Bacterial tail anchors can target to the mitochondrial outer membrane

Background

During the generation and evolution of the eukaryotic cell, a proteobacterial endosymbiont was re-fashioned into the mitochondrion, an organelle that appears to have been present in the ancestor of all present-day eukaryotes. Mitochondria harbor proteomes derived from coding information located both inside and outside the organelle, and the rate-limiting step toward the formation of eukaryotic cells may have been development of an import apparatus allowing protein entry to mitochondria. Currently, a widely conserved translocon allows proteins to pass from the cytosol into mitochondria, but how proteins encoded outside of mitochondria were first directed to these organelles at the dawn of eukaryogenesis is not clear. Because several proteins targeted by a carboxyl-terminal tail anchor (TA) appear to have the ability to insert spontaneously into the mitochondrial outer membrane (OM), it is possible that self-inserting, tail-anchored polypeptides obtained from bacteria might have formed the first gate allowing proteins to access mitochondria from the cytosol.

Results

Here, we tested whether bacterial TAs are capable of targeting to mitochondria. In a survey of proteins encoded by the proteobacterium Escherichia coli, predicted TA sequences were directed to specific subcellular locations within the yeast Saccharomyces cerevisiae. Importantly, TAs obtained from DUF883 family members ElaB and YqjD were abundantly localized to and inserted at the mitochondrial OM.

Conclusions

Our results support the notion that eukaryotic cells are able to utilize membrane-targeting signals present in bacterial proteins obtained by lateral gene transfer, and our findings make plausible a model in which mitochondrial protein translocation was first driven by tail-anchored proteins.

Reviewers

This article was reviewed by Michael Ryan and Thomas Simmen.

     

Bacterial expression,purification, and characterization of rat kidney-type mitochondrial glutaminase

The human gene that encodes the kidney-type glutaminase (KGA) spans 84-kb, contains 19 exons, and encodes two alternatively spliced mRNAs. Various segments of the rat KGA cDNA were PCR amplified and cloned into a bacterial expression vector to determine whether the N- and C- terminal ends of the glutaminase protein were essential for activity. A recombinant glutaminase, lacking the coding sequence contained in exon 1, was found to be fully active. In contrast, proteins that lacked sequences from exons 1 and 2 and exons 1-3 were inactive. An additional construct that corresponded to the sequence encoded by exons 2-14 also retained full activity. Both of the fully active, truncated proteins were purified to apparent homogeneity using an incorporated N-terminal His(6)-tag and Ni(2+)-affinity chromatography. The K(M) values for glutamine of the native and recombinant forms of glutaminase were nearly identical. However, the two truncated forms of the glutaminase exhibit the characteristic phosphate activation profile only when dialyzed into a buffer lacking phosphate. Dialysis versus 10mM Tris-phosphate was sufficient to form an active tetramer. Thus, the deleted N-terminal sequence may contribute to the phosphate-dependent oligomerization and activation of the native glutaminase.     

Bacterial proteins and CpG-rich extrachromosomal DNA in potential cancer therapy

Bacterial proteins such as azurin and Laz have recently been shown to enter preferentially to cancer cells and kill them by multiple mechanisms. Historically, bacterial DNA, particularly the unmethylated CpG dinucleotides, have been shown to trigger activation of specific Toll-like receptors (TLRs) in immune cells, leading to various cytokine and chemokine production that allows cancer cell death and their regression. However, the enhanced release of specific protein or extrachromosomal DNA by bacteria in response to exposure to cancer cells has not been previously demonstrated. In this review, we discuss how an opportunistic, extracellular pathogenic bacterium, Pseudomonas aeruginosa, senses the presence of cancer cells and releases a specific protein or extrachromosomal DNA with antitumor activity for inhibition of cancer cell growth.     

Long-term growth hormone therapy in mitochondrial cytopathy

OBJECTIVE: To describe in a 5-year-old Caucasian male with mitochondrial cytopathy, a biochemical growth hormone (GH) deficiency associated with normal GH biological activity as evaluated by Nb2 cell bioassay and normal serum IGF-I and IGFBP3 values increasing slightly after GH administration. METHOD: Serum GH concentrations were measured with a commercial immunofluorometric assay and with a biological assay, which uses the Nb2 cell line. Serum IGF-I and IGFBP3 concentrations were measured with RIA. RESULTS: The GH-supplementary therapy was initially effective in terms of growth gain, but no therapeutic benefit was observed over a long period of time. CONCLUSION: In patients suffering from mitochondrial cytopathy, short stature seems to be attributed more to a disease-related inadequate protein substrate than to the non-classical GH deficiency.     

Bacterial proteins with N-terminal leader sequences resembling mitochondrial targeting sequences of eukaryotes

Amphipathic, alpha-helical, leader sequences, analogous to those that direct nuclear-encoded eukaryotic proteins into mitochondria, have been found in one and only one class of bacterial integral membrane proteins. These bacterial proteins are the sugar permeases of the phosphoenolpyruvate-dependent phosphotransferase system. The amphipathic leader sequence in each of these proteins is terminated by a helix breaker, either a prolyl residue or 2 adjacent glycyl residues. Preliminary evidence suggests that these leader sequences function to target the proteins to the envelope fraction of the prokaryotic cell during their biosynthesis.     

Bacterial l-asparaginases for cancer therapy: Current knowledge and future perspectives

l -Asparaginases hydrolyzing plasma l -asparagine and l -glutamine has attracted tremendous attention in recent years owing to remarkable anticancer properties. This enzyme is efficiently used for acute lymphoblastic leukemia (ALL) and lymphosarcoma and emerged against ALL in children, neoplasia, and some other malignancies. Cancer cells reduce the expression of l -asparaginase leading to their elimination. The l -asparaginase anticancerous application approach has made incredible breakthrough in the field of modern oncology through depletion of plasma l -asparagine to inhibit the cancer cells growth; particularly among children. High level of l -asparaginase enzyme production by Escherichia coli, Erwinia species, Streptomyces, and Bacillus subtilis species is highly desirable as bacterial alternative enzyme sources for anticancer therapy. Thermal or harsh conditions stability of those from the two latter bacterial species is considerable. Some enzymes from marine bacteria have conferred stability in adverse conditions being more advantageous in cancer therapy. Several side effects exerted by l -asparaginases such as hypersensitivity should be hindered or decreased through alternative therapies or use of immune-suppressor drugs. The l -asparaginase from Erwinia species has displayed remarkable traits in children with this regard. Noticeably, Erwinia chrysanthemi l -asparaginase exhibited negligible glutaminase activity representing a promising efficiency mitigating related side effects. Application of software such as RSM would optimize conditions for higher levels of enzyme production. Additionally, genetic recombination of the encoding gene would indisputably help improving enzyme traits. Furthermore, the possibility of anticancer combination therapy using two or more l -asparaginases from various sources is plausible in future studies to achieve better therapeutic outcomes with lower side effects.     

Bacterial peptidoglycan muropeptides benefit mitochondrial homeostasis and animal physiology by acting as ATP synthase agonists

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Photodynamic therapy: a mitochondrial inducer of apoptosis

Photodamage to the mitochondria of murine leukemia P388 cells resulted in immediate loss of the mitochondrial membrane potential together with the release of cytochrome c into the cytosol. This was followed by a rapid activation of caspase 3-like proteases, as indicated by a marked rise in DEVDase activity. There was no significant effect on WEHDase or VEIDase activities, suggesting that only the late-stage caspases had been effected. The apoptotic response to mitochondrial photodamage was abolished by the broad-spectrum caspase inhibitor zVAD-fmk, but this did not prevent loss of viability after mitochondrial photodamage. These studies indicate that the release of cytochrome c from photodamaged mitochondria is sufficient to directly initiate a caspase-dependent apoptotic response.     

Delivering healthy mitochondria for the therapy of mitochondrial diseases and beyond

Mitochondrial transfer has been demonstrated to a play a physiological role in the rescuing of mitochondrial DNA deficient cells by co-culture with human mesenchymal stem cells. The successful replacement of mitochondria using microinjection into the embryo has been revealed to improve embryo maturation. Evidence of mitochondrial transfer has been shown to minimize injury of the ischemic-reperfusion rabbit heart model. In this mini review, the therapeutic strategies of mitochondrial diseases based on the concept of mitochondrial transfer are illustrated, as well as a novel approach to peptide-mediated mitochondrial delivery. The possible mechanism of peptide-mediated mitochondrial delivery in the treatment of the myoclonic epilepsy and ragged-red fiber disease is summarized. Understanding the feasibility of mitochondrial manipulation in cells facilitates novel therapeutic skills in the future clinical practice of mitochondrial disorder.     

The use of PNAs and their derivatives in mitochondrial gene therapy

Human mitochondria contain their own genome, mtDNA. This small molecule encodes 24 RNA species and 13 polypeptides, which are essential components of the mitochondrial respiratory chain. The mitochondrial genome is present in hundreds or thousands of copies in each cell and is believed to turnover throughout the life of the cell. Defects of the mitochondrial genome (mtDNA) cause a variety of multisystemic disorders routinely affecting the muscle and nervous system. There is currently no effective treatment for patients with defects of the mitochondrial genome. In many patients, defective cells harbour two sub-populations of mtDNA (a situation termed heteroplasmy), one being normal, the other containing the pathogenic mutation. The mutated copy is often recessive, with biochemical and clinical defects only becoming apparent when the levels of mutated mtDNA outweigh the normal copies. It has therefore been postulated that by selectively preventing replication of the mutated mtDNA, the normal copy will propagate, restoring biochemical function. The search has therefore been on during recent years to identify an antigenomic molecule that will fulfil this criterion. Following evidence that peptide nucleic acids could selectively inhibit replication of templates carrying a known pathogenic mtDNA mutation in vitro,we report on the progress of this approach and the various modificationsthat are now being used to improve the efficacy of PNA-based antigenomic inhibition.     

The use of PNAs and their derivatives in mitochondrial gene therapy

Summary Human mitochondria contain their own genome, mtDNA. This small molecule encodes 24 RNA species and 13 polypeptides, which are essential components of the mitochondrial respiratory chain. The mitochondrial genome is present in hundreds or thousands of copies in each cell and is believed to turnover throughout the life of the cell. Defects of the mitochondrial genome (mtDNA) cause a variety of multisystemic disorders routinely affecting the muscle and nervous system. There is currently no effective treatment for patients with defects of the mitochondrial genome. In many patients, defective cells harbour two sub-populations of mtDNA (a situation termed heteroplasmy), one being normal, the other containing the pathogenic mutation. The mutated copy is often recessive, with biochemical and clinical defects only becoming apparent when the levels of mutated mtDNA outweigh the normal copies. It has therefore been postulated that by selectively preventing replication of the mutated mtDNA, the normal copy will propagate, restoring biochemical function. The search has therefore been on during recent years to identify an antigenomic molecule that will fulfil this criterion. Following evidence that peptide nucleic acids could selectively inhibit replication of templates carrying a known pathogenic mtDNA mutation in vitro, we report on the progress of this approach and the various modifications that are now being used to improve the efficacy of PNA-based antigenomic inhibition.     

The use of PNAs and their derivatives in mitochondrial gene therapy

Summary Human mitochondria contain their own genome, mtDNA. This small molecule encodes 24 RNA species and 13 polypeptides, which are essential components of the mitochondrial respiratory chain. The mitochondrial genome is present in hundreds or thousands of copies in each cell and is believed to turnover throughout the life of the cell. Defects of the mitochondrial genome (mtDNA) cause a variety of multisystemic disorders routinely affecting the muscle and nervous system. There is currently no effective treatment for patients with defects of the mitochondrial genome. In many patients, defective cells harbour two sub-populations of mtDNA (a situation termed heteroplasmy), one being normal, the other containing the pathogenic mutation. The mutated copy is often recessive, with biochemical and clinical defects only becoming apparent when the levels of mutated mtDNA outweigh the normal copies. It has therefore been postulated that by selectively preventing replication of the mutated mtDNA, the normal copy will propagate, restoring biochemical function. The search has therefore been on during recent years to identify an antigenomic molecule that will fulfil this criterion. Following evidence that peptide nucleic acids could selectively inhibit replication of templates carrying a known pathogenic mtDNA mutation in vitro, we report on the progress of this approach and the various modifications that are now being used to improve the efficacy of PNA-based antigenomic inhibition.     

Bacterial replacement therapy: adapting 'germ warfare' to infection prevention

The individual bacterial members of our indigeneous microbiota are actively engaged in an on-going battle to prevent colonisation and overgrowth of their terrain by competing microbes, some of which might have pathogenic potential for the host. Humans have long attempted to intervene in these bacterial interactions. Ingestion of probiotic bacteria, particularly lactobacilli, is commonly practiced to promote well-balanced intestinal microflora. As bacterial resistance to antimicrobials has increased, so too has research into colonisation of human tissues with specific effector strains capable of out-competing known bacterial pathogens. Recent progress is particularly evident in the application of avirulent Streptococcus mutans to the control of dental caries, alpha hemolytic streptococci to reduction of otitis media recurrences and Streptococcus salivarius to streptococcal pharyngitis prevention.