COQ4 is required for the oxidative decarboxylation of the C1 carbon of coenzyme Q in eukaryotic cells.

Coenzyme Q (CoQ) is a redox lipid that fulfills critical functions in cellular bioenergetics and homeostasis. CoQ is synthesized by a multi-step pathway that involves several COQ proteins. Two steps of the eukaryotic pathway, the decarboxylation and hydroxylation of position C1, have remained uncharacterized. Here, we provide evidence that these two reactions occur in a single oxidative decarboxylation step catalyzed by COQ4. We demonstrate that COQ4 complements an Escherichia coli strain deficient for C1 decarboxylation and hydroxylation and that COQ4 displays oxidative decarboxylation activity in the non-CoQ producer Corynebacterium glutamicum. Overall, our results substantiate that COQ4 contributes to CoQ biosynthesis, not only via its previously proposed structural role but also via the oxidative decarboxylation of CoQ precursors. These findings fill a major gap in the knowledge of eukaryotic CoQ biosynthesis and shed light on the pathophysiology of human primary CoQ deficiency due to COQ4 mutations.

COQ4 is required for the oxidative decarboxylation of the C1 carbon of Coenzyme Q in eukaryotic cells.

Coenzyme Q (CoQ) is a redox lipid that fulfills critical functions in cellular bioenergetics and homeostasis. CoQ is synthesized by a multi-step pathway that involves several COQ proteins. Two steps of the eukaryotic pathway, the decarboxylation and hydroxylation of position C1, have remained uncharacterized. Here, we provide evidence that these two reactions occur in a single oxidative decarboxylation step catalyzed by COQ4. We demonstrate that COQ4 complements an Escherichia coli strain deficient for C1 decarboxylation and hydroxylation and that COQ4 displays oxidative decarboxylation activity in the non-CoQ producer Corynebacterium glutamicum. Overall, our results substantiate that COQ4 contributes to CoQ biosynthesis, not only via its previously proposed structural role, but also via oxidative decarboxylation of CoQ precursors. These findings fill a major gap in the knowledge of eukaryotic CoQ biosynthesis, and shed new light on the pathophysiology of human primary CoQ deficiency due to COQ4 mutations.

The mitochondrial calcium uniporter complex-A play in five acts.

Mitochondrial Ca2+ (mitCa2+) uptake controls both intraorganellar and cytosolic functions. Within the organelle, [Ca2+] increases regulate the activity of tricarboxylic acid (TCA) cycle enzymes, thus sustaining oxidative metabolism and ATP production. Reactive oxygen species (ROS) are also generated as side products of oxygen consumption. At the same time, mitochondria act as buffers of cytosolic Ca2+ (cytCa2+) increases, thus regulating Ca2+-dependent cellular processes. In pathological conditions, mitCa2+ overload triggers the opening of the mitochondrial permeability transition pore (mPTP) and the release of apoptotic cofactors. MitCa2+ uptake occurs in response of local [Ca2+] increases in sites of proximity between the endoplasmic reticulum (ER) and the mitochondria and is mediated by the mitochondrial Ca2+ uniporter (MCU), a highly selective channel of the inner mitochondrial membrane (IMM). Both channel and regulatory subunits form the MCU complex (MCUC). Cryogenic electron microscopy (Cryo-EM) and crystal structures revealed the correct assembly of MCUC and the function of critical residues for the regulation of Ca2+ conductance.

The ataxia-linked E1081Q mutation affects the sub-plasma membrane Ca2+-microdomains by tuning PMCA3 activity.

Calcium concentration must be finely tuned in all eukaryotic cells to ensure the correct performance of its signalling function. Neuronal activity is exquisitely dependent on the control of Ca2+ homeostasis: its alterations ultimately play a pivotal role in the origin and progression of many neurodegenerative processes. A complex toolkit of Ca2+ pumps and exchangers maintains the fluctuation of cytosolic Ca2+ concentration within the appropriate threshold. Two ubiquitous (isoforms 1 and 4) and two neuronally enriched (isoforms 2 and 3) of the plasma membrane Ca2+ATPase (PMCA pump) selectively regulate cytosolic Ca2+ transients by shaping the sub-plasma membrane (PM) microdomains. In humans, genetic mutations in ATP2B1, ATP2B2 and ATP2B3 gene have been linked with hearing loss, cerebellar ataxia and global neurodevelopmental delay: all of them were found to impair pump activity. Here we report three additional mutations in ATP2B3 gene corresponding to E1081Q, R1133Q and R696H amino acids substitution, respectively. Among them, the novel missense mutation (E1081Q) immediately upstream the C-terminal calmodulin-binding domain (CaM-BD) of the PMCA3 protein was present in two patients originating from two distinct families. Our biochemical and molecular studies on PMCA3 E1081Q mutant have revealed a splicing variant-dependent effect of the mutation in shaping the sub-PM [Ca2+]. The E1081Q substitution in the full-length b variant abolished the capacity of the pump to reduce [Ca2+] in the sub-PM microdomain (in line with the previously described ataxia-related PMCA mutations negatively affecting Ca2+ pumping activity), while, surprisingly, its introduction in the truncated a variant selectively increased Ca2+ extrusion activity in the sub-PM Ca2+ microdomains. These results highlight the importance to set a precise threshold of [Ca2+] by fine-tuning the sub-PM microdomains and the different contribution of the PMCA splice variants in this regulation.

Copper Binding and Oligomerization Studies of the Metal Resistance Determinant CrdA from Helicobacter pylori.

Within this research, the CrdA protein from Helicobacter pylori (HpCrdA), a putative copper-binding protein important for the survival of bacterium, was biophysically characterized in a solution, and its binding affinity toward copper was experimentally determined. Incubation of HpCrdA with Cu(II) ions favors the formation of the monomeric species in the solution. The modeled HpCrdA structure shows a conserved methionine-rich region, a potential binding site for Cu(I), as in the structures of similar copper-binding proteins, CopC and PcoC, from Pseudomonas syringae and from Escherichia coli, respectively. Within the conserved amino acid motif, HpCrdA contains two additional methionines and two glutamic acid residues (MMXEMPGMXXMXEM) in comparison to CopC and PcoC but lacks the canonical Cu(II) binding site (two His) since the sequence has no His residues. The methionine-rich site is in a flexible loop and can adopt different geometries for the two copper oxidation states. It could bind copper in both oxidation states (I and II), but with different binding affinities, micromolar was found for Cu(II), and less than nanomolar is proposed for Cu(I). Considering that CrdA is a periplasmic protein involved in chaperoning copper export and delivery in the H. pylori cell and that the affinity of the interaction corresponds to a middle or strong metal-protein interaction depending on the copper oxidation state, we conclude that the interaction also occurs in vivo and is physiologically relevant for H. pylori.

Exceptionally potent human monoclonal antibodies are effective for prophylaxis and treatment of tetanus in mice.

We used human monoclonal antibodies (humAbs) to study the mechanism of neuron intoxication by tetanus neurotoxin and to evaluate these antibodies as a safe preventive and therapeutic substitute for hyperimmune sera to treat tetanus in mice. By screening memory B cells from immune donors, we selected 2 tetanus neurotoxin-specific mAbs with exceptionally high neutralizing activities and extensively characterized them both structurally and functionally. We found that these antibodies interfered with the binding and translocation of the neurotoxin into neurons by interacting with 2 epitopes, whose identification pinpoints crucial events in the cellular pathogenesis of tetanus. Our observations explain the neutralization ability of these antibodies, which we found to be exceptionally potent in preventing experimental tetanus when injected into mice long before the toxin. Moreover, their Fab derivatives neutralized tetanus neurotoxin in post-exposure experiments, suggesting their potential for therapeutic use via intrathecal injection. As such, we believe these humAbs, as well as their Fab derivatives, meet the requirements to be considered for prophylactic and therapeutic use in human tetanus and are ready for clinical trials.

Structure of filamentous viruses.

Despite filamentous viruses represent an important portion of the universe of viruses, their 3D structures available are quite limited, particularly if compared to the large number of structures of icosahedral viruses present in the Protein Data Bank. As a matter of fact, flexible filamentous viruses cannot be grown as single crystals and past structural studies have mostly been limited to X-ray fiber diffraction or to the determination of the structure of isolated viral proteins. Only very recently, several structures of filamentous viruses have become available, owing to the recent development of cryo-electron microscopy. This technique has given a strong impulse to the field and has allowed the building of reliable molecular models of entire viruses, in some cases at a nearly atomic resolution level. In this paper we briefly describe the architecture of filamentous viruses that infect bacteria, archaea, plants and humans. It is easy to foresee that more new structures of filamentous viruses will become available soon and they will allow a better understanding of the rules underlying the structural organization of these organisms so relevant for the life on our planet.

Aldosterone synthase inhibitors for cardiovascular diseases: A comprehensive review of preclinical, clinical and in silico data.

Aldosterone, the main mineralocorticoid hormone, plays a fundamental role in maintaining blood pressure (BP)and volume under hypovolemic conditions. However, in numerous diseases, where it is produced in excess, it plays a detrimental role and contributes to cardiovascular events and ultimately to death in a multitude of patients. The seminal observation that the fungicide-derivative fadrozole blunted steroidogenesis has led to develop several agents to inhibit aldosterone synthase (AS, CYP11B2), the mitochondrial NADH-dependent enzyme that is necessary for aldosterone biosynthesis. Aldosterone synthase inhibitors (ASI) have, thereafter, been conceived and investigated in phase I and phase II studies. We herein reviewed the ASIs available so far considering their chemical structure, the related aldosterone synthase binding and pharmacodynamic properties. We also examined the promising results obtained with ASIs that have already been tested in phase II human studies.

High-Light versus Low-Light: Effects on Paired Photosystem II Supercomplex Structural Rearrangement in Pea Plants.

In plant grana thylakoid membranes Photosystem II (PSII) associates with a variable number of antenna proteins (LHCII) to form different types of supercomplexes (PSII-LHCII), whose organization is dynamically adjusted in response to light cues, with the C2S2 more abundant in high-light and the C2S2M2 in low-light. Paired PSII-LHCII supercomplexes interacting at their stromal surface from adjacent thylakoid membranes were previously suggested to mediate grana stacking. Here, we present the cryo-electron microscopy maps of paired C2S2 and C2S2M2 supercomplexes isolated from pea plants grown in high-light and low-light, respectively. These maps show a different rotational offset between the two supercomplexes in the pair, responsible for modifying their reciprocal interaction and energetic connectivity. This evidence reveals a different way by which paired PSII-LHCII supercomplexes can mediate grana stacking at diverse irradiances. Electrostatic stromal interactions between LHCII trimers almost completely overlapping in the paired C2S2 can be the main determinant by which PSII-LHCII supercomplexes mediate grana stacking in plants grown in high-light, whereas the mutual interaction of stromal N-terminal loops of two facing Lhcb4 subunits in the paired C2S2M2 can fulfil this task in plants grown in low-light. The high-light induced accumulation of the Lhcb4.3 protein in PSII-LHCII supercomplexes has been previously reported. Our cryo-electron microscopy map at 3.8 Å resolution of the C2S2 supercomplex isolated from plants grown in high-light suggests the presence of the Lhcb4.3 protein revealing peculiar structural features of this high-light-specific antenna important for photoprotection.

Functional analysis and cryo-electron microscopy of Campylobacterjejuni serine protease HtrA.

Campylobacter jejuni is a predominant zoonotic pathogen causing gastroenteritis and other diseases in humans. An important bacterial virulence factor is the secreted serine protease HtrA (HtrA Cj ), which targets tight and adherens junctional proteins in the gut epithelium. Here we have investigated the function and structure of HtrA Cj using biochemical assays and cryo-electron microscopy. Mass spectrometry analysis identified differences and similarities in the cleavage site specificity for HtrA Cj by comparison to the HtrA counterparts from Helicobacter pylori and Escherichia coli. We defined the architecture of HtrA Cj at 5.8 Å resolution as a dodecamer, built of four trimers. The contacts between the trimers are quite loose, a fact that explains the flexibility and mobility of the dodecameric assembly. This flexibility has also been studied through molecular dynamics simulation, which revealed opening of the dodecamer to expose the proteolytically active site of the protease. Moreover, we examined the rearrangements at the level of oligomerization in the presence or absence of substrate using size exclusion chromatography, which revealed hexamers, dodecamers and larger oligomeric forms, as well as remarkable stability of higher oligomeric forms (> 12-mers) compared to previously tested homologs from other bacteria. Extremely dynamic decay of the higher oligomeric forms into lower forms was observed after full cleavage of the substrate by the proteolytically active variant of HtrA Cj . Together, this is the first report on the in-depth functional and structural analysis of HtrA Cj , which may allow the construction of therapeutically relevant HtrA Cj inhibitors in the near future.

Experimentally Determined Long Intrinsically Disordered Protein Regions Are Now Abundant in the Protein Data Bank.

Intrinsically disordered protein regions are commonly defined from missing electron density in X-ray structures. Experimental evidence for long disorder regions (LDRs) of at least 30 residues was so far limited to manually curated proteins. Here, we describe a comprehensive and large-scale analysis of experimental LDRs for 3133 unique proteins, demonstrating an increasing coverage of intrinsic disorder in the Protein Data Bank (PDB) in the last decade. The results suggest that long missing residue regions are a good quality source to annotate intrinsically disordered regions and perform functional analysis in large data sets. The consensus approach used to define LDRs allows to evaluate context dependent disorder and provide a common definition at the protein level.

Benzylaminoethyureido-Tailed Benzenesulfonamides: Design, Synthesis, Kinetic and X-ray Investigations on Human Carbonic Anhydrases.

A drug design strategy of carbonic anhydrase inhibitors (CAIs) belonging to sulfonamides incorporating ureidoethylaminobenzyl tails is presented. A variety of substitution patterns on the ring and the tails, located on para- or meta- positions with respect to the sulfonamide warheads were incorporated in the new compounds. Inhibition of human carbonic anhydrases (hCA) isoforms I, II, IX and XII, involving various pathologies, was assessed with the new compounds. Selective inhibitory profile towards hCA II was observed, the most active compounds being low nM inhibitors (KIs of 2.8-9.2 nM, respectively). Extensive X-ray crystallographic analysis of several sulfonamides in an adduct with hCA I allowed an in-depth understanding of their binding mode and to lay a detailed structure-activity relationship.

Atomic structure of potato virus X, the prototype of the Alphaflexiviridae family.

Potato virus X (PVX) is a positive-sense single-stranded RNA (ssRNA) filamentous plant virus belonging to the Alphaflexiviridae family, considered in recent years as a tool for nanotechnology applications. We present the cryo-electron microscopy structure of the PVX particle at a resolution of 2.2 Å. The well-defined density of the coat proteins and of the genomic RNA allowed a detailed analysis of protein-RNA interactions, including those mediated by solvent molecules. The particle is formed by repeated segments made of 8.8 coat proteins, forming a left-handed helical structure. The RNA runs in an internal crevice along the virion, packaged in 5-nucleotide repeats in which the first four bases are stacked in the classical way, while the fifth is rotated and nearly perpendicular. The resolution of the structure described here suggests a mechanism for the virion assembly and potentially provides a platform for the rational design of antiviral compounds and for the use of PVX in nanotechnology.

Structure-activity relationships of flurbiprofen analogues as stabilizers of the amyloidogenic protein transthyretin.

The inherent amyloidogenic potentialof wild type transthyretin (TTR) is enhanced by a large number of point mutations, which destabilize the TTR tetramer, thereby promoting its disassembly and pathological aggregation responsible for TTR-related amyloidosis. TTR stabilizers are able to interact with the thyroxine-binding sites of TTR, stabilizing its tetrameric native state and inhibiting amyloidogenesis. Herein, we report on in vitro, ex vivo, and X-ray analyses to assess the TTR structural stabilization by analogues of flurbiprofen, a non-steroidal anti-inflammatory drug (NSAID). Overall, considering together binding selectivity and protective effects on TTR native structure by flurbiprofen analogues in the presence of plasma proteins, as determined by Western Blot,the aforementioned properties of analyzed compounds appear to be better (CHF5075 and CHF4802) or similar (CHF4795) or worse (CHF5074, also known as CSP-1103) as compared to those of diflunisal, used as a reference TTR stabilizer. Molecular details of the determinants affecting the interactionsof CHF5075, CHF4802, and CHF4795 with wild type TTRand of CHF5074 withtheamyloidogenic A25TTTR variant havebeen elucidated by X-ray analysis. Distinct interactions with TTR appear to characterize flurbiprofen analogues and the NSAID diflunisal and its analogues as TTR stabilizers. Relationships between stabilizing effect on TTR by flurbiprofen analogues determined experimentally and molecular details of their interactions with TTR have been established, providing the rationale for their protective effects on the native protein structure.

Structural Aspects of Helicobacter pylori Antibiotic Resistance.

Resistance to antibiotics of Helicobacter pylori infections is growing rapidly together with the need for more potent antimicrobials or novel strategies to recover the efficacy of the existing ones. Despite the main mechanisms according to which H. pylori acquires resistance are common to other microbial infections affecting humans, H. pylori has its own peculiarities, mostly due to the unique conditions experienced by the bacterium in the gastric niche. Possibly the most used of the antibiotics for H. pylori are those molecules that bind to the ribosome or to the DNA and RNA machinery, and in doing so they interfere with protein synthesis. Another important class is represented by molecules that binds to some enzyme essential for the bacterium survival, as in the case of enzymes involved in the bacterial wall biosynthesis. The mechanism used by the bacterium to fight antibiotics can be grouped in three classes: (i) mutations of some key residues in the protein that binds the inhibitor, (ii) regulation of the efflux systems or of the membrane permeability in order to reduce the uptake of the antibiotic, and (iii) other more complex indirect effects. Interestingly, the production of enzymes that degrade the antibiotics (as in the case of ß-lactamases in many other bacteria) has not been clearly detected in H. pylori. The structural aspects of resistance players have not been object of extensive studies yet and the structure of very few H. pylori proteins involved in the resistance mechanisms are determined till now. Models of the proteins that play key roles in reducing antimicrobials susceptibility and their implications will be discussed in this chapter.

The lipoprotein HP1454 of Helicobacter pylori regulates T-cell response by shaping T-cell receptor signalling.

Helicobacter pylori (HP) is a Gram-negative bacterium that chronically infects the stomach of more than 50% of human population and represents a major cause of gastric cancer, gastric lymphoma, gastric autoimmunity, and peptic ulcer. It still remains to be elucidated, which HP virulence factors are important in the development of gastric disorders. Here, we analysed the role of the HP protein HP1454 in the host-pathogen interaction. We found that a significant proportion of T cells isolated from HP patients with chronic gastritis and gastric adenocarcinoma proliferated in response to HP1454. Moreover, we demonstrated in vivo that HP1454 protein drives Th1/Th17 inflammatory responses. We further analysed the in vitro response of human T cells exposed either to an HP wild-type strain or to a strain with a deletion of the hp1454 gene, and we revealed that HP1454 triggers the T-cell antigen receptor-dependent signalling and lymphocyte proliferation, as well as the CXCL12-dependent cell adhesion and migration. Our study findings prove that HP1454 is a crucial bacterial factor that exerts its proinflammatory activity by directly modulating the T-cell response. The relevance of these results can be appreciated by considering that compelling evidence suggest that chronic gastric inflammation, a condition that paves the way to HP-associated diseases, is dependent on T cells.

Author Correction: A family of archaea-like carboxylesterases preferentially expressed in the symbiotic phase of the mycorrhizal fungus Tuber melanosporum.

A correction to this article has been published and is linked from the HTML and the PDF versions of this paper. The error has been fixed in the paper.

A V1143F mutation in the neuronal-enriched isoform 2 of the PMCA pump is linked with ataxia.

The fine regulation of intracellular calcium is fundamental for all eukaryotic cells. In neurons, Ca2+ oscillations govern the synaptic development, the release of neurotransmitters and the expression of several genes. Alterations of Ca2+ homeostasis were found to play a pivotal role in neurodegenerative progression. The maintenance of proper Ca2+ signaling in neurons demands the continuous activity of Ca2+ pumps and exchangers to guarantee physiological cytosolic concentration of the cation. The plasma membrane Ca2+ATPases (PMCA pumps) play a key role in the regulation of Ca2+ handling in selected sub-plasma membrane microdomains. Among the four basic PMCA pump isoforms existing in mammals, isoforms 2 and 3 are particularly enriched in the nervous system. In humans, genetic mutations in the PMCA2 gene in association with cadherin 23 mutations have been linked to hearing loss phenotypes, while those occurring in the PMCA3 gene were associated with X-linked congenital cerebellar ataxias. Here we describe a novel missense mutation (V1143F) in the calmodulin binding domain (CaM-BD) of the PMCA2 protein. The mutant pump was present in a patient showing congenital cerebellar ataxia but no overt signs of deafness, in line with the absence of mutations in the cadherin 23 gene. Biochemical and molecular dynamics studies on the mutated PMCA2 have revealed that the V1143F substitution alters the binding of calmodulin to the CaM-BD leading to impaired Ca2+ ejection.

Structural and dynamics evidence for scaffold asymmetric flexibility of the human transthyretin tetramer.

The molecular symmetry of multimeric proteins is generally determined by using X-ray diffraction techniques, so that the basic question as to whether this symmetry is perfectly preserved for the same protein in solution remains open. In this work, human transthyretin (TTR), a homotetrameric plasma transport protein with two binding sites for the thyroid hormone thyroxine (T4), is considered as a case study. Based on the crystal structure of the TTR tetramer, a hypothetical D2 symmetry is inferred for the protein in solution, whose functional behavior reveals the presence of two markedly different Kd values for the two T4 binding sites. The latter property has been ascribed to an as yet uncharacterized negative binding cooperativity. A triple mutant form of human TTR (F87M/L110M/S117E TTR), which is monomeric in solution, crystallizes as a tetrameric protein and its structure has been determined. The exam of this and several other crystal forms of human TTR suggests that the TTR scaffold possesses a significant structural flexibility. In addition, TTR tetramer dynamics simulated using normal modes analysis exposes asymmetric vibrational patterns on both dimers and thermal fluctuations reveal small differences in size and flexibility for ligand cavities at each dimer-dimer interface. Such small structural differences between monomers can lead to significant functional differences on the TTR tetramer dynamics, a feature that may explain the functional heterogeneity of the T4 binding sites, which is partially overshadowed by the crystal state.

Structural characterization of FlgE2 protein from Helicobacter pylori hook.

The Helicobacter pylori flagellum is a complex rotatory nanomachine fundamental for the bacterium's survival in the human stomach. Protein FlgE is a component of the hook, a flexible junction exposed on the cell surface. In the H. pylori genome two different genes are present in different positions coding for hypothetical FlgE. The first protein, FlgE1, is the actual component of the flagellum hook, whilst the second, FlgE2, shares only 26% of the sequence identity with the other and its physiological function is still undefined. We have cloned, purified and crystallized FlgE2, whose structure, determined by the single-wavelength anomalous diffraction method, shows that in overall organization, the protein is composed of three distinct domains, two of them relatively similar to those of FlgE from other Gram-negative bacteria, whilst the third is peculiar to H. pylori. The crystal structure, along with the detected interaction with the regulatory cap protein FlgD, suggests a complementary function of FlgE1 and FlgE2 in the H. pylori flagellum, possibly typical of polar flagella, confirming the role of both proteins in the flagellar hook organization. Although some general features are shared with other Gram-negative bacteria, the presence of two different hook proteins indicates that the molecular organization of H. pylori flagellum has its own peculiarities. DATABASE: Atomic coordinates and structural factors have been deposited in the Protein Data Bank as 5NPY.