s underwent a conformational change that produced a slower mobility indicative of partial unfolding. Furthermore, although ANS did not bind appreciably to the WT holoenzyme, incubation of metal-deficient WT or mutant SOD1s with ANS increased the ANS fluorescence and shifted its peak toward shorter wavelengths. This increased interaction with ANS was greater for the mutant SOD1s and could be reversed by the addition of metal ions, especially Cu²⁺, even for SOD1 variants incapable of forming the disulfide bond. Overall, our findings support the notion that misfolding associated with metal deficiency may facilitate aberrant interactions of SOD1 with itself or with other cellular constituents and may thereby contribute to neuronal toxicity.The sequence of events by which more than 100 mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD1)³ cause familial forms of amyotrophic lateral sclerosis (ALS) is unknown. Studies of purified SOD1 proteins and cellular or rodent models of SOD1-linked ALS suggest that impaired metal ion binding or misfolding of mutant SOD1 proteins in the cellular environment may be related to their toxicity (1–10). Available evidence suggests that partially unfolded mutant SOD1 species could contribute to motor neuron death by promoting abnormal interactions that produce cellular dysfunction (11–16).In previous studies we characterized physicochemical properties of 14 different biologically metallated ALS SOD1 mutants (17) and demonstrated altered thermal stabilities of these mutants compared with wild-type (WT) SOD1 (18). These “as-isolated” SOD1 proteins, which contain variable amounts of copper and zinc, were broadly grouped into two classes based on their ability to incorporate and retain metal ions with high affinity. WT-like SOD1 mutants retain the ability to bind copper and zinc ions and exhibit dismutase activity similar to the normal enzyme, whereas metal binding region (MBR) mutants are significantly deficient in copper and/or zinc (17, 19). We also observed that ALS-associated SOD1 mutants were more susceptible than the WT enzyme to reduction of the intrasubunit disulfide bond between Cys-57 and Cys-146 (20). The significance of these results is that even WT-like mutants, which exhibit a nearly normal backbone structure (21–23), may be vulnerable to destabilizing influences in vivo. Our group and others subsequently showed that the mutant SOD1 proteins share a susceptibility to increased hydrophobicity under conditions that reduce disulfide bonds and/or chelate metal ions (5) and that similar hydrophobic species exist in tissue lysates from mutant SOD1 transgenic mice (4–6). One consequence of such hydrophobic exposure could be the facilitation of abnormal interactions between the mutant enzymes and other cellular constituents (e.g. chaperones, mitochondrial components, or other targets), which might influence pathways leading to motor neuron death (15, 16, 24–27).Accumulating evidence suggests that metal deficiency of SOD1 is an important factor that can influence SOD1 aggregation or neurotoxicity (4, 28–33), but the metal-deficient states of SOD1 that are most relevant to ALS remain unclear. Zinc-deficient, copper-replete SOD1 species, which can be produced in vitro by adding copper to SOD1 that has been stripped of its metal ions at acidic pH, were shown to be toxic to motor neurons in culture (28). However, it has not been shown that zinc-deficient, copper-replete SOD1 is produced in vivo as a consequence of ALS mutations, and loading of copper into SOD1 by the copper chaperone for SOD1 (CCS) is not required for toxicity (34, 35). Furthermore, the MBR mutants have a disrupted copper site and have been found to be severely deficient in both zinc and copper (17, 30), yet expression of these SOD1s still produces motor neuron disease (1, 2, 30, 34, 36, 37).When recombinant human SOD1 was overexpressed in insect cells, we instead observed zinc-replete but copper-deficient species for most WT-like mutants, probably because the capacity of the copper-loading mechanism was exceeded (17). These preparations indicate that zinc can be efficiently incorporated into many WT-like mutants in vivo, and much of it is retained after purification. Furthermore, these copper-deficient biologically metallated proteins may be useful reagents to assess the influence of copper binding upon other properties of SOD1 mutants that may be relevant to their neurotoxicity.We previously observed that reduction of the Cys-57—Cys-146 disulfide bond facilitates the ability of metal chelators to alter the electrophoretic mobility and to increase the hydrophobicity of SOD1 mutants (5). This is consistent with the known properties of this linkage to stabilize the dimeric interface, to orient Arg-143 via a hydrogen bond from the carbonyl oxygen of Cys-57 to Arg-143-NH₂, and to prevent metal ion loss (38–40). However, it remains unclear whether the Cys-57—Cys-146 bond is required to prevent abnormal SOD1 hydrophobic exposure or whether the aberrant conformational change primarily results from metal ion loss. Ablation of the disulfide bond by the experimental (non-ALS) mutants C57S and C146S provides useful reagents to test the relative influence of the disulfide bond and copper binding upon SOD1 properties.In this study we sought to correlate the consequences of copper deficiency, copper and zinc deficiency, and disulfide reduction upon the hydrodynamic behavior and surface hydrophobicity of WT and representative mutant SOD1 enzymes (Fig. 1A). We quantitated the metal contents of as-isolated SOD1 proteins, detected changes in conformation or metal occupancy using native PAGE to assess their electrophoretic mobility, a measure of global conformational change, and correlated these changes to hydrophobic exposure using 1-anilinonaphthalene-8-sulfonic acid (ANS), which is very sensitive to local conformational changes. ANS is a small amphipathic dye (Fig. 1B) that has been used as a sensitive probe to detect hydrophobic pockets on protein surfaces (41–44). Free ANS exhibits only weak fluorescence that is maximal near 520 nm, but when ANS binds to a hydrophobic site in a partially or fully folded protein, the fluorescence peak increases in amplitude and shifts to a shorter wavelength (42). ANS also has an anionic sulfonate group that can interact with cationic groups (e.g. Arg or Lys residues) through ion-pair formation which may be further strengthened by hydrophobic interactions (43–46).Open in a separate windowFIGURE 1.A, WT SOD1 structure showing the position of the C57-C146 intrasubunit disulfide bond (S–S, yellow), bound copper and zinc ions, and ALS mutant residues. The residues altered in A4V, G85R, G93A, D124V, and S134N SOD1s are indicated as green spheres. The backbone of the β-barrel core and the loops is shown in a rainbow color, from blue at the amino terminus to red at the carboxyl terminus. The figure was generated using PyMOL (84) and PDB entry 1HL5 (22). B, chemical structure of ANS fluorophore.To evaluate further the importance of metal ion binding, we measured spectral changes related to the binding of cobalt and copper to the same SOD1 proteins. We observed that as-isolated WT-like mutants containing zinc could interact with copper ions to produce an electrophoretic mobility and decreased hydrophobicity resembling that of the fully metalated holo-WT SOD1. In contrast, we saw no evidence for copper binding to MBR mutants in a manner that alters their hydrodynamic properties or their hydrophobicity. Our data suggest that binding of both copper and zinc are important determinants of SOD1 conformation and that perturbation of such binding may be relevant to the ALS disease process.     

The N Terminus of Connexin37 Contains an ��-Helix That Is Required for Channel Function

The cytoplasmic N-terminal domain of connexins has been implicated in multiple aspects of gap junction function, including connexin trafficking/assembly and channel gating. A synthetic peptide corresponding to the first 23 amino acids of human connexin37 was prepared, and circular dichroism and nuclear magnetic resonance studies showed that this N-terminal peptide was predominantly α-helical between glycine 5 and glutamate 16. The importance of this structure for localization of the protein at appositional membranes and channel function was tested by expression of site-directed mutants of connexin37 in which amino acids leucine 10 and glutamine 15 were replaced with prolines or alanines. Wild type connexin37 and both substitution mutants localized to appositional membranes between transfected HeLa cells. The proline mutant did not allow intercellular transfer of microinjected neurobiotin; the alanine mutant allowed transfer, but less extensively than wild type connexin37. When expressed alone in Xenopus oocytes, wild type connexin37 produced hemichannel currents, but neither of the double substitution mutants produced detectable currents. The proline mutant (but not the alanine mutant) inhibited co-expressed wild type connexin37. Taken together, our data suggest that the α-helical structure of the connexin37 N terminus may be dispensable for protein localization, but it is required for channel and hemichannel function.Gap junction channels allow intercellular passage of ions and small molecules up to 1000 Da. They are oligomeric assemblies of members of a family of related proteins called connexins (CX)² (reviewed in Ref. 1). Six connexin monomers assemble to form a hemichannel or connexon (Fig. 1, top panel), which, in turn, forms a complete gap junction channel by docking with a hemichannel from an adjacent cell. Based on sequence similarities, connexins have been separated into subfamilies designated by Greek characters (2, 3). The majority of connexins are members of the α- and β-subfamilies. Connexin polypeptides span the plasma membrane four times and have three cytoplasmic regions: the N terminus (NT), a cytoplasmic loop between the second and third transmembrane domains, and the C terminus (Fig. 1, middle panel). Structural studies of gap junctions have revealed that each hemichannel contains a ring of 24 transmembrane spanning helices (4, 5). Most topological models suggest that the NT of α-subfamily connexins contains 23 amino acids (illustrated for connexin37, CX37, in Fig. 1, bottom panel) and that of β-subfamily connexins contains 22 amino acids.Open in a separate windowFIGURE 1.Diagrams depicting the relationships between a gap junction hemichannel (top), the connexin polypeptide (middle), and the amino acid sequence of the CX37 N-terminal domain (bottom). Thick vertical lines represent the boundaries of the plasma membrane; the intracellular and extracellular spaces are indicated. The transmembrane (M1–M4), extracellular (E1 and E2), and cytoplasmic (NT, N terminus; CL, cytoplasmic loop; and CT, C terminus) domains within a connexin are indicated.The importance of the connexin NT has been emphasized by the identification of a number of connexin mutants that cause amino acid substitutions within this region and are linked to diseases including sensorineural deafness (CX26, CX30, and CX31), Charcot-Marie-Tooth disease (CX32), oculodentodigital dysplasia (CX43), and congenital cataracts (CX46 and CX50). Among the disease-linked mutants that have been studied, some show impaired protein trafficking to the cell surface, whereas others traffic properly, but show loss or alterations of channel function (6–16). Heterologous expression of site-directed mutants and chimeric connexins has demonstrated the influence of NT amino acids upon channel properties, including transjunctional voltage (V_j)-dependent gating, unitary conductance, permeability, and sensitivity to regulation by polyamines (17–22). Lagree et al. (23) have provided evidence that the NT influences the compatibility of connexin hetero-oligomerization.The structure of the NT domain of a β-group connexin, Cx26, has been investigated through circular dichroism (CD) and nuclear magnetic resonance (NMR) of a synthetic peptide corresponding to part of the predicted CX26NT (24, 25). Based on their data, Purnick et al. (24) proposed a model for the NT of CX26 with an α-helix extending from position 1 to 10 and a critical bend at positions 11 and 12 that was suggested to act as a “hinge” allowing the first 10 amino acids to swing into the pore and block the channel. Oshima et al. (5) have published structural studies of a “permeability” mutant (M34A) of CX26 (26) showing a density within the pore of the channel that they suggested might represent a bundle of N termini acting as a “plug” to close the channel.We have been studying CX37, an α-group connexin that is expressed in endothelial cells (27), which may be important for development of atherosclerotic disease (28) and that can form large conductance channels and hemichannels (27, 29). We have shown that as much as half the length of the CX37NT can be deleted without affecting formation of gap junction plaques, but a full-length N terminus is required for hemichannel gating and intercellular communication (30). These observations suggested that the CX37NT may have a structure that is required for function. Therefore, the present experiments were designed to determine the structure of the NT of CX37 and the importance of that structure for protein localization and formation of functional channels and hemichannels. Differences between our data and those previously reported in studies of CX26 suggest that the structure of the NT in α-group connexins may differ from that in β-group connexins.     

A cis-Proline in ��-Hemoglobin Stabilizing Protein Directs the Structural Reorganization of ��-Hemoglobin

α-Hemoglobin (αHb) stabilizing protein (AHSP) is expressed in erythropoietic tissues as an accessory factor in hemoglobin synthesis. AHSP forms a specific complex with αHb and suppresses the heme-catalyzed evolution of reactive oxygen species by converting αHb to a conformation in which the heme is coordinated at both axial positions by histidine side chains (bis-histidyl coordination). Currently, the detailed mechanism by which AHSP induces structural changes in αHb has not been determined. Here, we present x-ray crystallography, NMR spectroscopy, and mutagenesis data that identify, for the first time, the importance of an evolutionarily conserved proline, Pro³⁰, in loop 1 of AHSP. Mutation of Pro³⁰ to a variety of residue types results in reduced ability to convert αHb. In complex with αHb, AHSP Pro³⁰ adopts a cis-peptidyl conformation and makes contact with the N terminus of helix G in αHb. Mutations that stabilize the cis-peptidyl conformation of free AHSP, also enhance the αHb conversion activity. These findings suggest that AHSP loop 1 can transmit structural changes to the heme pocket of αHb, and, more generally, highlight the importance of cis-peptidyl prolyl residues in defining the conformation of regulatory protein loops.Mammalian adult hemoglobin (HbA)⁵ is a tetramer of two αHb and two βHb subunits, which is produced to extremely high concentrations (∼340 mg/ml) in red blood cells. Numerous mechanisms exist to balance and coordinate HbA synthesis in normal erythropoiesis, and problems with the production of either HbA subunit give rise to thalassemia, a common cause of anemia worldwide. Previously, we identified α-hemoglobin stabilizing protein (AHSP) as an accessory factor in normal HbA production (1). AHSP forms a dimeric complex with αHb (see Fig. 1A) (2) but does not interact with βHb or HbA. AHSP also binds heme-free (apo) αHb (3) and may serve functions in both the folding of nascent αHb (4) and the detoxification of excess αHb that remains following HbA assembly (2, 5). Mice carrying an Ahsp gene knock-out display mild anemia, ineffective erythropoiesis, and enhanced sensitivity to oxidative stress (1, 6), features also observed in β-thalassemia patients due to the cytotoxic effects of free αHb.Open in a separate windowFIGURE 1.Summary of αHb·AHSP interactions. A, the αHb·AHSP complex(PDB code 1Z8U) (2). The interface is formed from helices 1 and 2 and the intervening loop 1 (green) of AHSP, together with helices G-H and the B-C corner of αHb (cyan). B, detailed views of the heme binding site of αHb as it appears in oxy-HbA (PDB code 1GZX) (69) and the final bis-histidyl αHb·AHSP complex (PDB code 1Z8U) with two histidine ligands to the iron. Typical visible absorption spectra in the region 450–700 nm are shown.Free αHb promotes the formation of harmful reactive oxygen species as a result of reduction/oxidation reactions involving the heme iron (7, 8). Reactive oxygen species can damage heme, αHb, and other cellular structures, resulting in hemoglobin precipitates and death of erythroid precursor cells (9–12). The presence of AHSP may explain how cells tolerate the slight excess of αHb that is observed in normal erythropoiesis, which is postulated to inhibit the formation of non-functional βHb tetramers, thus providing a robust mechanism for achieving the correct subunit stoichiometry during HbA assembly (13).Structural and biochemical studies have begun to elucidate the molecular mechanism by which AHSP detoxifies αHb. AHSP binds to oxygenated αHb to generate an initial complex that retains the oxy-heme, as evidenced by a characteristic visible absorption spectrum (see Fig. 1B, middle) and resonance Raman spectrum (5). This initial oxy-αHb·AHSP complex then converts to a low spin Fe³⁺ complex (2), in which the heme iron is bound at both axial positions by the side chains of His⁵⁸ and His⁸⁷ from αHb (see Fig. 1B, right). The formation of this complex inhibits αHb peroxidase activity and heme loss (2). Bis-histidyl heme coordination is becoming increasingly recognized as a feature of numerous vertebrate and non-vertebrate globins (14) and has been shown previously to confer a relative stabilization of the Fe³⁺ over the Fe²⁺ oxidation state (15–17). Although bis-histidyl heme coordination has previously been detected in solutions of met-Hb, formed through spontaneous autoxidation of Hb (18–21), the bishis-αHb·AHSP complex provides the first evidence that the bis-histidyl heme may play a positive functional role in Hb biochemistry by inhibiting the production of harmful reactive oxygen species.Despite its potential importance, the mechanism by which AHSP influences heme coordination in its binding partner is still unknown. As shown in Fig. 1A, AHSP binds αHb at a surface away from the heme pocket, and thus structural changes must somehow be transmitted through the αHb protein. It is intriguing that the free AHSP protein switches between two alternative conformations linked to cis/trans isomerization of the Asp²⁹-Pro³⁰ peptide bond in loop 1 (22) and that, in complex with αHb, this loop is located at the αHb·AHSP interface (see Fig. 1A). Peptide bonds preceding proline residues are unique in that the cis or trans bonding conformations have relatively similar stabilities (23), allowing an interconversion between these conformations that can be important for protein function (24, 25). Previous x-ray crystal structures of αHb·AHSP complexes have been obtained only with a P30A mutant of AHSP, in which isomerization is abolished and the Asp²⁹-Ala³⁰ peptide bond adopts a trans conformation, leaving the potential structural and functional significance of the evolutionarily conserved Pro³⁰ undisclosed. Here, we demonstrate a functional role for AHSP Pro³⁰ in conversion of oxy-αHb to the bis-histidyl form and identify a specific structural role for a cis Asp²⁹-Pro³⁰ peptide bond in this process. From a mechanistic understanding of how AHSP promotes formation of bis-histidyl αHb, we may eventually be able to engineer AHSP function as a tool in new treatments for Hb diseases such as β-thalassemia.