MALDI Imaging Mass Spectrometry: STATE OF THE ART TECHNOLOGY IN CLINICAL PROTEOMICS*

A decade after its inception, MALDI imaging mass spectrometry has become a unique technique in the proteomics arsenal for biomarker hunting in a variety of diseases. At this stage of development, it is important to ask whether we can consider this technique to be sufficiently developed for routine use in a clinical setting or an indispensable technology used in translational research. In this report, we consider the contributions of MALDI imaging mass spectrometry and profiling technologies to clinical studies. In addition, we outline new directions that are required to align these technologies with the objectives of clinical proteomics, including: 1) diagnosis based on profile signatures that complement histopathology, 2) early detection of disease, 3) selection of therapeutic combinations based on the individual patient''s entire disease-specific protein network, 4) real time assessment of therapeutic efficacy and toxicity, 5) rational redirection of therapy based on changes in the diseased protein network that are associated with drug resistance, and 6) combinatorial therapy in which the signaling pathway itself is viewed as the target rather than any single “node” in the pathway.MS has become a versatile tool that we are familiar with in large part due to important electronic and informatics advancements. The ability to obtain the molecular weight is one of the first steps in the identification of a molecule. With the addition of primary structural information mass spectrometry has become a useful technique to identify molecules within complex mixtures.Biological specimens, such as tissues, urine, or plasma, are complex and highly heterogeneous, which makes them inherently difficult to analyze. Further research and developments are necessary to achieve reliable biological models for understanding and studying pathologies. Therefore, it is of primary importance to identify the constituents of these systems and subsequently understand how they function within the framework of the tissue. With regard to clinical proteomics, there is the added dimension of disease, and therefore, the main goal is to characterize the cellular circuitry with a focus on the impact of the disease and/or therapy on these cellular networks.Mass spectrometry has become a centerpiece technology predominantly in the field of proteomics. Nonetheless a more comprehensive understanding of the constituents of biological systems will be aided by determining the constituent distribution. This anatomical dimension has been added through mass spectrometry imaging (MSI)¹ especially using MALDI-MSI.MALDI is an ion source that is well compatible with the introduction of raw materials and surfaces. Shortly after its introduction, MALDI was used for direct tissue profiling. The first applications were neurobiological studies on dissected organs from the mollusk Lymnaea stagnalis (1–8), crustaceans (9), and other mollusks (10, 11). More recently, MALDI was used to generate profiles from tissue sections and ion images using a scanning method to analyze the surface (12) (Fig. 1). This led to the first MALDI MS tissue section imaging micrographs in 1997 (13–15). These studies were followed by 10 years of intense efforts to improve the sensitivity, reproducibility, data processing, tissue preservation, and preparation treatments to fully characterize the proteome leading to a clear improvement of molecular images (16–39) (Fig. 2).Open in a separate windowFig. 1.Schematic representation of the MALDI-MSI work flow. After tissue sectioning and transfer onto a conductive and transparent sample plate, the MALDI matrix is deposited, and data are acquired by recording mass spectra according to a raster of points covering the surface to be analyzed. Mass spectra recorded with their coordinates on the tissue are processed, and molecular images of the localization of molecules can be reconstructed. a.u., arbitrary units; ITO, idium tin oxide.Open in a separate windowFig. 2.Ten years'' evolution from one of the first MALDI images presented in 1999 at the 47th ASMS Conference on Mass Spectrometry and Allied Topics (left) (reprinted with permission of Caprioli and co-workers (84)) and molecular images obtained by our group for mouse stem cells injected in brain tissue sections (right) (M. Wisztorski, C. Meriaux, M. Salzet, and I. Fournier, unpublished results).These developments led to clinical studies using MALDI-MSI technology. Clinical proteomics has many objectives including 1) diagnosis based on signatures as a complement to histopathology, 2) early disease detection, 3) individualized selection of therapeutic combinations that best target the patient''s entire disease-specific protein network, 4) real time assessment of therapeutic efficacy and toxicity, 5) rational redirection of therapy based on changes in the diseased protein network that are associated with drug resistance, and 6) combinatorial therapy in which the signaling pathway itself is viewed as the target rather than any single “node” in the pathway.Based on these key objectives, can we consider MALDI-MSI a mature technology for use in clinical studies? What is the potential impact of this technology in anatomy/pathology and disease? By reviewing each objective, do we have sufficient evidence that MALDI-MSI satisfies the criteria imposed by clinical proteomics? We will now specifically address each of these key points.     

Contribution of Flavin Covalent Linkage with Histidine 99 to the Reaction Catalyzed by Choline Oxidase

The FAD-dependent choline oxidase has a flavin cofactor covalently attached to the protein via histidine 99 through an 8α-N(3)-histidyl linkage. The enzyme catalyzes the four-electron oxidation of choline to glycine betaine, forming betaine aldehyde as an enzyme-bound intermediate. The variant form of choline oxidase in which the histidine residue has been replaced with asparagine was used to investigate the contribution of the 8α-N(3)-histidyl linkage of FAD to the protein toward the reaction catalyzed by the enzyme. Decreases of 10-fold and 30-fold in the k_cat/K_m and k_cat values were observed as compared with wild-type choline oxidase at pH 10 and 25 °C, with no significant effect on k_cat/K_O using choline as substrate. Both the k_cat/K_m and k_cat values increased with increasing pH to limiting values at high pH consistent with the participation of an unprotonated group in the reductive half-reaction and the overall turnover of the enzyme. The pH independence of both ^D(k_cat/K_m) and ^Dk_cat, with average values of 9.2 ± 3.3 and 7.4 ± 0.5, respectively, is consistent with absence of external forward and reverse commitments to catalysis, and the chemical step of CH bond cleavage being rate-limiting for both the reductive half-reaction and the overall enzyme turnover. The temperature dependence of the ^Dk_red values suggests disruption of the preorganization in the asparagine variant enzyme. Altogether, the data presented in this study are consistent with the FAD-histidyl covalent linkage being important for the optimal positioning of the hydride ion donor and acceptor in the tunneling reaction catalyzed by choline oxidase.A number of enzymes, including dehydrogenases (1–3), monooxygenases (4–7), halogenases (8–11), and oxidases (7, 12, 13), employ flavin cofactors (FAD or FMN) for their catalytic processes. About a tenth of all flavoproteins have been shown to contain a covalently attached cofactor, which may be linked at the C8M position via histidyl, tyrosyl, or cysteinyl side chains or at the C6M position via a cysteinyl side chain (14). Glucooligosaccharide oxidase (15, 16), hexose oxidase (17), and berberine bridge enzyme (18, 19) are examples of flavoproteins (FAD as cofactor) with both linkages present in one flavin molecule. The covalent linkages in flavin-dependent enzymes have been shown to stabilize protein structure (20–22), prevent loss of loosely bound flavin cofactors (23), modulate the redox potential of the flavin microenvironment (20, 23–27), facilitate electron transfer reactions (28), and contribute to substrate binding as in the case of the cysteinyl linkage (20). However, no study has implicated a mechanistic role of the flavin covalent linkages in enzymatic reactions in which a hydride ion is transferred by quantum mechanical tunneling.The discovery of quantum mechanical tunneling in enzymatic reactions, in which hydrogen atoms, protons, and hydride ions are transferred, has attracted considerable interest in enzyme studies geared toward understanding the mechanisms underlying the several orders of magnitudes in the rate enhancements of protein-catalyzed reactions compared with non-enzymatic ones. Tunneling mechanisms have been shown in a wide array of cofactor-dependent enzymes, including flavoenzymes. Examples of flavoenzymes in which the tunneling mechanisms have been demonstrated include morphinone reductase (29, 30), pentaerythritol tetranitrate reductase (29), glucose oxidase (31–33), and choline oxidase (34). Mechanistic data on Class 2 dihydroorotate dehydrogenases, also with a flavin cofactor (FMN) covalently linked to the protein moiety (35, 36), could only propose a mechanism that is either stepwise or concerted with significant quantum mechanical tunneling for the hydride transfer from C6 and the deprotonation at C5 in the oxidation of dihydroorotate to orotate (37). This leaves choline oxidase as the only characterized enzyme with a covalently attached flavin cofactor (12, 38), where the oxidation of its substrate occurs unequivocally by quantum mechanical tunneling.Choline oxidase from Arthrobacter globiformis catalyzes the two-step FAD-dependent oxidation of the primary alcohol substrate choline to glycine betaine with betaine aldehyde, which is predominantly bound to the enzyme and forms a gem-diol species, as intermediate (Scheme 1). Glycine betaine accumulates in the cytoplasm of plants and bacteria as a defensive mechanism against stress conditions, thus making genetic engineering of relevant plants of economic interest (39–45), and the biosynthetic pathway for the osmolyte is a potential drug target in human microbial infections of clinical interest (46–48). The first oxidation step catalyzed by choline oxidase involves the transfer of a hydride ion from a deprotonated choline to the protein-bound flavin followed by reaction of the anionic flavin hydroquinone with molecular oxygen to regenerate the oxidized FAD (for a recent review see Ref. 50). The gem-diol choline, i.e. hydrated betaine aldehyde, is the substrate for the second oxidation step (49), suggesting that the reaction may follow a similar mechanism. The isoalloxazine ring of the flavin cofactor, which is buried within the protein, is physically constrained through a covalent linkage via the C(8) methyl of the flavin and the N(3) atom of the histidine side chain at position 99 (Fig. 1) (12). Also contributing to the physical constrain are the proximity of Ile-103 to the pyrimidine ring and the interactions of the backbone atoms of residues His-99 through Ile-103 with the isoalloxazine ring. The rigid positioning of the isoalloxazine ring could only permit a solvent-excluded cavity of ∼125 ų adjacent to the re face of the FAD to accommodate a 93-ų choline molecule in the substrate binding domain (12). Mechanistic data thus far obtained on choline oxidase, coupled with the crystal structure of the wild-type enzyme resolved to 1.86 Å, are consistent with a quantum tunneling mechanism for the hydride ion transfer occurring within a highly preorganized enzyme-substrate complex (Scheme 2) (12, 34, 50). Exploitation of the tunneling mechanism requires minimal independent movement of the hydride ion donor and acceptor, with the only dynamic motions permitted being the ones that promote the hydride transfer reaction.Open in a separate windowSCHEME 1.Two-step, four-electron oxidation of choline catalyzed by choline oxidase.Open in a separate windowFIGURE 1.x-ray crystal structure of the active site of wild-type choline oxidase resolved to 1.86 Å (PDB 2jbv). Note the significant distortion of the flavin ring at the C(4a) atom, which is due to the presence of a C(4a) adduct (69).Open in a separate windowSCHEME 2.The hydride ion transfer reaction from the α-carbon of the activated choline alkoxide species to the N(5) atom of the isoalloxazine ring of the enzyme-bound flavin in choline oxidase.In the present study, the contribution of the physically constrained flavin isoalloxazine ring to the reaction catalyzed by choline oxidase has been investigated in a variant enzyme in which the histidine residue at position 99 was replaced with an asparagine. The results suggest that, although not being required per se, the covalent linkage in choline oxidase contributes to the hydride tunneling reaction by either preventing independent movement or contributing to the optimal positioning of the flavin acting as hydride ion acceptor with respect to the alkoxide species acting as a donor. However, the covalent linkage is not required for the reaction.     

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).     

Spatial and Developmental Differentiation of Mannitol Dehydrogenase and Mannitol-1-Phosphate Dehydrogenase in Aspergillus niger

The presence of a mannitol cycle in fungi has been subject to discussion for many years. Recent studies have found no evidence for the presence of this cycle and its putative role in regenerating NADPH. However, all enzymes of the cycle could be measured in cultures of Aspergillus niger. In this study we have analyzed the localization of two enzymes from the pathway, mannitol dehydrogenase and mannitol-1-phosphate dehydrogenase, and the expression of their encoding genes in nonsporulating and sporulating cultures of A. niger. Northern analysis demonstrated that mpdA was expressed in both sporulating and nonsporulating mycelia, while expression of mtdA was expressed only in sporulating mycelium. More detailed studies using green fluorescent protein and dTomato fused to the promoters of mtdA and mpdA, respectively, demonstrated that expression of mpdA occurs in vegetative hyphae while mtdA expression occurs in conidiospores. Activity assays for MtdA and MpdA confirmed the expression data, indicating that streaming of these proteins is not likely to occur. These results confirm the absence of the putative mannitol cycle in A. niger as two of the enzymes of the cycle are not present in the same part of A. niger colonies. The results also demonstrate the existence of spore-specific genes and enzymes in A. niger.Mannitol has been described as one of the main compatible solutes in fungi (20) and may play a role as a storage carbon source (3) or a protectant against a variety of stresses (10, 16, 20, 22). Mannitol metabolism in fungi has been the subject of study for decades. It was proposed to exist in the form of a cyclic pathway, the mannitol cycle (9). This cycle consists of four steps enabling the conversion of fructose into mannitol and back to fructose (Fig. 1). The main role proposed for this cycle was regenerating NADPH (9, 10). Subsequently, many studies have questioned the existence of a mannitol cycle (reviewed in reference 20), and it has been shown that a mannitol cycle is not involved in NADPH regeneration in Stagonospora nodorum (19), Aspergillus niger (16), and Alternaria alternata (21). However, all enzymes of the cycle were detected in both sporulating and nonsporulating mycelia in A. niger (16), suggesting that a cycle could operate in this fungus. Fungi are able to use mannitol as a sole carbon source but do so in various ways (7).Open in a separate windowFig. 1.Putative mannitol cycle in fungi as proposed by Hult and Gatenbeck (9). HXK, hexokinase (EC 2.7.1.1); MTD, mannitol dehydrogenase (EC 1.1.1.138); MPD, mannitol-1-phosphate dehydrogenase (EC 1.1.1.17); MPP, mannitol-1-phosphate phosphatase (EC 3.1.3.22).d-Mannitol plays an important role in germination of Aspergillus conidia. In A. niger (23) and Aspergillus oryzae (8), mannitol accumulates in conidiospores and is utilized during the initial stages of germination. Production of mannitol appears to be largely dependent on mannitol-1-phosphate dehydrogenase (MPD) while mannitol dehydrogenase (MTD) contributes to a lesser extent (16, 19, 20).In this study we demonstrate that MTD and MPD as well as the expression of the corresponding genes (mtdA and mpdA) are spatially separated in colonies of A. niger. This demonstrates that a mannitol cycle does not exist in this fungus and shows that spores express specific genes that are involved in germination.     

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.     

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.     

Novel Inhibitors Complexed with Glutamate Dehydrogenase: ALLOSTERIC REGULATION BY CONTROL OF PROTEIN DYNAMICS*

Mammalian glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate using NAD(P)⁺ as coenzyme. Unlike its counterparts from other animal kingdoms, mammalian GDH is regulated by a host of ligands. The recently discovered hyperinsulinism/hyperammonemia disorder showed that the loss of allosteric inhibition of GDH by GTP causes excessive secretion of insulin. Subsequent studies demonstrated that wild-type and hyperinsulinemia/hyperammonemia forms of GDH are inhibited by the green tea polyphenols, epigallocatechin gallate and epicatechin gallate. This was followed by high throughput studies that identified more stable inhibitors, including hexachlorophene, GW5074, and bithionol. Shown here are the structures of GDH complexed with these three compounds. Hexachlorophene forms a ring around the internal cavity in GDH through aromatic stacking interactions between the drug and GDH as well as between the drug molecules themselves. In contrast, GW5074 and bithionol both bind as pairs of stacked compounds at hexameric 2-fold axes between the dimers of subunits. The internal core of GDH contracts when the catalytic cleft closes during enzymatic turnover. None of the drugs cause conformational changes in the contact residues, but all bind to key interfaces involved in this contraction process. Therefore, it seems likely that the drugs inhibit enzymatic turnover by inhibiting this transition. Indeed, this expansion/contraction process may play a major role in the inter-subunit communication and allosteric regulation observed in GDH.Glutamate dehydrogenase (GDH)² is found in all living organisms and catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate using NAD(P)⁺ as coenzyme (1). In eukaryotic organisms, GDH resides within the inner mitochondrial matrix where it catabolizes glutamate to feed 2-oxoglutarate to the Krebs cycle. Although there is some debate as to the directionality of the reaction, the high K_m for ammonium in the reductive amination reaction seems to prohibit the reverse reaction under normal conditions in most organisms (2). GDH from animals, but not other kingdoms (3), is allosterically regulated by a wide array of ligands (3–9). GTP (9–11), and with ∼100-fold lower affinity, ATP (3), is a potent inhibitor of the reaction and acts by increasing the binding affinity for the product, thereby slowing down enzymatic turnover (11). Hydrophobic compounds such as palmitoyl-CoA (12), steroid hormones (13), and steroid hormone analogs such as diethylstilbestrol (5) are also potent inhibitors. ADP is an activator of GDH (3, 6, 10, 11, 14) that acts in an opposite manner to GTP by facilitating product release. Leucine is a poor substrate for GDH but is also an allosteric activator for the enzyme (8). Its activation is akin to ADP but acts at site distinct from ADP (15).The crystal structures of the bacterial (16–18) and animal forms (19, 20) of GDH have shown that the general architecture and the locations of the catalytically important residues have remained unchanged throughout evolution. The structure of GDH (Fig. 1) is essentially two trimers of subunits stacked directly on top of each other with each subunit being composed of at least three domains (19–22). The bottom domain makes extensive contacts with a subunit from the other trimer. Resting on top of this domain is the “NAD binding domain” that has the conserved nucleotide binding motif. Animal GDH has a long protrusion, “antenna,” rising above the NAD binding domain that is not found in bacteria, plants, fungi, and the vast majority of protists. The antenna from each subunit lies immediately behind the adjacent, counterclockwise neighbor within the trimer. Because these intertwined antennae are only found in the forms of GDH that are allosterically regulated by numerous ligands, it is reasonable to speculate that it plays a role in regulation.Open in a separate windowFIGURE 1.Conformational transitions and locations of ligand binding sites in bovine glutamate dehydrogenase. A, a ribbon diagram of apo-bovine glutamate dehydrogenase with each of the identical subunits represented by different colors. The subunit arrangement is that of a trimer of dimers where anti-parallel β-strands form extensive interactions between the subunits stacked on top of each other. This pairing is represented by different shades of the same color. The conformational changes that during substrate binding are shown by the numbered arrows. As substrate binds, the NAD⁺ binding domain closes (1). The ascending helix of the antenna moves toward the pivot helix of the adjacent subunit (2). The short helix of the descending strand of the antenna becomes extended and distorted at the carboxyl end (3). Finally, the internal cavity of the helix compresses, bringing the three pairs closer together (4). B shows the structure of ADP (green spheres) bound to the apo-form of GDH and the location of Arg-463 (mauve spheres) that is involved in ADP activation (22). C shows the location of the inhibitor, GTP (mauve spheres), bound to the NADH (gray spheres), and glutamate (orange spheres) abortive complex. The green arrow notes the approximate location of one of the two sites (Lys-420) modified by 5′-FSBA (48). Comparing B and C, the closing of the catalytic cleft and the movement of the pivot helix is evident.From the structures GDH alone and complexed with various ligands, it is clear that GDH undergoes large conformational changes during each catalytic cycle (19–22) (the locations of these changes are summarized in Fig. 1). Substrate binds to the deep recesses of the cleft between the coenzyme binding domain and the lower domain. Coenzyme binds along the coenzyme binding domain surface of the cleft. Upon binding, the coenzyme binding domain rotates by ∼18° to firmly close down upon the substrate and coenzyme (Fig. 1, arrow 1). As the catalytic cleft closes, the base of each of the long ascending helices in the antenna appears to rotate out in a counterclockwise manner to push against the “pivot” helix of the adjacent subunit (Fig. 1, arrow 2). There is a short helix in the descending loop of the antenna that becomes distended and shorter as the mouth closes in a manner akin to an extending spring (Fig. 1, arrow 3). The pivot helix rotates in a counterclockwise manner along the helical axes as well as rotating counterclockwise around the trimer 3-fold axis. Finally, the entire hexamer seems to compress as the mouth closes (Fig. 1, arrow 4). The three pairs of subunits that sit on top of each other move as a rigid units toward each other, compressing the cavity at the core of the hexamer. This last conformational change will be further examined in this work. Allosteric regulation is likely exacted by controlling some or all of these conformational changes.The reason for complex animal regulation came from studies that linked GDH regulation with insulin and ammonia homeostasis. The connection between GDH and insulin regulation was initially established using a non-metabolizable analog of leucine (7, 23), BCH (β-2-aminobicycle[2.2.1]-heptane-2-carboxylic acid). These studies demonstrated that activation of GDH was tightly correlated with increased glutaminolysis and release of insulin. In addition, it has also been noted that factors that regulate GDH also affect insulin secretion (24). The in vivo importance of GDH in glucose homeostasis was demonstrated by the discovery that a genetic hypoglycemic disorder, the hyperinsulinemia/hyperammonemia (HHS) syndrome, is caused by loss of GTP regulation of GDH (25–27). Children with HHS have increased β-cell responsiveness to leucine and susceptibility to hypoglycemia following high protein meals (28). This is likely due to uncontrolled catabolism of amino acids yielding high ATP levels that stimulate insulin secretion as well as high serum ammonium levels. During glucose-stimulated insulin secretion in normal individuals, it has been proposed that the generation of high energy phosphates inhibits GDH and promotes conversion of glutamate to glutamine, which, alone or combined, might amplify the release of insulin (29, 30). Further support for this contention came from studies on the inhibitory effects of the polyphenolic compounds from green tea on BCH-stimulated insulin secretion (31). This not only lent support for the contention that GDH plays a significant role in insulin homeostasis, but also suggests that the HHS disorder might be directly controlled pharmaceutically. The role of GDH in insulin homeostasis is summarized in Fig. 2.Open in a separate windowFIGURE 2.Link between GDH and insulin homeostasis. This figure shows the role of GDH in BCH stimulated insulin secretion and how GDH inhibitors affect this process (29, 30). In energy-depleted β-cells, a BCH ramp stimulates insulin secretion. Here, the major energy source is glutaminolysis via phosphate-dependent glutaminase and GDH, because the concentration of GDH inhibitors (ATP/GTP) have been depleted and the phosphate-dependent glutaminase activator P_i (inorganic phosphate) has been increased. BCH stimulates glutamine utilization via GDH activation, thus providing the ATP signal necessary for insulin secretion. GDH inhibitors block this process by inhibiting GDH activity.To both find a more stable pharmaceutical agent to control HHS and to better understand the allosteric regulation of GDH, a high throughput screen was performed to identify new GDH inhibitors (32). Of the ∼30,000 compounds tested, ∼20 demonstrated significant activity. Three of the most active compounds, hexachlorophene, GW5074, and bithionol, were chosen for further analysis in this study. As shown here, all three compounds exhibit essentially non-competitive inhibition of the reaction and therefore do not compete with either substrate or coenzyme. Structural studies are presented here that demonstrate that six hexachlorophene (HCP) molecules bind to the inner core of the GDH hexamer, forming an internal ring via aromatic interactions. In contrast, bithionol and GW5074 bind as pairs between dimers of GDH subunits further away from the core of the enzyme. None of these compounds induce significant conformational changes in their immediate vicinity, and the mechanism of action is not clear from the location of their binding sites. However, detailed analysis of the various GDH complexes shows all of the drugs are binding to contact areas in the core of the hexamer that appear to be expanding and contracting during catalytic turnover. Therefore, inhibition is likely due interference with this “breathing” process.     

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.     

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.     

Comparative Studies of Class IIa Bacteriocins of Lactic Acid Bacteria

Four class IIa bacteriocins (pediocin PA-1, enterocin A, sakacin P, and curvacin A) were purified to homogeneity and tested for activity toward a variety of indicator strains. Pediocin PA-1 and enterocin A inhibited more strains and had generally lower MICs than sakacin P and curvacin A. The antagonistic activity of pediocin-PA1 and enterocin A was much more sensitive to reduction of disulfide bonds than the antagonistic activity of sakacin P and curvacin A, suggesting that an extra disulfide bond that is present in the former two may contribute to their high levels of activity. The food pathogen Listeria monocytogenes was among the most sensitive indicator strains for all four bacteriocins. Enterocin A was most effective in inhibiting Listeria, having MICs in the range of 0.1 to 1 ng/ml. Sakacin P had the interesting property of being very active toward Listeria but not having concomitant high levels of activity toward lactic acid bacteria. Strains producing class IIa bacteriocins displayed various degrees of resistance toward noncognate class IIa bacteriocins; for the sakacin P producer, it was shown that this resistance is correlated with the expression of immunity genes. It is hypothesized that variation in the presence and/or expression of such immunity genes accounts in part for the remarkably large variation in bacteriocin sensitivity displayed by lactic acid bacteria.Many lactic acid bacteria (LAB), including members of the genera Lactococcus, Lactobacillus, Carnobacterium, Enterococcus, and Pediococcus, are known to secrete small, ribosomally synthesized antimicrobial peptides called bacteriocins (26, 29, 34). Some of these peptides undergo posttranslational modifications (class I bacteriocins), whereas others are not modified (class II bacteriocins) (29, 34). Class II bacteriocins contain between 30 and 60 residues and are usually positively charged at a neutral pH. Studies of a large number of class II bacteriocins have led to subgrouping of these compounds (29, 34). One of the subgroups, class IIa, contains bacteriocins that are characterized by the presence of YGNG and CXXXXCXV sequence motifs in their N-terminal halves as well as by their strong inhibitory effect on Listeria (e.g., 3, 4, 22, 23, 27, 28, 31, 38, 45) (Fig. ​(Fig.1).1). Because of their effectiveness against the food pathogen Listeria, class IIa bacteriocins have potential as antimicrobial agents in food and feed. Open in a separate windowFIG. 1Sequence alignment of class IIa bacteriocins. Residue numbering is according to the sequence of pediocin PA-1. Cysteine residues are printed in boldface; the two known class IIa bacteriocins with four cysteine residues are in the upper group. No attempt was made to optimize the alignment in the C-terminal halves of the peptides. Piscicolin 126 is identical to piscicocin V1a (4). Carnobacteriocin BM1 most probably is identical to piscicocin V1b (4). Sakacin P most probably is identical to bavaricin A (30). Curvacin A is identical to sakacin A (2). The consensus sequence includes residues conserved in at least 8 of the 12 sequences shown; 100% conserved residues are underlined.Class IIa bacteriocins act by permeabilizing the membrane of their target cells (1, 5, 6, 9, 10, 26, 28). The most recent studies on the mode of action of these bacteriocins indicate that antimicrobial activity does not require a specific receptor and is enhanced by (but not fully dependent on) a membrane potential (9, 28). Little is known about bacteriocin structure, and unravelling the relationships between structure and function is one of the great challenges in current bacteriocin research. A logical starting point for structure-function studies is a thorough study of the differences in activity and target cell specificity between naturally occurring homologous bacteriocins. A few such studies have been described, but these suffer from either a very limited number of tested indicator strains or the use of culture supernatants instead of purified bacteriocins (3, 4, 17, 45). The use of purified bacteriocins for comparative analyses is absolutely essential, since it is becoming increasingly evident that bacteriocin producers produce more than one bacteriocin (4, 8, 38, 48; this study).In the present study, the activities of four pure class IIa bacteriocins (pediocin PA-1, enterocin A, curvacin A, and sakacin P) (Fig. ​(Fig.1)1) were tested against a large number of LAB as well as several strains of the food pathogen Listeria monocytogenes. The bacteriocins were purified from their respective producer strains by use of an optimized purification protocol yielding highly pure samples. The contribution of disulfide formation was assessed and found to be important for activity. The effects of the purified bacteriocins on (noncognate) class IIa bacteriocin-producing strains are described, and the implications of our findings for immunity and resistance are discussed.     

Structure and Function of Glycosylated Tandem Repeats from Candida albicans Als Adhesins

Tandem repeat (TR) regions are common in yeast adhesins, but their structures are unknown, and their activities are poorly understood. TR regions in Candida albicans Als proteins are conserved glycosylated 36-residue sequences with cell-cell aggregation activity (J. M. Rauceo, R. De Armond, H. Otoo, P. C. Kahn, S. A. Klotz, N. K. Gaur, and P. N. Lipke, Eukaryot. Cell 5:1664–1673, 2006). Ab initio modeling with either Rosetta or LINUS generated consistent structures of three-stranded antiparallel β-sheet domains, whereas randomly shuffled sequences with the same composition generated various structures with consistently higher energies. O- and N-glycosylation patterns showed that each TR domain had exposed hydrophobic surfaces surrounded by glycosylation sites. These structures are consistent with domain dimensions and stability measurements by atomic force microscopy (D. Alsteen, V. Dupres, S. A. Klotz, N. K. Gaur, P. N. Lipke, and Y. F. Dufrene, ACS Nano 3:1677–1682, 2009) and with circular dichroism determination of secondary structure and thermal stability. Functional assays showed that the hydrophobic surfaces of TR domains supported binding to polystyrene surfaces and other TR domains, leading to nonsaturable homophilic binding. The domain structures are like “classic” subunit interaction surfaces and can explain previously observed patterns of promiscuous interactions between TR domains in any Als proteins or between TR domains and surfaces of other proteins. Together, the modeling techniques and the supporting data lead to an approach that relates structure and function in many kinds of repeat domains in fungal adhesins.Yeast adhesins are a diverse set of cell adhesion proteins that mediate adhesion to host cells, environmental substrates, other fungi, and coinfecting bacteria (6, 8, 20, 21, 23, 29). The adhesins share common features, including compact N-terminal domains similar to Ig or lectin domains, Thr-rich midpieces, often in tandem repeats, and long highly glycosylated Ser/Thr-rich C-terminal regions that extend the functional domains out from the cell surface. No structures for the Thr-rich midpieces are known, but they can mediate aggregation of fungal cells (33, 35, 47). The prevalence and conservation of such repeats argue that they are functionally important, despite limited data on their structure and function.In Candida albicans, the Als adhesins are homologous proteins, products of 8 loci that encode numerous alleles of cell surface adhesins (16). In each mature Als protein, there are, from the N terminus, three tandem Ig-like domains, a β-sheet-rich conserved 127-residue amyloid-forming T region, a variable number of 36-residue tandem repeats (TRs), and a highly glycosylated stalk region that extends the N-terminal domains away from the cell surface (Fig. 1) (16, 33, 41). The C termini of these and other wall-associated adhesins are covalently cross-linked into the cell wall through transglycosylation of a modified glycosylphosphatidylinositol (GPI) anchor (18, 25). This modular design, including tandem repeats, is typical of fungal adhesins (8).Open in a separate windowFig. 1.Schematic diagram of the sequence of Als5p. The regions are named above, and the number of amino acid residues in each region is shown below. The modeled sequences are in the TR region.The Als protein Ig-like region, T region, and TR region all have protein-protein interaction activities (26, 33, 35). The Ig-like regions can interact with diverse mammalian proteins, presumably in a way analogous to antibody-antigen binding, as has been shown in the homologous protein α-agglutinin from Saccharomyces cerevisiae (8, 24, 26, 35). The T regions interact through formation of amyloid-like structures both in vivo and in vitro (33, 34a, 36). An insight into the function of the tandem repeats followed from observations that Als proteins initiate and maintain cell-to-cell aggregations, either spontaneously (“autoaggregation”) or following adhesion to a bead-bound defined ligand (10, 11, 36). Aggregation is more extensive for Als proteins with more tandem repeats (26, 35). This result suggested that the tandem repeats are uniquely structured to facilitate or mediate the aggregative function. Circular dichroism spectroscopy of the TR region of Als5p shows a β-sheet-rich structure in the soluble protein (35).In support of their direct involvement in aggregation, the repeat region of the C. albicans adhesin Als5p mediates cell-cell aggregation in the absence of the Ig-like and T domains (35). Moreover, the repeats can also potentiate binding of Als5p to fibronectin (35). Thus, the TR domains mediate cellular aggregation and increased binding to fibronectin. In addition, TR domains and their amino acid sequences are highly conserved across several Candida species (3). These properties need to be explained by their three-dimensional structure.Because there are no homologous structures known, we modeled by two independent ab initio methods. Rosetta assembles structures by combining short peptide structures extracted from the protein structural database PDB (38), then combines structures in a Monte Carlo approach, and assesses energetics of assembled structures. Rosetta has recently been shown to generate accurate models for protein-sized domains (40). We also predicted structures with LINUS, which generates randomized structures and rapidly estimates energetics to choose low-energy models (45). The models were supported by structural analyses with atomic force microscopy and circular dichroism spectroscopy. Functional assays showed that the TR domains can mediate binding activities predicted from the calculated structures.     

A Simple and Effective Cleavable Linker for Chemical Proteomics Applications

The study of metabolically labeled or probe-modified proteins is an important area in chemical proteomics. Isolation and purification of the protein targets is a necessary step before MS identification. The biotin-streptavidin system is widely used in this process, but the harsh denaturing conditions also release natively biotinylated proteins and non-selectively bound proteins. A cleavable linker strategy is a promising approach for solving this problem. Though several cleavable linkers have been developed and tested, an efficient, easily synthesized, and inexpensive cleavable linker is a desirable addition to the proteomics toolbox. Here, we describe the chemical proteomics application of a vicinal diol cleavable linker. Through easy-to-handle chemistry we incorporate this linker into an activity-based probe and a biotin alkyne tag amenable for bioorthogonal ligation. With these reagents, background protein identifications are significantly reduced relative to standard on-bead digestion.The covalent modification of proteins by small molecules within a complex proteome is a major theme in chemical biology and proteomics. An effective method for the detection of posttranslational modifications of proteins is the metabolic incorporation of modified biomolecules such as tagged carbohydrates or lipids (1). Reversible interactions of enzyme inhibitors, natural products, or drugs can be detected when one appends photocrosslinking agents, thereby facilitating target discovery (2, 3). A particularly interesting example of protein labeling is activity-based protein profiling (ABPP)¹ (4, 5), which utilizes the intrinsic catalytic activity of a target enzyme for the covalent attachment of an affinity or visualization tag. ABPP makes use of small molecules (activity-based probes (ABPs)) that react with the active form of a specific enzyme or enzyme class by means of a “warhead,” which is often derived from a mechanism-based enzyme inhibitor (Fig. 1A). DCG-04, for example, is based on the naturally occurring inhibitor E-64 and targets the papain family of cysteine proteases via covalent attachment of the epoxysuccinate group to the active site cysteine (Fig. 1B) (6).Open in a separate windowFig. 1.The cleavable linker strategy in ABPP. A, the elements of an ABP. B, the example ABP DCG-04, an epoxysuccinate-containing probe for clan CA cysteine proteases. DCG-04 is based on the naturally occurring protease inhibitor E-64. C, schematic strategy of cleavable linker-mediated target identification. D, the cleavage mechanism of a vicinal diol.Bulky fluorophore or biotin tags on chemical probes might interfere with efficient protein binding. Moreover, they can negatively influence the cell permeability of probes, which therefore limits their applicability in in vitro experiments. Bioorthogonal chemistries, such as the Bertozzi-Staudinger ligation (7) and the 1,3-bipolar cycloaddition of an azide and an alkyne (click chemistry) (8), allow tandem labeling strategies in which a biotin or a fluorophore is attached to an enzyme probe complex in a separate step. Consequently, the probes themselves only carry azide or alkyne groups as “mini-tags.” Tandem labeling using bioorthogonal chemistry has now become a widely used strategy to label biomolecules in lysates and in live cells (9–11).An essential step in ABPP, as well as in other chemical proteomics approaches, is the elucidation of the tagged proteins. This usually involves a biotin-mediated enrichment step followed by mass-spectrometry-based identification. Although the streptavidin-biotin interaction allows efficient enrichment as a result of the strong binding affinity (K_d ∼ 10⁻¹⁵ m), it also has limitations. The quantitative elution of biotinylated proteins requires harsh conditions (12), which lead to contamination of the sample by endogenous biotinylated and non-specifically bound proteins. These other proteins will be identified together with the real protein targets. Given that subsequent target validation with secondary assays can be a costly and time-consuming process, a reduction in false positive identifications is highly desirable. For cleaner protein identification, cleavable linker strategies (13) that allow the selective release of target proteins have been developed (Fig. 1C). The commercially available disulfide linker can be cleaved under mild conditions, but it suffers from premature cleavage in reducing media such as the intracellular environment and reducing buffers used for click chemistry and in vitro reactions of cysteine proteases. Therefore, a variety of alternative linkers for proteomics applications have been reported, including a sterically hindered disulfide (14), diazobenzenes (15–19), hydrazones (20, 21), silanes (22), light sensitive linkers (23–25), tobacco etch virus protease sensitive linkers (26, 27), and a levulinoyl-based linker (28). The synthesis of some of these linkers is lengthy or difficult to scale up, which limits their general application in chemical proteomics.Ideally, a cleavable linker is stable under a wide variety of conditions, is efficiently and selectively cleaved, and can be synthesized in a low number of easy chemical transformations. We aimed to meet these requirements by using a vicinal diol as a cleavable linker system. When vicinal diols are treated with sodium periodate (NaIO₄), the carbon–carbon bond is cleaved (Fig. 1D). Periodate treatment of proteins can result in side-reactions, such as the cleavage of linked carbohydrates or the oxidation of N-terminal serine and threonine residues. However, these N-termini rarely occur in proteins and are therefore of minor concern. In general, the mild, neutral conditions of periodate cleavage are compatible with proteins. This has been illustrated in the past, for example, by its application in the detection of protein–protein interactions (29) and the creation of unliganded MHC class I molecules (30). In this article, we report the chemical proteomics application of diol cleavable linker probes. We show that the synthesis of the linker and its probe derivatives is straightforward, that the linker is compatible with tandem click labeling, that enrichment and release of probe targets is efficient, and that the identification of targets takes place with significantly lower background than in on-bead digestion protocols.     

Genetic Analysis of the nuo Locus,Which Encodes the Proton-Translocating NADH Dehydrogenase in Escherichia coli

Complex I (EC 1.6.99.3) of the bacterium Escherichia coli is considered to be the minimal form of the type I NADH dehydrogenase, the first enzyme complex in the respiratory chain. Because of its small size and relative simplicity, the E. coli enzyme has become a model used to identify and characterize the mechanism(s) by which cells regulate the synthesis and assembly of this large respiratory complex. To begin dissecting the processes by which E. coli cells regulate the expression of nuo and the assembly of complex I, we undertook a genetic analysis of the nuo locus, which encodes the 14 Nuo subunits comprising E. coli complex I. Here we present the results of studies, performed on an isogenic collection of nuo mutants, that focus on the physiological, biochemical, and molecular consequences caused by the lack of or defects in several Nuo subunits. In particular, we present evidence that NuoG, a peripheral subunit, is essential for complex I function and that it plays a role in the regulation of nuo expression and/or the assembly of complex I.

Complex I (NADH:ubiquinone oxidoreductase; EC 1.6.99.3), a type I NADH dehydrogenase that couples the oxidation of NADH to the generation of a proton motive force, is the first enzyme complex of the respiratory chain (2, 35, 47). The Escherichia coli enzyme, considered to be the minimal form of complex I, consists of 14 subunits instead of the 40 to 50 subunits associated with the homologous eukaryotic mitochondrial enzyme (17, 29, 30, 48–50). E. coli also possesses a second NADH dehydrogenase, NDH-II, which does not generate a proton motive force (31). E. coli complex I resembles eukaryotic complex I in many ways (16, 17, 30, 49): it performs the same enzymatic reaction and is sensitive to a number of the same inhibitors, it consists of subunits homologous to those found in all proton-translocating NADH:ubiquinone oxidoreductases studied thus far, it is comprised of a large number of subunits relative to the number that comprise other respiratory enzymes, and it contains flavin mononucleotide and FeS center prosthetic groups. Additionally, it possesses an L-shaped topology (14, 22) like that of its Neurospora crassa homolog (27), and it consists of distinct fragments or subcomplexes. Whereas eukaryotic complex I can be dissected into a peripheral arm and a membrane arm, the E. coli enzyme consists of three subcomplexes referred to as the peripheral, connecting, and membrane fragments (29) (Fig. ​(Fig.1A).1A). The subunit composition of these three fragments correlates approximately with the organization of the 14 structural genes (nuoA to nuoN) (49) of the nuo (for NADH:ubiquinone oxidoreductase) locus (Fig. ​(Fig.1B),1B), an organization that is conserved in several other bacteria, including Salmonella typhimurium (3), Paracoccus denitrificans (53), Rhodobacter capsulatus (12), and Thermus thermophilus (54). The 5′ half of the locus contains a promoter (nuoP), previously identified and located upstream of nuoA (8, 49), and the majority of genes that encode subunits homologous to the nucleus-encoded subunits of eukaryotic complex I and to subunits of the Alcaligenes eutrophus NAD-reducing hydrogenase (17, 29, 30, 49). In contrast, the 3′ half contains the majority of the genes that encode subunits homologous to the mitochondrion-encoded subunits of eukaryotic complex I and to subunits of the E. coli formate-hydrogen lyase complex (17, 29, 30, 49). Whereas the nuclear homologs NuoE, NuoF, and NuoG constitute the peripheral fragment (also referred to as the NADH dehydrogenase fragment [NDF]), the nuclear homologs NuoB, NuoC, NuoD, and NuoI constitute the connecting fragment. The mitochondrial homologs NuoA, NuoH, NuoJ, NuoK, NuoL, NuoM, and NuoN constitute the membrane fragment (29). E. coli complex I likely evolved by fusion of preexisting protein assemblies constituting modules for electron transfer and proton translocation (17–19, 30). Open in a separate windowFIG. 1Schematic of E. coli complex I and the nuo locus. Adapted with permission of the publisher (17, 29, 30, 49). (A) E. coli complex I is comprised of three distinct fragments: the peripheral (light gray), connecting (white), and membrane (dark gray) fragments (17, 29). The peripheral fragment (NDF) is comprised of the nuclear homologs NuoE, -F, and -G and exhibits NADH dehydrogenase activity that oxidizes NADH to NAD⁺; the connecting fragment is comprised of the nuclear homologs NuoB, -C, -D, and -I; and the membrane fragment is comprised of the mitochondrial homologs NuoA, -H, and -J to -N and catalyzes ubiquinone (Q) to its reduced form (QH₂). FMN, flavin mononucleotide. (B) The E. coli nuo locus encodes the 14 Nuo subunits that constitute complex I. The 5′ half of the locus contains a previously identified promoter (nuoP) and the majority of genes that encode the peripheral and connecting subunits (light gray and white, respectively). The 3′ half of the locus contains the majority of the genes encoding the membrane subunits (dark gray). The 3′ end of nuoG encodes a C-Terminal region (CTR) of the NuoG subunit (hatched).Because of its smaller size and relative simplicity, researchers recently have begun to utilize complex I of E. coli, and that of its close relative S. typhimurium, to identify and characterize the mechanism(s) by which cells regulate the synthesis and assembly of this large respiratory complex (3, 8, 46) and to investigate the diverse physiological consequences caused by defects in this enzyme (4, 6, 10, 40, 59). Such defects affect the ability of cells to perform chemotaxis (40), to grow on certain carbon sources (4, 6, 10, 40, 57), to survive stationary phase (59), to perform energy-dependent proteolysis (4), to regulate the expression of at least one gene (32), and to maintain virulence (5).To begin dissecting the processes by which E. coli cells regulate the expression of nuo and the assembly of complex I, we undertook a genetic analysis of the nuo locus. Here, we present the results of studies, performed on an isogenic collection of nuo mutants, that focus on the physiological, biochemical, and molecular consequences caused by the lack of or defects in several Nuo subunits. In particular, we present evidence that NuoG, a peripheral subunit, is essential for complex I function and that it plays a role in the regulation of nuo expression and/or the assembly of complex I.     

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.     

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.