Polyphosphate and tyrosine phosphorylation in the N-terminal domain of the human mitochondrial Lon protease disrupts its functions.

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.

Characterization of a newly discovered putative DNA replication initiator from Paenibacillus polymyxa phage phiBP.

The bacteriophage phiBP contains a newly discovered putative replisome organizer, a helicase loader, and a beta clamp, which together may serve to replicate its DNA. Bioinformatics analysis of the phiBP replisome organizer sequence showed that it belongs to a recently identified family of putative initiator proteins. We prepared and isolated a wild type-like recombinant protein, gpRO-HC, and a mutant protein gpRO-HCK8A, containing a lysine to alanine substitution at position 8. gpRO-HC had low ATPase activity regardless of the presence of DNA, while the ATPase activity of the mutant was significantly higher. gpRO-HC bound to both single- and double-stranded DNA substrates. Different methods showed that gpRO-HC forms higher oligomers containing about 12 subunits. This work provides the first information about another group of phage initiator proteins, which trigger DNA replication in phages infecting low GC Gram-positive bacteria.

Extracting the Dynamic Motion of Proteins Using Normal Mode Analysis.

Normal mode analysis (NMA) is a technique for describing the conformational states accessible to a protein in a minimum energy conformation. NMA gives results similar to those produced by principal components analysis of a molecular dynamics simulation, but with only a fraction of the computational effort. Here, we provide a brief overview of the theory and describe three methods for carrying out NMA, including the use of one of the on-line services, the use of off-line software for calculating the projection of the modes calculated from one conformation onto another, and an all-atom NMA calculated using GROMACS. For all three methods, we will use the E1·2Ca2+ form of the Ca2+-ATPase as a concrete example.

Glucose Oxidase, an Enzyme "Ferrari": Its Structure, Function, Production and Properties in the Light of Various Industrial and Biotechnological Applications.

Glucose oxidase (GOx) is an important oxidoreductase enzyme with many important roles in biological processes. It is considered an "ideal enzyme" and is often called an oxidase "Ferrari" because of its fast mechanism of action, high stability and specificity. Glucose oxidase catalyzes the oxidation of ß-d-glucose to d-glucono-δ-lactone and hydrogen peroxide in the presence of molecular oxygen. d-glucono-δ-lactone is sequentially hydrolyzed by lactonase to d-gluconic acid, and the resulting hydrogen peroxide is hydrolyzed by catalase to oxygen and water. GOx is presently known to be produced only by fungi and insects. The current main industrial producers of glucose oxidase are Aspergillus and Penicillium. An important property of GOx is its antimicrobial effect against various pathogens and its use in many industrial and medical areas. The aim of this review is to summarize the structure, function, production strains and biophysical and biochemical properties of GOx in light of its various industrial, biotechnological and medical applications.

Mitochondrial Processing Peptidases-Structure, Function and the Role in Human Diseases.

Mitochondrial proteins are encoded by both nuclear and mitochondrial DNA. While some of the essential subunits of the oxidative phosphorylation (OXPHOS) complexes responsible for cellular ATP production are synthesized directly in the mitochondria, most mitochondrial proteins are first translated in the cytosol and then imported into the organelle using a sophisticated transport system. These proteins are directed mainly by targeting presequences at their N-termini. These presequences need to be cleaved to allow the proper folding and assembly of the pre-proteins into functional protein complexes. In the mitochondria, the presequences are removed by several processing peptidases, including the mitochondrial processing peptidase (MPP), the inner membrane processing peptidase (IMP), the inter-membrane processing peptidase (MIP), and the mitochondrial rhomboid protease (Pcp1/PARL). Their proper functioning is essential for mitochondrial homeostasis as the disruption of any of them is lethal in yeast and severely impacts the lifespan and survival in humans. In this review, we focus on characterizing the structure, function, and substrate specificities of mitochondrial processing peptidases, as well as the connection of their malfunctions to severe human diseases.

The yeast mitochondrial succinylome: Implications for regulation of mitochondrial nucleoids.

Acylation modifications, such as the succinylation of lysine, are post-translational modifications and a powerful means of regulating protein activity. Some acylations occur nonenzymatically, driven by an increase in the concentration of acyl group donors. Lysine succinylation has a profound effect on the corresponding site within the protein, as it dramatically changes the charge of the residue. In eukaryotes, it predominantly affects mitochondrial proteins because the donor of succinate, succinyl-CoA, is primarily generated in the tricarboxylic acid cycle. Although numerous succinylated mitochondrial proteins have been identified in Saccharomyces cerevisiae, a more detailed characterization of the yeast mitochondrial succinylome is still lacking. Here, we performed a proteomic MS analysis of purified yeast mitochondria and detected 314 succinylated mitochondrial proteins with 1763 novel succinylation sites. The mitochondrial nucleoid, a complex of mitochondrial DNA and mitochondrial proteins, is one of the structures whose protein components are affected by succinylation. We found that Abf2p, the principal component of mitochondrial nucleoids responsible for compacting mitochondrial DNA in S. cerevisiae, can be succinylated in vivo on at least thirteen lysine residues. Abf2p succinylation in vitro inhibits its DNA-binding activity and reduces its sensitivity to digestion by the ATP-dependent ScLon protease. We conclude that changes in the metabolic state of a cell resulting in an increase in the concentration of tricarboxylic acid intermediates may affect mitochondrial functions.

Mitochondrial HSP70 Chaperone System-The Influence of Post-Translational Modifications and Involvement in Human Diseases.

Since their discovery, heat shock proteins (HSPs) have been identified in all domains of life, which demonstrates their importance and conserved functional role in maintaining protein homeostasis. Mitochondria possess several members of the major HSP sub-families that perform essential tasks for keeping the organelle in a fully functional and healthy state. In humans, the mitochondrial HSP70 chaperone system comprises a central molecular chaperone, mtHSP70 or mortalin (HSPA9), which is actively involved in stabilizing and importing nuclear gene products and in refolding mitochondrial precursor proteins, and three co-chaperones (HSP70-escort protein 1-HEP1, tumorous imaginal disc protein 1-TID-1, and Gro-P like protein E-GRPE), which regulate and accelerate its protein folding functions. In this review, we summarize the roles of mitochondrial molecular chaperones with particular focus on the human mtHsp70 and its co-chaperones, whose deregulated expression, mutations, and post-translational modifications are often considered to be the main cause of neurological disorders, genetic diseases, and malignant growth.

Mitochondrial Kinases and the Role of Mitochondrial Protein Phosphorylation in Health and Disease.

The major role of mitochondria is to provide cells with energy, but no less important are their roles in responding to various stress factors and the metabolic changes and pathological processes that might occur inside and outside the cells. The post-translational modification of proteins is a fast and efficient way for cells to adapt to ever changing conditions. Phosphorylation is a post-translational modification that signals these changes and propagates these signals throughout the whole cell, but it also changes the structure, function and interaction of individual proteins. In this review, we summarize the influence of kinases, the proteins responsible for phosphorylation, on mitochondrial biogenesis under various cellular conditions. We focus on their role in keeping mitochondria fully functional in healthy cells and also on the changes in mitochondrial structure and function that occur in pathological processes arising from the phosphorylation of mitochondrial proteins.

Structure and Function of the Human Ryanodine Receptors and Their Association with Myopathies-Present State, Challenges, and Perspectives.

Cardiac arrhythmias are serious, life-threatening diseases associated with the dysregulation of Ca2+ influx into the cytoplasm of cardiomyocytes. This dysregulation often arises from dysfunction of ryanodine receptor 2 (RyR2), the principal Ca2+ release channel. Dysfunction of RyR1, the skeletal muscle isoform, also results in less severe, but also potentially life-threatening syndromes. The RYR2 and RYR1 genes have been found to harbor three main mutation "hot spots", where mutations change the channel structure, its interdomain interface properties, its interactions with its binding partners, or its dynamics. In all cases, the result is a defective release of Ca2+ ions from the sarcoplasmic reticulum into the myocyte cytoplasm. Here, we provide an overview of the most frequent diseases resulting from mutations to RyR1 and RyR2, briefly review some of the recent experimental structural work on these two molecules, detail some of the computational work describing their dynamics, and summarize the known changes to the structure and function of these receptors with particular emphasis on their N-terminal, central, and channel domains.

Mitochondrial HMG-Box Containing Proteins: From Biochemical Properties to the Roles in Human Diseases.

Mitochondrial DNA (mtDNA) molecules are packaged into compact nucleo-protein structures called mitochondrial nucleoids (mt-nucleoids). Their compaction is mediated in part by high-mobility group (HMG)-box containing proteins (mtHMG proteins), whose additional roles include the protection of mtDNA against damage, the regulation of gene expression and the segregation of mtDNA into daughter organelles. The molecular mechanisms underlying these functions have been identified through extensive biochemical, genetic, and structural studies, particularly on yeast (Abf2) and mammalian mitochondrial transcription factor A (TFAM) mtHMG proteins. The aim of this paper is to provide a comprehensive overview of the biochemical properties of mtHMG proteins, the structural basis of their interaction with DNA, their roles in various mtDNA transactions, and the evolutionary trajectories leading to their rapid diversification. We also describe how defects in the maintenance of mtDNA in cells with dysfunctional mtHMG proteins lead to different pathologies at the cellular and organismal level.

Disease-associated mutations alter the dynamic motion of the N-terminal domain of the human cardiac ryanodine receptor.

The human cardiac ryanodine receptor (hRyR2), the ion channel responsible for the release of Ca2+ ions from the sarcoplasmic reticulum into the cytosol, plays an important role in cardiac muscle contraction. Mutations to this channel are associated with inherited cardiac arrhythmias. These mutations appear to cluster in distinct parts of the N-terminal, central and C-terminal areas of the channel. Here, we used molecular dynamics simulation to examine the effects three disease-associated mutations to the N-terminal region, R414L, I419F and R420W, have on the dynamics of a model of residues 1-655 of hRyR2. We find that the R414L and I419F mutations diminish the overall amplitude of motion without greatly changing the direction of motion of the individual domains, whereas R420W both enhances the amplitude and changes the direction of motion. Based on these results, we hypothesize that R414L and I419F hinder channel closing, whereas R420W may enhance channel opening. Overall, it appears that the wild-type protein possesses a moderate level of flexibility which allows the gate to close and not easily open without an opening signal. These mutations, however, disrupt this balance by making the gate either too rigid or too loose, causing closing to become difficult or less effective. Small-angle X-ray scattering studies of the same 1-655 residue fragment are in agreement with the molecular dynamics results and also suggest that the rest of the protein is needed to keep the entire domain properly folded.Communicated by Ramaswamy H. Sarma.

Normal Mode Analysis as a Routine Part of a Structural Investigation.

Normal mode analysis (NMA) is a technique that can be used to describe the flexible states accessible to a protein about an equilibrium position. These states have been shown repeatedly to have functional significance. NMA is probably the least computationally expensive method for studying the dynamics of macromolecules, and advances in computer technology and algorithms for calculating normal modes over the last 20 years have made it nearly trivial for all but the largest systems. Despite this, it is still uncommon for NMA to be used as a component of the analysis of a structural study. In this review, we will describe NMA, outline its advantages and limitations, explain what can and cannot be learned from it, and address some criticisms and concerns that have been voiced about it. We will then review the most commonly used techniques for reducing the computational cost of this method and identify the web services making use of these methods. We will illustrate several of their possible uses with recent examples from the literature. We conclude by recommending that NMA become one of the standard tools employed in any structural study.

Yeast phosphatidylinositol transfer protein Pdr17 does not require high affinity phosphatidylinositol binding for its cellular function.

Yeast phosphatidylinositol transfer protein (PITP) Pdr17 is an essential component of the complex required for decarboxylation of phosphatidylserine (PS) to phosphatidylethanolamine (PE) at a non-mitochondrial location. According to current understanding, this process involves the transfer of PS from the endoplasmic reticulum to the Golgi/endosomes. We generated a Pdr17E237A, K269A mutant protein to better understand the mechanism by which Pdr17p participates in the processes connected to the decarboxylation of PS to PE. We show that the Pdr17E237A, K269A mutant protein is not capable of binding phosphatidylinositol (PI) using permeabilized human cells, but still retains the ability to transfer PI between two membrane compartments in vitro. We provide data together with molecular models showing that the mutations E237A and K269A changed only the lipid binding cavity of Pdr17p and not its surface properties. In contrast to Pdr16p, a close homologue, the ability of Pdr17p to bind PI is not required for its major cellular function in the inter-membrane transfer of PS. We hypothesize that these two closely related yeast PITPs, Pdr16p and Pdr17p, have evolved from a common ancestor. Pdr16p fulfills those role(s) in which the ability to bind and transfer PI is required, while Pdr17p appears to have adapted to a different role which does not require the high affinity binding of PI, although the protein retains the capacity to transfer PI. Our results indicate that PITPs function in complex ways in vivo and underscore the need to consider multiple PITP parameters when studying these proteins in vitro.

The role of Lon-mediated proteolysis in the dynamics of mitochondrial nucleic acid-protein complexes.

Mitochondrial nucleoids consist of several different groups of proteins, many of which are involved in essential cellular processes such as the replication, repair and transcription of the mitochondrial genome. The eukaryotic, ATP-dependent protease Lon is found within the central nucleoid region, though little is presently known about its role there. Aside from its association with mitochondrial nucleoids, human Lon also specifically interacts with RNA. Recently, Lon was shown to regulate TFAM, the most abundant mtDNA structural factor in human mitochondria. To determine whether Lon also regulates other mitochondrial nucleoid- or ribosome-associated proteins, we examined the in vitro digestion profiles of the Saccharomyces cerevisiae TFAM functional homologue Abf2, the yeast mtDNA maintenance protein Mgm101, and two human mitochondrial proteins, Twinkle helicase and the large ribosomal subunit protein MrpL32. Degradation of Mgm101 was also verified in vivo in yeast mitochondria. These experiments revealed that all four proteins are actively degraded by Lon, but that three of them are protected from it when bound to a nucleic acid; the Twinkle helicase is not. Such a regulatory mechanism might facilitate dynamic changes to the mitochondrial nucleoid, which are crucial for conducting mitochondrial functions and maintaining mitochondrial homeostasis.

The structure and DNA-binding properties of Mgm101 from a yeast with a linear mitochondrial genome.

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.

Structure of the catalytic domain of the human mitochondrial Lon protease: proposed relation of oligomer formation and activity.

ATP-dependent proteases are crucial for cellular homeostasis. By degrading short-lived regulatory proteins, they play an important role in the control of many cellular pathways and, through the degradation of abnormally misfolded proteins, protect the cell from a buildup of aggregates. Disruption or disregulation of mammalian mitochondrial Lon protease leads to severe changes in the cell, linked with carcinogenesis, apoptosis, and necrosis. Here we present the structure of the proteolytic domain of human mitochondrial Lon at 2 A resolution. The fold resembles those of the three previously determined Lon proteolytic domains from Escherichia coli, Methanococcus jannaschii, and Archaeoglobus fulgidus. There are six protomers in the asymmetric unit, four arranged as two dimers. The intersubunit interactions within the two dimers are similar to those between adjacent subunits of the hexameric ring of E. coli Lon, suggesting that the human Lon proteolytic domain also forms hexamers. The active site contains a 3(10) helix attached to the N-terminal end of alpha-helix 2, which leads to the insertion of Asp852 into the active site, as seen in M. jannaschii. Structural considerations make it likely that this conformation is proteolytically inactive. When comparing the intersubunit interactions of human with those of E. coli Lon taken with biochemical data leads us to propose a mechanism relating the formation of Lon oligomers with a conformational shift in the active site region coupled to a movement of a loop in the oligomer interface, converting the proteolytically inactive form seen here to the active one in the E. coli hexamer.

Three-dimensional structure of YaaE from Bacillus subtilis, a glutaminase implicated in pyridoxal-5'-phosphate biosynthesis.

The structure of YaaE from Bacillus subtilis was determined at 2.5-A resolution. YaaE is a member of the triad glutamine aminotransferase family and functions in a recently identified alternate pathway for the biosynthesis of vitamin B(6). Proposed active residues include conserved Cys-79, His-170, and Glu-172. YaaE shows similarity to HisH, a glutaminase involved in histidine biosynthesis. YaaD associates with YaaE. A homology model of this protein was constructed. YaaD is predicted to be a (beta/alpha)(8) barrel on the basis of sequence comparisons. The predicted active site includes highly conserved residues 211-216 and 233-235. Finally, a homology model of a putative YaaD-YaaE complex was prepared using the structure of HisH-F as a model. This model predicts that the ammonia molecule generated by YaaE is channeled through the center of the YaaD barrel to the putative YaaD active site.