Lucia Banci

Opposing cardioprotective actions and parallel hypertrophic effects of δPKC and ɛPKC

Conflicting roles for protein kinase C (PKC) isozymes in cardiac disease have been reported. Here, δPKC-selective activator and inhibitor peptides were designed rationally, based on molecular modeling and structural homology analyses. Together with previously identified activator and inhibitor peptides of ɛPKC, δPKC peptides were used to identify cardiac functions of these isozymes. In isolated cardiomyocytes, perfused hearts, and transgenic mice, δPKC and ɛPKC had opposing actions on protection from ischemia-induced damage. Specifically, activation of ɛPKC caused cardioprotection whereas activation of δPKC increased damage induced by ischemia in vitro and in vivo. In contrast, δPKC and ɛPKC caused identical nonpathological cardiac hypertrophy; activation of either isozyme caused nonpathological hypertrophy of the heart. These results demonstrate that two related PKC isozymes have both parallel and opposing effects in the heart, indicating the danger in the use of therapeutics with nonselective isozyme inhibitors and activators. Moreover, reduction in cardiac damage caused by ischemia by perfusion of selective regulator peptides of PKC through the coronary arteries constitutes a major step toward developing a therapeutic agent for acute cardiac ischemia.

Affinity gradients drive copper to cellular destinations

Copper is an essential trace element for eukaryotes and most prokaryotes. However, intracellular free copper must be strictly limited because of its toxic side effects. Complex systems for copper trafficking evolved to satisfy cellular requirements while minimizing toxicity. The factors driving the copper transfer between protein partners along cellular copper routes are, however, not fully rationalized. Until now, inconsistent, scattered and incomparable data on the copper-binding affinities of copper proteins have been reported. Here we determine, through a unified electrospray ionization mass spectrometry (ESI-MS)-based strategy, in an environment that mimics the cellular redox milieu, the apparent Cu(I)-binding affinities for a representative set of intracellular copper proteins involved in enzymatic redox catalysis, in copper trafficking to and within various cellular compartments, and in copper storage. The resulting thermodynamic data show that copper is drawn to the enzymes that require it by passing from one copper protein site to another, exploiting gradients of increasing copper-binding affinity. This result complements the finding that fast copper-transfer pathways require metal-mediated protein–protein interactions and therefore protein–protein specific recognition. Together with Cu,Zn-SOD1, metallothioneins have the highest affinity for copper(I), and may play special roles in the regulation of cellular copper distribution; however, for kinetic reasons they cannot demetallate copper enzymes. Our study provides the thermodynamic basis for the kinetic processes that lead to the distribution of cellular copper.

MIA40 is an oxidoreductase that catalyzes oxidative protein folding in mitochondria

MIA40 has a key role in oxidative protein folding in the mitochondrial intermembrane space. We present the solution structure of human MIA40 and its mechanism as a catalyst of oxidative folding. MIA40 has a 66-residue folded domain made of an α-helical hairpin core stabilized by two structural disulfides and a rigid N-terminal lid, with a characteristic CPC motif that can donate its disulfide bond to substrates. The CPC active site is solvent-accessible and sits adjacent to a hydrophobic cleft. Its second cysteine (Cys55) is essential in vivo and is crucial for mixed disulfide formation with the substrate. The hydrophobic cleft functions as a substrate binding domain, and mutations of this domain are lethal in vivo and abrogate binding in vitro. MIA40 represents a thioredoxin-unrelated, minimal oxidoreductase, with a facile CPC redox active site that ensures its catalytic function in oxidative folding in mitochondria.

Metal-free superoxide dismutase forms soluble oligomers under physiological conditions: A possible general mechanism for familial ALS

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder selectively affecting motor neurons; 90% of the total cases are sporadic, but 2% are associated with mutations in the gene coding for the antioxidant enzyme copper–zinc superoxide dismutase (SOD1). The causes of motor neuron death in ALS are poorly understood in general, but for SOD1-linked familial ALS, aberrant oligomerization of SOD1 mutant proteins has been strongly implicated. In this work, we show that wild-type human SOD1, when lacking both its metal ions, forms large, stable, soluble protein oligomers with an average molecular mass of ≈650 kDa under physiological conditions, i.e., 37°C, pH 7.0, and 100 μM protein concentration. It further is shown here that intermolecular disulfide bonds are formed during oligomerization and that Cys-6 and Cys-111 are implicated in this bonding. The formation of the soluble oligomers was monitored by their ability to enhance the fluorescence of thioflavin T, a benzothiazole dye that increases in fluorescence intensity upon binding to amyloid fibers, and by disruption of this binding upon addition of the chaotropic agent guanidine hydrochloride. Our results suggest a general, unifying picture of SOD1 aggregation that could operate when wild-type or mutant SOD1 proteins lack their metal ions. Although we cannot exclude other mechanisms in SOD1-linked familial ALS, the one proposed here has the strength of explaining how a large and diverse set of SOD1 mutant proteins all could lead to disease through the same mechanism.

High‐resolution NMR studies of the zinc‐binding site of the Alzheimer's amyloid β‐peptide

Metal binding to the amyloid β‐peptide is suggested to be involved in the pathogenesis of Alzheimers disease. We used high‐resolution NMR to study zinc binding to amyloid β‐peptide 1–40 at physiologic pH. Metal binding induces a structural change in the peptide, which is in chemical exchange on an intermediate rate, between the apo‐form and the holo‐form, with respect to the NMR timescale. This causes loss of NMR signals in the resonances affected by the binding. Heteronuclear correlation experiments, 15N‐relaxation and amide proton exchange experiments on amyloid β‐peptide 1–40 revealed that zinc binding involves the three histidines (residues 6, 13 and 14) and the N‐terminus, similar to a previously proposed copper‐binding site [Syme CD, Nadal RC, Rigby SE, Viles JH (2004) J Biol Chem279, 18169–18177]. Fluorescence experiments show that zinc shares a common binding site with copper and that the metals have similar affinities for amyloid β‐peptide. The dissociation constant Kd of zinc for the fragment amyloid β‐peptide 1–28 was measured by fluorescence, using competitive binding studies, and that for amyloid β‐peptide 1–40 was measured by NMR. Both methods gave Kd values in the micromolar range at pH 7.2 and 286 K. Zinc also has a second, weaker binding site involving residues between 23 and 28. At high metal ion concentrations, the metal‐induced aggregation should mainly have an electrostatic origin from decreased repulsion between peptides. At low metal ion concentrations, on the other hand, the metal‐induced structure of the peptide counteracts aggregation.

Structural properties of peroxidases

Peroxidases are heme proteins which are able to catalyze the oxidation of a large variety of substrates through the reaction with hydrogen peroxide. The specific biological function, the reduction potential of the iron and the nature of the substrates which can be oxidized, are strongly determined by the structural features of the protein matrix around the prosthetic group. In particular, two main features are considered to be responsible of the specificity of the biological function: the strong anionic character of the fifth, proximal ligand to the iron, which is able to stabilize high oxidation states, and the hydrophilic nature of the residues in the distal pocket. Beside the correct reduction potential for the oxidation reaction, the specificity towards different substrates also depends on the protein structural arrangement which can determine specific binding sites for substrates and mediators. Particularly, in the case of MnP,the Mn2+ binding site has been individuated in the X-ray structure. NMR studies were previously reported which provided an iron-manganese distance consistent with that from the X-ray structure. This information can help in defining the possible pathway for the electron transfer from the Mn2+ ion to the iron. On the contrary, in the case of LiP no information is available on the possible binding site of veratryl alcohol as well as of other aromatic substrates. This article reviews these structural properties of peroxidases with particular emphasis to their implications in the catalytic process. Finally, the calcium ions have been located in the structure of LiP and the MnP: their structural relevance will be discussed on the light of the possible role in determining the optimal arrangement of residues in the distal cavity for the enzymatic reaction.

A redox switch in CopC: an intriguing copper trafficking protein that binds copper(I) and copper(II) at different sites.

The protein CopC from Pseudomonas syringae has been found capable of binding copper(I) and copper(II) at two different sites, occupied either one at a time or simultaneously. The protein, consisting of 102 amino acids, is known to bind copper(II) in a position that is now found consistent with a coordination arrangement including His-1, Glu-27, Asp-89, and His-91. A full solution structure analysis is reported here for Cu(I)-CopC. The copper(I) site is constituted by His-48 and three of the four Met residues (40, 43, 46, 51), which are clustered in a Met-rich region. Both copper binding sites have been characterized through extended x-ray absorption fine structure studies. They represent novel coordination environments for copper in proteins. The two sites are ≈30 Å far apart and have little affinity for the ion in the other oxidation state. Oxidation of Cu(I)-CopC or reduction of Cu(II)-CopC causes migration of copper from one site to the other. This behavior is observed both in NMR and EXAFS studies and indicates that CopC can exchange copper between two sites activated by a redox switch. CopC resides in the periplasm of Gram-negative bacteria where there is a multicopper oxidase, CopA, which may modulate the redox state of copper. CopC and CopA are coded in the same operon, responsible for copper resistance. These peculiar and novel properties of CopC are discussed with respect to their relevance for copper homeostasis.

SOD1 and Amyotrophic Lateral Sclerosis: Mutations and Oligomerization

There are about 100 single point mutations of copper, zinc superoxide dismutase 1 (SOD1) which are reported (http://alsod.iop.kcl.ac.uk/Als/index.aspx) to be related to the familial form (fALS) of amyotrophic lateral sclerosis (ALS). These mutations are spread all over the protein. It is well documented that fALS produces protein aggregates in the motor neurons of fALS patients, which have been found to be associated to mitochondria. We selected eleven SOD1 mutants, most of them reported as pathological, and characterized them investigating their propensity to aggregation using different techniques, from circular dichroism spectra to ThT-binding fluorescence, size-exclusion chromatography and light scattering spectroscopy. We show here that these eleven SOD1 mutants, only when they are in the metal-free form, undergo the same general mechanism of oligomerization as found for the WT metal-free protein. The rates of oligomerization are different but eventually they give rise to the same type of soluble oligomeric species. These oligomers are formed through oxidation of the two free cysteines of SOD1 (6 and 111) and stabilized by hydrogen bonds, between beta strands, thus forming amyloid-like structures. SOD1 enters the mitochondria as demetallated and mitochondria are loci where oxidative stress may easily occur. The soluble oligomeric species, formed by the apo form of both WT SOD1 and its mutants through an oxidative process, might represent the precursor toxic species, whose existence would also suggest a common mechanism for ALS and fALS. The mechanism here proposed for SOD1 mutant oligomerization is absolutely general and it provides a common unique picture for the behaviors of the many SOD1 mutants, of different nature and distributed all over the protein.

A novel intermembrane space–targeting signal docks cysteines onto Mia40 during mitochondrial oxidative folding

A nine-residue intermembrane-targeting signal brings the active Cys of substrate proteins into contact with Mia40 oxidase for folding and import into mitochondria.

Rational Design of a Meningococcal Antigen Inducing Broad Protective Immunity

A single chimeric protein induces protective immunity against all meningococcal B strains with implications for producing broadly protective vaccines. All for One and One for All The three musketeers were a formidable team, but imagine combining all of their skills and valor into just one musketeer. That is precisely the approach that Rappuoli and his colleagues have taken with their design of a vaccine against meningococcus B, the bacterial pathogen that causes meningitis. Although mining of the genome sequence of this pathogen has yielded excellent targets that could be used in a vaccine, many of these antigens show a high degree of variation that has stymied attempts to use them as vaccine immunogens. For example, factor H binding protein is essential for the survival of meningococcus B in the human host because it protects the pathogen from the onslaught of the human immune system’s complement pathway. Because it is essential for survival, factor H binding protein should be a valuable immunogen, but because it has at least 300 sequence variants, it is impractical to make one vaccine that contains all of these variants. Rappuoli and his colleagues have tackled this problem by dividing the 300 sequence variants of factor H binding protein into three major groups. Using detailed structural information about these three major variants, they engineered variant 1 to carry patches of amino acids from the surfaces of variants 2 and 3. They then introduced groups of point mutations into the amino acids of these transplanted patches to mimic the natural variation of variant 2 and 3 strains of meningococcus B. They then tested which of the 54 engineered single chimeric immunogens could elicit bactericidal antibodies against many different strains of meningococcus B. To do this, they injected the immunogens into mice and assayed mouse sera in vitro for bactericidal activity against multiple bacterial strains. One chimeric immunogen, called G1, was capable of inducing bactericidal antibodies that could kill all strains of meningococcus B, suggesting that it could be used to produce a broadly protective vaccine. This structure-based approach to vaccine design may be useful not only for meningococcus B but also for other pathogens like HIV that show a high degree of antigenic variation. The sequence variability of protective antigens is a major challenge to the development of vaccines. For Neisseria meningitidis, the bacterial pathogen that causes meningitis, the amino acid sequence of the protective antigen factor H binding protein (fHBP) has more than 300 variations. These sequence differences can be classified into three distinct groups of antigenic variants that do not induce cross-protective immunity. Our goal was to generate a single antigen that would induce immunity against all known sequence variants of N. meningitidis. To achieve this, we rationally designed, expressed, and purified 54 different mutants of fHBP and tested them in mice for the induction of protective immunity. We identified and determined the crystal structure of a lead chimeric antigen that was able to induce high levels of cross-protective antibodies in mice against all variant strains tested. The new fHBP antigen had a conserved backbone that carried an engineered surface containing specificities for all three variant groups. We demonstrate that the structure-based design of multiple immunodominant antigenic surfaces on a single protein scaffold is possible and represents an effective way to create broadly protective vaccines.