RESUMO
The yeast Rcf1 protein is a member of the conserved family of proteins termed the hypoxia-induced gene (domain) 1 (Hig1 or HIGD1) family. Rcf1 interacts with components of the mitochondrial oxidative phosphorylation system, in particular the cytochrome bc1 (complex III)-cytochrome c oxidase (complex IV) supercomplex (termed III-IV) and the ADP/ATP carrier proteins. Rcf1 plays a role in the assembly and modulation of the activity of complex IV; however, the molecular basis for how Rcf1 influences the activity of complex IV is currently unknown. Hig1 type 2 isoforms, which include the Rcf1 protein, are characterized in part by the presence of a conserved motif, (Q/I)X3(R/H)XRX3Q, termed here the QRRQ motif. We show that mutation of conserved residues within the Rcf1 QRRQ motif alters the interactions between Rcf1 and partner proteins and results in the destabilization of complex IV and alteration of its enzymatic properties. Our findings indicate that Rcf1 does not serve as a stoichiometric component, i.e. as a subunit of complex IV, to support its activity. Rather, we propose that Rcf1 serves to dynamically interact with complex IV during its assembly process and, in doing so, regulates a late maturation step of complex IV. We speculate that the Rcf1/Hig1 proteins play a role in the incorporation and/or remodeling of lipids, in particular cardiolipin, into complex IV and. possibly, other mitochondrial proteins such as ADP/ATP carrier proteins.
Assuntos
Motivos de Aminoácidos/genética , Análise Mutacional de DNA , Complexo IV da Cadeia de Transporte de Elétrons/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/enzimologia , Cardiolipinas/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Mitocondriais/metabolismo , Mutação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The mitochondrial inner membrane plays central roles in bioenergetics and metabolism and contains several established membrane protein complexes. Here, we report the identification of a mega-complex of the inner membrane, termed mitochondrial multifunctional assembly (MIMAS). Its large size of 3 MDa explains why MIMAS has escaped detection in the analysis of mitochondria so far. MIMAS combines proteins of diverse functions from respiratory chain assembly to metabolite transport, dehydrogenases, and lipid biosynthesis but not the large established supercomplexes of the respiratory chain, ATP synthase, or prohibitin scaffold. MIMAS integrity depends on the non-bilayer phospholipid phosphatidylethanolamine, in contrast to respiratory supercomplexes whose stability depends on cardiolipin. Our findings suggest that MIMAS forms a protein-lipid mega-assembly in the mitochondrial inner membrane that integrates respiratory biogenesis and metabolic processes in a multifunctional platform.
Assuntos
Mitocôndrias , Membranas Mitocondriais , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Fosfolipídeos/metabolismo , Transporte de Elétrons , Cardiolipinas/metabolismoRESUMO
Uncontrolled complement activation contributes to multiple immune pathologies. Although synthetic compstatin derivatives targeting C3 and C3b are robust inhibitors of complement activation, their physicochemical and molecular properties may limit access to specific organs, development of bifunctional moieties, and therapeutic applications requiring transgenic expression. Complement-targeting therapeutics containing only natural amino acids could enable multifunctional pharmacology, gene therapies, and targeted delivery for underserved diseases. A Nanofitin library of hyperthermophilic protein scaffolds was screened using ribosome display for C3/C3b-targeting clones mimicking compstatin pharmacology. APL-1030, a recombinant 64-residue Nanofitin, emerged as the lead candidate. APL-1030 is thermostable, binds C3 (KD, 1.59 nM) and C3b (KD, 1.11 nM), and inhibits complement activation via classical (IC50 = 110.8 nM) and alternative (IC50 = 291.3 nM) pathways in Wieslab assays. Pharmacologic activity (determined by alternative pathway inhibition) was limited to primate species of tested sera. C3b-binding sites of APL-1030 and compstatin were shown to overlap by X-ray crystallography of C3b-bound APL-1030. APL-1030 is a novel, high-affinity inhibitor of primate C3-mediated complement activation developed from natural amino acids on the hyperthermophilic Nanofitin platform. Its properties may support novel drug candidates, enabling bifunctional moieties, gene therapy, and tissue-targeted C3 pharmacologics for diseases with high unmet need.
Assuntos
Ativação do Complemento , Complemento C3 , Aminoácidos , Animais , Sítios de Ligação , Complemento C3/metabolismo , Cristalografia por Raios XRESUMO
The mitochondrial respiratory chain enzymes are organized as individual complexes and supercomplexes, whose biogenesis remains to be fully understood. To disclose the role of the human Hypoxia Inducible Gene Domain family proteins HIGD1A and HIGD2A in these processes, we generate and characterize HIGD-knockout (KO) cell lines. We show that HIGD2A controls and coordinates the modular assembly of isolated and supercomplexed complex IV (CIV) by acting on the COX3 assembly module. In contrast, HIGD1A regulates CIII and CIII-containing supercomplex biogenesis by supporting the incorporation of UQCRFS1. HIGD1A also clusters with COX4-1 and COX5A CIV subunits and, when overexpressed, suppresses the CIV biogenesis defect of HIGD2A-KO cells. We conclude that HIGD1A and HIGD2A have both independent and overlapping functions in the biogenesis of respiratory complexes and supercomplexes. Our data illuminate the existence of multiple pathways to assemble these structures by dynamic HIGD-mediated CIV biogenesis, potentially to adapt to changing environmental and nutritional conditions.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , HumanosRESUMO
We report that Rcf1 (formerly Aim31), a member of the conserved hypoxia-induced gene 1 (Hig1) protein family, represents a novel component of the yeast cytochrome bc(1)-cytochrome c oxidase (COX) supercomplex. Rcf1 (respiratory supercomplex factor 1) partitions with the COX complex, and evidence that it may act as a bridge to the cytochrome bc(1) complex is presented. Rcf1 interacts with the Cox3 subunit and can do so prior to their assembly into the COX complex. A close proximity of Rcf1 and members of the ADP/ATP carrier (AAC) family was also established. Rcf1 displays overlapping function with another Hig1-related protein, Rcf2 (formerly Aim38), and their joint presence is required for optimal COX enzyme activity and the correct assembly of the cytochrome bc(1)-COX supercomplex. Rcf1 and Rcf2 can independently associate with the cytochrome bc(1)-COX supercomplex, indicating that at least two forms of this supercomplex exist within mitochondria. We provide evidence that the association with the cytochrome bc(1)-COX supercomplex and regulation of the COX complex are a conserved feature of Hig1 family members. Based on our findings, we propose a model where the Hig1 proteins regulate the COX enzyme activity through Cox3 and associated Cox12 protein, in a manner that may be influenced by the neighboring AAC proteins.