The application of gold in medicine is traceable for several thousand years and Au(i) compounds have been used clinically to treat rheumatoid arthritis since the last century. Recently research into ...gold-based drugs for a range of human diseases has seen a renaissance. Old as well as new Au(i) and Au(iii) compounds have been used and designed with an aim of targeting cellular components that are implicated in the onset or progression of cancers, rheumatoid arthiritis, viral and parasitic diseases. In addition, new disease targets have been found for gold compounds that have given insight into the mechanism of action of these compounds, as well as in the molecular pathophysiology of human diseases. Here we discuss the rationale for the design and use of gold compounds that have specific and selective targets in cells to alleviate the symptoms of a range of human diseases. We summarise the most recent findings in this research and our own discoveries to show that gold compounds can be developed to become versatile and powerful drugs for diseases caused by dysfunction of selenol and thiol containing proteins.
Dysfunction of the oxidative phosphorylation (OXPHOS) system is a major cause of human disease and the cellular consequences are highly complex. Here, we present comparative analyses of mitochondrial ...proteomes, cellular transcriptomes and targeted metabolomics of five knockout mouse strains deficient in essential factors required for mitochondrial DNA gene expression, leading to OXPHOS dysfunction. Moreover, we describe sequential protein changes during post-natal development and progressive OXPHOS dysfunction in time course analyses in control mice and a middle lifespan knockout, respectively. Very unexpectedly, we identify a new response pathway to OXPHOS dysfunction in which the intra-mitochondrial synthesis of coenzyme Q (ubiquinone, Q) and Q levels are profoundly decreased, pointing towards novel possibilities for therapy. Our extensive omics analyses provide a high-quality resource of altered gene expression patterns under severe OXPHOS deficiency comparing several mouse models, that will deepen our understanding, open avenues for research and provide an important reference for diagnosis and treatment.
Prioritisation of gene ontology terms from differential gene expression analyses in a two-dimensional format remains a challenge with exponentially growing data volumes. Typically, gene ontology ...terms are represented as tree-maps that enclose all data into defined space. However, large datasets make this type of visualisation appear cluttered and busy, and often not informative as some labels are omitted due space limits, especially when published in two-dimensional (2D) figures.
Here we present an open source CirGO (Circular Gene Ontology) software that visualises non-redundant two-level hierarchically structured ontology terms from gene expression data in a 2D space. Gene ontology terms based on statistical significance were summarised with a semantic similarity algorithm and grouped by hierarchical clustering. This software visualises the most enriched gene ontology terms in an informative, comprehensive and intuitive format that is achieved by organising data from the most relevant to the least, as well as the appropriate use of colours and supporting information. Additionally, CirGO is an easy to use software that supports researchers with little computational background to present their gene ontology data in a publication ready format.
Our easy to use open source CirGO Python software package provides biologists with a succinct presentation of terms and functions that are most represented in a specific gene expression data set in a visually appealing 2D format (e.g. for reporting research results in scientific articles). CirGO is freely available at https://github.com/IrinaVKuznetsova/CirGO.git .
Mitochondrial ribosomes are specialized for the synthesis of membrane proteins responsible for oxidative phosphorylation. Mammalian mitoribosomes have diverged considerably from the ancestral ...bacterial ribosomes and feature dramatically reduced ribosomal RNAs. The structural basis of the mammalian mitochondrial ribosome assembly is currently not well understood. Here we present eight distinct assembly intermediates of the human large mitoribosomal subunit involving seven assembly factors. We discover that the NSUN4-MTERF4 dimer plays a critical role in the process by stabilizing the 16S rRNA in a conformation that exposes the functionally important regions of rRNA for modification by the MRM2 methyltransferase and quality control interactions with the conserved mitochondrial GTPase MTG2 that contacts the sarcin-ricin loop and the immature active site. The successive action of these factors leads to the formation of the peptidyl transferase active site of the mitoribosome and the folding of the surrounding rRNA regions responsible for interactions with tRNAs and the small ribosomal subunit.
It was thought that the proteins produced by ribosomes were dictated only by the sequences of the mRNAs they translated, however it is becoming apparent that subpopulations of ribosomes can have ...unique properties that influence the functions of the proteins they produce. Ribosomes have been engineered to discriminate between different mRNA templates or with unique decoding properties, and many new applications of unnatural ribosomes can be foreseen. In natural systems ribosomes with alternate protein and RNA composition have been shown to selectively translate specific mRNAs. As more is learned about ribosome structure and the mechanisms of translation, new opportunities to engineer ribosomes for applications in biotechnology and synthetic biology can be developed and new examples of ribosome-mediated regulation of translation are likely to emerge in nature.
Mitochondria maintain their own specialized protein synthesis machinery, which in mammals is used exclusively for the synthesis of the membrane proteins responsible for oxidative phosphorylation
. ...The initiation of protein synthesis in mitochondria differs substantially from bacterial or cytosolic translation systems. Mitochondrial translation initiation lacks initiation factor 1, which is essential in all other translation systems from bacteria to mammals
. Furthermore, only one type of methionyl transfer RNA (tRNA
) is used for both initiation and elongation
, necessitating that the initiation factor specifically recognizes the formylated version of tRNA
(fMet-tRNA
). Lastly, most mitochondrial mRNAs do not possess 5' leader sequences to promote mRNA binding to the ribosome
. There is currently little mechanistic insight into mammalian mitochondrial translation initiation, and it is not clear how mRNA engagement, initiator-tRNA recruitment and start-codon selection occur. Here we determine the cryo-EM structure of the complete translation initiation complex from mammalian mitochondria at 3.2 Å. We describe the function of an additional domain insertion that is present in the mammalian mitochondrial initiation factor 2 (mtIF2). By closing the decoding centre, this insertion stabilizes the binding of leaderless mRNAs and induces conformational changes in the rRNA nucleotides involved in decoding. We identify unique features of mtIF2 that are required for specific recognition of fMet-tRNA
and regulation of its GTPase activity. Finally, we observe that the ribosomal tunnel in the initiating ribosome is blocked by insertion of the N-terminal portion of mitochondrial protein mL45, which becomes exposed as the ribosome switches to elongation mode and may have an additional role in targeting of mitochondrial ribosomes to the protein-conducting pore in the inner mitochondrial membrane.
Transcription of the human mitochondrial genome and correct processing of the two long polycistronic transcripts are crucial for oxidative phosphorylation. According to the tRNA punctuation model, ...nucleolytic processing of these large precursor transcripts occurs mainly through the excision of the tRNAs that flank most rRNAs and mRNAs. However, some mRNAs are not punctuated by tRNAs, and it remains largely unknown how these non-canonical junctions are resolved. The FASTK family proteins are emerging as key players in non-canonical RNA processing. Here, we have generated human cell lines carrying single or combined knockouts of several FASTK family members to investigate their roles in non-canonical RNA processing. The most striking phenotypes were obtained with loss of FASTKD4 and FASTKD5 and with their combined double knockout. Comprehensive mitochondrial transcriptome analyses of these cell lines revealed a defect in processing at several canonical and non-canonical RNA junctions, accompanied by an increase in specific antisense transcripts. Loss of FASTKD5 led to the most severe phenotype with marked defects in mitochondrial translation of key components of the electron transport chain complexes and in oxidative phosphorylation. We reveal that the FASTK protein family members are crucial regulators of non-canonical junction and non-coding mitochondrial RNA processing.
Directed evolution emulates the process of natural selection to produce proteins with improved or altered functions. These approaches have proven to be very powerful but are technically challenging ...and particularly time and resource intensive. To bypass these limitations, we constructed a system to perform the entire process of directed evolution in silico. We employed iterative computational cycles of mutation and evaluation to predict mutations that confer high-affinity binding activities for DNA and RNA to an initial de novo designed protein with no inherent function. Beneficial mutations revealed modes of nucleic acid recognition not previously observed in natural proteins, highlighting the ability of computational directed evolution to access new molecular functions. Furthermore, the process by which new functions were obtained closely resembles natural evolution and can provide insights into the contributions of mutation rate, population size and selective pressure on functionalization of macromolecules in nature.
The expression of the compact mammalian mitochondrial genome requires transcription, RNA processing, translation and RNA decay, much like the more complex chromosomal systems, and here we use it as a ...model system to understand the fundamental aspects of gene expression. Here we combine RNase footprinting with PAR-CLIP at unprecedented depth to reveal the importance of RNA-protein interactions in dictating RNA folding within the mitochondrial transcriptome. We show that LRPPRC, in complex with its protein partner SLIRP, binds throughout the mitochondrial transcriptome, with a preference for mRNAs, and its loss affects the entire secondary structure and stability of the transcriptome. We demonstrate that the LRPPRC-SLIRP complex is a global RNA chaperone that stabilizes RNA structures to expose the required sites for translation, stabilization, and polyadenylation. Our findings reveal a general mechanism where extensive RNA-protein interactions ensure that RNA is accessible for its biological functions.
Pentatricopeptide repeat (PPR) domain proteins are a large family of RNA-binding proteins that are involved in the maturation and translation of organelle transcripts in eukaryotes. They were first ...identified in plant organelles and their important role in mammalian mitochondrial gene regulation is now emerging. Mammalian PPR proteins, like their plant counterparts, have diverse roles in mitochondrial transcription, RNA metabolism and translation and consequently are important for mitochondrial function and cell health. Here we discuss the current knowledge about the seven mammalian PPR proteins identified to date and their roles in the regulation of mitochondrial gene expression. Furthermore we discuss the mitochondrial RNA targets of the mammalian PPR proteins and methods to investigate the RNA targets of these mitochondrial RNA-binding proteins. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.
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► PPR proteins are a large family of RNA-binding proteins that regulate organelle gene expression. ► We discuss the diverse roles of mammalian PPR proteins in mitochondrial RNA metabolism. ► We discuss methods to investigate the RNA targets of mitochondrial PPR proteins.
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