RESUMO
Guanidine is a chemically stable nitrogen compound that is excreted in human urine and is widely used in manufacturing of plastics, as a flame retardant and as a component of propellants, and is well known as a protein denaturant in biochemistry1-3. Guanidine occurs widely in nature and is used by several microorganisms as a nitrogen source, but microorganisms growing on guanidine as the only substrate have not yet been identified. Here we show that the complete ammonia oxidizer (comammox) Nitrospira inopinata and probably most other comammox microorganisms can grow on guanidine as the sole source of energy, reductant and nitrogen. Proteomics, enzyme kinetics and the crystal structure of a N. inopinata guanidinase homologue demonstrated that it is a bona fide guanidinase. Incubation experiments with comammox-containing agricultural soil and wastewater treatment plant microbiomes suggested that guanidine serves as substrate for nitrification in the environment. The identification of guanidine as a growth substrate for comammox shows an unexpected niche of these globally important nitrifiers and offers opportunities for their isolation.
RESUMO
The spectrin superfamily of proteins plays key roles in assembling the actin cytoskeleton in various cell types, crosslinks actin filaments, and acts as scaffolds for the assembly of large protein complexes involved in structural integrity and mechanosensation, as well as cell signaling. α-actinins in particular are the major actin crosslinkers in muscle Z-disks, focal adhesions, and actin stress fibers. We report a complete high-resolution structure of the 200 kDa α-actinin-2 dimer from striated muscle and explore its functional implications on the biochemical and cellular level. The structure provides insight into the phosphoinositide-based mechanism controlling its interaction with sarcomeric proteins such as titin, lays a foundation for studying the impact of pathogenic mutations at molecular resolution, and is likely to be broadly relevant for the regulation of spectrin-like proteins.
Assuntos
Actinina/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Músculo Esquelético/química , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Difração de Raios XRESUMO
Sarcomeres, the basic contractile units of striated muscle cells, contain arrays of thin (actin) and thick (myosin) filaments that slide past each other during contraction. The Ig-like domain-containing protein myotilin provides structural integrity to Z-discs-the boundaries between adjacent sarcomeres. Myotilin binds to Z-disc components, including F-actin and α-actinin-2, but the molecular mechanism of binding and implications of these interactions on Z-disc integrity are still elusive. To illuminate them, we used a combination of small-angle X-ray scattering, cross-linking mass spectrometry, and biochemical and molecular biophysics approaches. We discovered that myotilin displays conformational ensembles in solution. We generated a structural model of the F-actin:myotilin complex that revealed how myotilin interacts with and stabilizes F-actin via its Ig-like domains and flanking regions. Mutant myotilin designed with impaired F-actin binding showed increased dynamics in cells. Structural analyses and competition assays uncovered that myotilin displaces tropomyosin from F-actin. Our findings suggest a novel role of myotilin as a co-organizer of Z-disc assembly and advance our mechanistic understanding of myotilin's structural role in Z-discs.
Assuntos
Actinas/metabolismo , Multimerização Proteica , Sarcômeros/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/genética , Animais , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Humanos , Camundongos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Contração Muscular/genética , Músculo Esquelético/metabolismo , Ligação Proteica/genética , Domínios e Motivos de Interação entre Proteínas/genética , Multimerização Proteica/genética , Sarcômeros/genética , Tropomiosina/química , Tropomiosina/genética , Tropomiosina/metabolismoRESUMO
The actin cytoskeleton, a dynamic network of actin filaments and associated F-actin-binding proteins, is fundamentally important in eukaryotes. α-Actinins are major F-actin bundlers that are inhibited by Ca2+ in nonmuscle cells. Here we report the mechanism of Ca2+-mediated regulation of Entamoeba histolytica α-actinin-2 (EhActn2) with features expected for the common ancestor of Entamoeba and higher eukaryotic α-actinins. Crystal structures of Ca2+-free and Ca2+-bound EhActn2 reveal a calmodulin-like domain (CaMD) uniquely inserted within the rod domain. Integrative studies reveal an exceptionally high affinity of the EhActn2 CaMD for Ca2+, binding of which can only be regulated in the presence of physiological concentrations of Mg2+ Ca2+ binding triggers an increase in protein multidomain rigidity, reducing conformational flexibility of F-actin-binding domains via interdomain cross-talk and consequently inhibiting F-actin bundling. In vivo studies uncover that EhActn2 plays an important role in phagocytic cup formation and might constitute a new drug target for amoebic dysentery.
Assuntos
Actinina/metabolismo , Cálcio/farmacologia , Entamoeba histolytica/metabolismo , Actinina/química , Actinina/genética , Domínio Catalítico , Entamoeba histolytica/genética , Regulação da Expressão Gênica , Modelos Moleculares , Conformação Proteica , Domínios ProteicosRESUMO
Stable anchoring of titin within the muscle Z-disk is essential for preserving muscle integrity during passive stretching. One of the main candidates for anchoring titin in the Z-disk is the actin cross-linker α-actinin. The calmodulin-like domain of α-actinin binds to the Z-repeats of titin. However, the mechanical and kinetic properties of this important interaction are still unknown. Here, we use a dual-beam optical tweezers assay to study the mechanics of this interaction at the single-molecule level. A single interaction of α-actinin and titin turns out to be surprisingly weak if force is applied. Depending on the direction of force application, the unbinding forces can more than triple. Our results suggest a model where multiple α-actinin/Z-repeat interactions cooperate to ensure long-term stable titin anchoring while allowing the individual components to exchange dynamically.
Assuntos
Actinina/metabolismo , Conectina/metabolismo , Actinina/química , Sequência de Aminoácidos , Animais , Conectina/química , Cisteína/química , Cistina/química , Humanos , Mutagênese Sítio-Dirigida , Pinças Ópticas , Domínios Proteicos , Mapeamento de Interação de Proteínas , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Sequências Repetitivas de Aminoácidos , Sarcômeros/química , Sarcômeros/ultraestrutura , Estresse MecânicoRESUMO
The BAR domain is the eponymous domain of the "BAR-domain protein superfamily", a large and diverse set of mostly multi-domain proteins that play eminent roles at the membrane cytoskeleton interface. BAR domain homodimers are the functional units that peripherally associate with lipid membranes and are involved in membrane sculpting activities. Differences in their intrinsic curvatures and lipid-binding properties account for a large variety in membrane modulating properties. Membrane activities of BAR domains are further modified and regulated by intramolecular or inter-subunit domains, by intermolecular protein interactions, and by posttranslational modifications. Rather than providing detailed cell biological information on single members of this superfamily, this review focuses on biochemical, biophysical, and structural aspects and on recent findings that paradigmatically promote our understanding of processes driven and modulated by BAR domains.
Assuntos
Membrana Celular/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Animais , Humanos , Fosforilação , Ligação Proteica , Domínios Proteicos , Mapeamento de Interação de ProteínasRESUMO
To study the mechanisms involved in the maintenance of a linear mitochondrial genome we investigated the biochemical properties of the recombination protein Mgm101 from Candida parapsilosis. We show that CpMgm101 complements defects associated with the Saccharomyces cerevisiae mgm101-1(ts) mutation and that it is present in both the nucleus and mitochondrial nucleoids of C. parapsilosis. Unlike its S. cerevisiae counterpart, CpMgm101 is associated with the entire nucleoid population and is able to bind to a broad range of DNA substrates in a non-sequence specific manner. CpMgm101 is also able to catalyze strand annealing and D-loop formation. CpMgm101 forms a roughly C-shaped trimer in solution according to SAXS. Electron microscopy of a complex of CpMgm101 with a model mitochondrial telomere revealed homogeneous, ring-shaped structures at the telomeric single-stranded overhangs. The DNA-binding properties of CpMgm101, together with its DNA recombination properties, suggest that it can play a number of possible roles in the replication of the mitochondrial genome and the maintenance of its telomeres.
Assuntos
Candida/genética , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Genoma Mitocondrial , Proteínas Mitocondriais/genética , Proteínas de Saccharomyces cerevisiae/genética , Telômero/química , Candida/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Clonagem Molecular , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Teste de Complementação Genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mutação , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinação Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Telômero/metabolismo , Homeostase do TelômeroRESUMO
Two mechanisms have emerged as major regulators of membrane shape: BAR domain-containing proteins, which induce invaginations and protrusions, and nuclear promoting factors, which cause generation of branched actin filaments that exert mechanical forces on membranes. While a large body of information exists on interactions of BAR proteins with membranes and regulatory proteins of the cytoskeleton, little is known about connections between these two processes. Here, we show that the F-BAR domain protein pacsin2 is able to associate with actin filaments using the same concave surface employed to bind to membranes, while some other tested N-BAR and F-BAR proteins (endophilin, CIP4 and FCHO2) do not associate with actin. This finding reveals a new level of complexity in membrane remodeling processes.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Citoesqueleto de Actina/química , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Membrana Celular/metabolismo , Galinhas , Proteínas Associadas aos Microtúbulos/metabolismo , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Ligação ProteicaRESUMO
Chlorite dismutases (Clds) are heme b containing oxidoreductases that convert chlorite to chloride and molecular oxygen. In order to elucidate the role of conserved heme cavity residues in the catalysis of this reaction comprehensive mutational and biochemical analyses of Cld from "Candidatus Nitrospira defluvii" (NdCld) were performed. Particularly, point mutations of the cavity-forming residues R173, K141, W145, W146, and E210 were performed. The effect of manipulation in 12 single and double mutants was probed by UV-vis spectroscopy, spectroelectrochemistry, pre-steady-state and steady-state kinetics, and X-ray crystallography. Resulting biochemical data are discussed with respect to the known crystal structure of wild-type NdCld and the variants R173A and R173K as well as the structures of R173E, W145V, W145F, and the R173Q/W146Y solved in this work. The findings allow a critical analysis of the role of these heme cavity residues in the reaction mechanism of chlorite degradation that is proposed to involve hypohalous acid as transient intermediate and formation of an OâO bond. The distal R173 is shown to be important (but not fully essential) for the reaction with chlorite, and, upon addition of cyanide, it acts as a proton acceptor in the formation of the resulting low-spin complex. The proximal H-bonding network including K141-E210-H160 keeps the enzyme in its ferric (E°' = -113 mV) and mainly five-coordinated high-spin state and is very susceptible to perturbation.
Assuntos
Heme/química , Oxirredutases/química , Oxirredutases/genética , Oxirredutases/metabolismo , Cloretos/metabolismo , Cristalografia por Raios X , Cianetos/química , Eletroquímica , Cinética , Modelos Moleculares , OxirreduçãoRESUMO
The S-adenosyl-L-methionine (SAM)-dependent methyltransferase CcbJ from Streptomyces caelestis catalyzes one of the final steps in the biosynthesis of the antibiotic celesticetin, methylation of the N atom of its proline moiety, which greatly enhances the activity of the antibiotic. Since several celesticetin variants exist, this enzyme may be able to act on a variety of substrates. The structures of CcbJ determined by MAD phasing at 3.0â Å resolution, its native form at 2.7â Å resolution and its complex with S-adenosyl-L-homocysteine (SAH) at 2.9â Å resolution are reported here. Based on these structures, three point mutants, Y9F, Y17F and F117G, were prepared in order to study its behaviour as well as docking simulations of both CcbJ-SAM-substrate and CcbJ-SAH-product complexes. The structures show that CcbJ is a class I SAM-dependent methyltransferase with a wide active site, thereby suggesting that it may accommodate a number of different substrates. The mutation results show that the Y9F and F117G mutants are almost non-functional, while the Y17F mutant has almost half of the wild-type activity. In combination with the docking studies, these results suggest that Tyr9 and Phe117 are likely to help to position the substrate for the methyl-transfer reaction and that Tyr9 may also facilitate the reaction by removing an H(+) ion. Tyr17, on the other hand, seems to operate by helping to stabilize the SAM cofactor.
Assuntos
Metiltransferases/química , Streptomyces/enzimologia , Metiltransferases/genética , Modelos Moleculares , Mutação , Estrutura Terciária de ProteínaRESUMO
Treatment of cultured vertebrate neurons with nitric oxide leads to growth-cone collapse, axon retraction and the reconfiguration of axonal microtubules. We show that the light chain of microtubule-associated protein (MAP) 1B is a substrate for S-nitrosylation in vivo, in cultured cells and in vitro. S-nitrosylation occurs at Cys 2457 in the COOH terminus. Nitrosylation of MAP1B leads to enhanced interaction with microtubules and correlates with the inhibition of neuroblastoma cell differentiation. We further show, in dorsal root ganglion neurons, that MAP1B is necessary for neuronal nitric oxide synthase control of growth-cone size, growth-cone collapse and axon retraction. These results reveal an S-nitrosylation-dependent signal-transduction pathway that is involved in regulation of the axonal cytoskeleton and identify MAP1B as a major component of this pathway. We propose that MAP1B acts by inhibiting a microtubule- and dynein-based mechanism that normally prevents axon retraction.
Assuntos
Axônios/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais/fisiologia , Animais , Axônios/ultraestrutura , Células Cultivadas , Cisteína/metabolismo , Gânglios Espinais/citologia , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Compostos Nitrosos , Conformação Proteica , RatosRESUMO
Expression in Escherichia coli represents the simplest and most cost effective means for the production of recombinant proteins. This is a routine task in structural biology and biochemistry where milligrams of the target protein are required in high purity and monodispersity. To achieve these criteria, the user often needs to screen several constructs in different expression and purification conditions in parallel. We describe a pipeline, implemented in the Center for Optimized Structural Studies, that enables the systematic screening of expression and purification conditions for recombinant proteins and relies on a series of logical decisions. We first use bioinformatics tools to design a series of protein fragments, which we clone in parallel, and subsequently screen in small scale for optimal expression and purification conditions. Based on a scoring system that assesses soluble expression, we then select the top ranking targets for large-scale purification. In the establishment of our pipeline, emphasis was put on streamlining the processes such that it can be easily but not necessarily automatized. In a typical run of about 2 weeks, we are able to prepare and perform small-scale expression screens for 20-100 different constructs followed by large-scale purification of at least 4-6 proteins. The major advantage of our approach is its flexibility, which allows for easy adoption, either partially or entirely, by any average hypothesis driven laboratory in a manual or robot-assisted manner.
Assuntos
Proteínas Recombinantes/isolamento & purificação , Automação Laboratorial , Cromatografia em Gel/métodos , Clonagem Molecular , Clonagem de Organismos , Biologia Computacional , Escherichia coli/genética , Escherichia coli/metabolismo , Filaminas/genética , Filaminas/isolamento & purificação , Proteínas Recombinantes/biossínteseRESUMO
The trypanosomes are a family of parasitic protists of which the African trypanosome, Trypanosoma brucei, is the best characterized. The complex and highly ordered cytoskeleton of T. brucei has been shown to play vital roles in its biology but remains difficult to study, in large part owing to the intractability of its constituent proteins. Existing methods of protein identification, such as bioinformatic analysis, generation of monoclonal antibody panels, proteomics, affinity purification, and yeast two-hybrid screens, all have drawbacks. Such deficiencies-troublesome proteins and technical limitations-are common not only to T. brucei but also to many other protists, many of which are even less well studied. Proximity-dependent biotin identification (BioID) is a recently developed technique that allows forward screens for interaction partners and near neighbors in a native environment with no requirement for solubility in nonionic detergent. As such, it is extremely well suited to the exploration of the cytoskeleton. In this project, BioID was adapted for use in T. brucei. The trypanosome bilobe, a discrete cytoskeletal structure with few known protein components, represented an excellent test subject. Use of the bilobe protein TbMORN1 as a probe resulted in the identification of seven new bilobe constituents and two new flagellum attachment zone proteins. This constitutes the first usage of BioID on a largely uncharacterized structure, and demonstrates its utility in identifying new components of such a structure. This remarkable success validates BioID as a new tool for the study of unicellular eukaryotes in particular and the eukaryotic cytoskeleton in general.
Assuntos
Biotinilação , Proteínas do Citoesqueleto/metabolismo , Mapeamento de Interação de Proteínas , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Proteínas do Citoesqueleto/isolamento & purificação , Ligação Proteica , Transporte Proteico , Proteínas de Protozoários/isolamento & purificaçãoRESUMO
Phosphorylation plays a crucial role in the regulation of many fundamental cellular processes. Phosphorylation levels are increased in many cancer cells where they may promote changes in mitochondrial homeostasis. Proteomic studies on various types of cancer identified 17 phosphorylation sites within the human ATP-dependent protease Lon, which degrades misfolded, unassembled and oxidatively damaged proteins in mitochondria. Most of these sites were found in Lon's N-terminal (NTD) and ATPase domains, though little is known about the effects on their function. By combining the biochemical and cryo-electron microscopy studies, we show the effect of Tyr186 and Tyr394 phosphorylations in Lon's NTD, which greatly reduce all Lon activities without affecting its ability to bind substrates or perturbing its tertiary structure. A substantial reduction in Lon's activities is also observed in the presence of polyphosphate, whose amount significantly increases in cancer cells. Our study thus provides an insight into the possible fine-tuning of Lon activities in human diseases, which highlights Lon's importance in maintaining proteostasis in mitochondria.
Assuntos
Mitocôndrias , Polifosfatos , Protease La , Tirosina , Humanos , Fosforilação , Protease La/metabolismo , Polifosfatos/metabolismo , Mitocôndrias/metabolismo , Tirosina/metabolismo , Microscopia Crioeletrônica , Domínios ProteicosRESUMO
Chlorite dismutases (Cld) are unique heme b containing oxidoreductases that convert chlorite to chloride and dioxygen. Recent phylogenetic and structural analyses demonstrated that these metalloproteins significantly differ in oligomeric and subunit structure. Here we have analyzed two representatives of two phylogenetically separated lineages, namely pentameric Cld from Candidatus "Nitrospira defluvii" and dimeric Cld from Nitrobacter winogradskyi having a similar enzymatic activity at room temperature. By application of a broad set of techniques including differential scanning calorimetry, electronic circular dichroism, UV-vis and fluorescence spectroscopy the temperature-mediated and chemical unfolding of both recombinant proteins were analyzed. Significant differences in thermal and conformational stability are reported. The pentameric enzyme is very stable between pH 3 and 10 (T(m)=92°C at pH 7.0) and active at high temperatures thus being an interesting candidate for bioremediation of chlorite. By contrast the dimeric protein starts to unfold already at 53°C. The observed unfolding pathways are discussed with respect to the known subunit structure and subunit interaction.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , Oxirredutases/química , Proteínas de Bactérias/genética , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Modelos Moleculares , Nitrobacter/enzimologia , Oxirredutases/genética , Filogenia , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Temperatura , TermodinâmicaRESUMO
Chlorite dismutases (Clds) are heme b-containing oxidoreductases that convert chlorite to chloride and dioxygen. In this work, the thermodynamics of the one-electron reduction of the ferric high-spin forms and of the six-coordinate low-spin cyanide adducts of the enzymes from Nitrobacter winogradskyi (NwCld) and Candidatus "Nitrospira defluvii" (NdCld) were determined through spectroelectrochemical experiments. These proteins belong to two phylogenetically separated lineages that differ in subunit (21.5 and 26 kDa, respectively) and oligomeric (dimeric and pentameric, respectively) structure but exhibit similar chlorite degradation activity. The E°' values for free and cyanide-bound proteins were determined to be -119 and -397 mV for NwCld and -113 and -404 mV for NdCld, respectively (pH 7.0, 25 °C). Variable-temperature spectroelectrochemical experiments revealed that the oxidized state of both proteins is enthalpically stabilized. Molecular dynamics simulations suggest that changes in the protein structure are negligible, whereas solvent reorganization is mainly responsible for the increase in entropy during the redox reaction. Obtained data are discussed with respect to the known structures of the two Clds and the proposed reaction mechanism.
Assuntos
Oxirredutases/química , Domínio Catalítico , Cloretos , Espectroscopia de Ressonância de Spin Eletrônica , Entropia , Compostos Férricos/química , Compostos Ferrosos/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Nitrobacter/enzimologia , Oxirredução , Oxirredutases/metabolismo , Conformação Proteica , Estrutura Quaternária de Proteína , TermodinâmicaRESUMO
Chlorite dismutase (Cld) is a unique heme enzyme catalyzing the conversion of ClO(2)(-) to Cl(-) and O(2). Cld is usually found in perchlorate- or chlorate-reducing bacteria but was also recently identified in a nitrite-oxidizing bacterium of the genus Nitrospira. Here we characterized a novel Cld-like protein from the chemolithoautotrophic nitrite oxidizer Nitrobacter winogradskyi which is significantly smaller than all previously known chlorite dismutases. Its three-dimensional (3D) crystal structure revealed a dimer of two identical subunits, which sharply contrasts with the penta- or hexameric structures of other chlorite dismutases. Despite a truncated N-terminal domain in each subunit, this novel enzyme turned out to be a highly efficient chlorite dismutase (K(m) = 90 µM; k(cat) = 190 s(-1); k(cat)/K(m) = 2.1 × 10(6) M(-1) s(-1)), demonstrating a greater structural and phylogenetic diversity of these enzymes than was previously known. Based on comparative analyses of Cld sequences and 3D structures, signature amino acid residues that can be employed to assess whether uncharacterized Cld-like proteins may have a high chlorite-dismutating activity were identified. Interestingly, proteins that contain all these signatures and are phylogenetically closely related to the novel-type Cld of N. winogradskyi exist in a large number of other microbes, including other nitrite oxidizers.
Assuntos
Variação Genética , Nitrobacter/enzimologia , Oxirredutases/química , Oxirredutases/metabolismo , Cloretos/metabolismo , Cristalografia por Raios X , Cinética , Modelos Moleculares , Nitrobacter/genética , Nitrobacter/metabolismo , Oxirredutases/genética , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
OBJECTIVE: To determine whether a new indel mutation in the dimerization domain of filamin C (FLNc) causes a hereditary myopathy with protein aggregation in muscle fibers, we clinically and molecularly studied a German family with autosomal dominant myofibrillar myopathy (MFM). METHODS: We performed mutational analysis in 3 generations, muscle histopathology, and proteomic studies of IM protein aggregates. Functional consequences of the FLNC mutation were investigated with interaction and transfection studies and biophysics molecular analysis. RESULTS: Eight patients revealed clinical features of slowly progressive proximal weakness associated with a heterozygous c.8025_8030delCAAGACinsA (p.K2676Pfs*3) mutation in FLNC. Two patients exhibited a mild cardiomyopathy. MRI of skeletal muscle revealed lipomatous changes typical for MFM with FLNC mutations. Muscle biopsies showed characteristic MFM findings with protein aggregation and lesion formation. The proteomic profile of aggregates was specific for MFM-filaminopathy and indicated activation of the ubiquitin-proteasome system (UPS) and autophagic pathways. Functional studies revealed that mutant FLNc is misfolded, unstable, and incapable of forming homodimers and heterodimers with wild-type FLNc. CONCLUSIONS: This new MFM-filaminopathy family confirms that expression of mutant FLNC leads to an adult-onset muscle phenotype with intracellular protein accumulation. Mutant FLNc protein is biochemically compromised and leads to dysregulation of protein quality control mechanisms. Proteomic analysis of MFM protein aggregates is a potent method to identify disease-relevant proteins, differentiate MFM subtypes, evaluate the relevance of gene variants, and identify novel MFM candidate genes.
RESUMO
In sarcomeres, α-actinin cross-links actin filaments and anchors them to the Z-disk. FATZ (filamin-, α-actinin-, and telethonin-binding protein of the Z-disk) proteins interact with α-actinin and other core Z-disk proteins, contributing to myofibril assembly and maintenance. Here, we report the first structure and its cellular validation of α-actinin-2 in complex with a Z-disk partner, FATZ-1, which is best described as a conformational ensemble. We show that FATZ-1 forms a tight fuzzy complex with α-actinin-2 and propose an interaction mechanism via main molecular recognition elements and secondary binding sites. The obtained integrative model reveals a polar architecture of the complex which, in combination with FATZ-1 multivalent scaffold function, might organize interaction partners and stabilize α-actinin-2 preferential orientation in Z-disk. Last, we uncover FATZ-1 ability to phase-separate and form biomolecular condensates with α-actinin-2, raising the question whether FATZ proteins can create an interaction hub for Z-disk proteins through membraneless compartmentalization during myofibrillogenesis.
RESUMO
Chlorite dismutase (Cld) is a unique heme enzyme which transforms chlorite to chloride and molecular oxygen (reaction: ClO(2)(-)âCl(-)+O(2)). Since bacteria with Cld play significant roles in the bioremediation of industrially contaminated sites and also in wastewater treatment, it is of high interest to understand the molecular mechanism of chlorite detoxification. Here we investigate a highly active Cld from Candidatus Nitrospira defluvii (NdCld), a key nitrifier in biological wastewater treatment, using a comprehensive structural, biochemical and bioinformatics approach. We determined the crystal structure of Cld from Candidatus Nitrospira defluvii and showed that functional NdCld is a homopentamer possessing a fold found in other Clds and Cld-like enzymes. To investigate the Cld function in more detail, site-directed mutagenesis of a catalytically important residue (Arg173) was performed and two enzyme mutants were structurally and biochemically characterized. Arginine 173 is demonstrated to play a key role in (i) controlling of ligand and substrate access and binding and (ii) in chlorite dismutation reaction. The flexible residue modulates the electrostatic potential and size of the active site entrance and might be involved in keeping transiently formed hypochlorite in place for final molecular oxygen and chloride formation. Furthermore, using a structure-based sequence alignment, we show that the residue corresponding to Arg173 is conserved in all known active forms of Cld and propose it as a marker for Cld activity in yet uncharacterized Cld-like proteins. Finally, our analysis indicates that all Clds and Cld-like enzymes employ a non-covalently bound heme as a cofactor.