Mitochondria as semiautonomic structures with major roles in energy metabolism and

The mitochondria are responsible for efficient energy accumulation and management within the organism and thus have intimate connections with most biosynthetic pathways.

The process of oxidative phosphorylation produces cellular energy, regulates mitochondrial and cellular redox status, generates most of the ROS and regulates Ca²⁺

concentration (Hebe, Blackburn, and Miller 2011). The oxidative phosphorylation chain is further described in the Section 1.3.

Mitochondria on the one hand influence nuclear gene expression via messaging systems termed retrograde mitochondrial signalling and on the other, require a nuclear contribution to produce most of their structural and functional protein, since most of the required genes are located in nuclear DNA, comprising those responsible for mitochondrial structural elements, those for glycolysis and most of the genes for oxidative metabolism. They also include genes for the transcriptional machinery needed for

Introduction

(Whelan and Zuckerbraun 2013). Proteins that are coded for by nuclear genes that are destined for the mitochondria are tagged with a presequence that is a 20- 35-mer peptide.

They are maintained in an unfolded state by chaperone proteins to enable passage across the mitochondrial membranes (Coope and Hausman 2006). The normal circular human mitochondrial genome, or mitochondrial DNA (mtDNA) encodes the structural genes for 12S and 16S ribosomal rRNAs, some subunits of NADH-coenzyme Q oxidoreductase, cyt c oxidase, cyt b, ATP synthase, and 22 tRNAs. Transcription takes place from both of the complementary strands, termed heavy (H, guanine-rich) and light, (L, cytosine- rich). Synthesis of mRNA starts from three promotors: two H promotors, heavy strand promotors 1 & 2 (HSP 1 & HSP 2) and one L — light strand promotor, LSP (Figure 5).

Figure 5. Gene sequences within mitochondrial circular DNA (taken from Asin-Cayuela &

Gustafsson (2007)).

The mitochondrion needs to synthesise enough rRNA to fulfil the simultaneous translation requirements of the 13 mRNAs within the polycistronic transcripts. This is the reason that transcription takes place in two versions — besides synthesis of the full length polycistronic H2 strand transcript, promoted by HSP 2, which covers almost all the length of the heavy strand, a shorter transcript, H1, promoted by HSP 1, ends at the 16S rRNA, and is transcribed at 20 times the level of H2. This ensures that a sufficient amount of 12S and 16S rRNA is available for protein translation. The shorter H1, with well-defined start

T/BS

LSP

12SrRNA D-loop

Cytb ND6

ND 5

ND4

COIII ATP6 COII

COI ND

2 ND1

16SrRNA

OH

HSP1 mTERF*

ND 4L ND

AT 3

P8

O

L

HSP2

Human mtDNA 16 569 bp mTERF

Image redrawn

Introduction

These promotors are found in the mtDNA non-coding region and recruit mitochondrial RNA polymerase to drive this transcription, leading to the production of three polycistronic transcription units. Transcription from the H-strand results in an RNA unit that is processed into two mt-rRNAs, fourteen mt-tRNAs and mt-mRNAs that encode twelve proteins. Transcription of the L-strand produces RNA that is cleaved to produce eight mt-tRNAs and only one mt-mRNA that encodes a single protein. Full polycistronic transcription from the L strand has never been observed under experimental conditions (Temperley et al. 2010). These initial polycistronic precursors are next cleaved by endogenous nucleases at the 5’ and 3’ends. The process of ‘tRNA editing’ is possible thanks to the tRNA sequence between rRNA and mRNA having a cloverleaf structure that is recognised by the enzyme processing the primary transcript. The mRNA and rRNA became polyadenylated by mitochondrial polymerase, which stabilises their structure and is needed to create a stop codon (Schapira and Dimauro 1994), (Asin-Cayuela and Gustafsson 2007), (Temperley et al. 2010). Figure 6 presents the mitochondrial genome translation mechanism.

Nuclear DNA gene expression is also controlled epigenetically, meaning that it is sensitive to the environment. The processes of gene expression are compound-dependent, controlled by a set of nuclear-encoded transcription factors. For example, nuclear respiratory factor 1, NRF-1, has specific binding sites in the promotors of the genes for the electron transport chain and cyt c. Further, these nuclear factors are coordinated by coactivators, some of the family members of which provide a link between the products of mitochondrial function or malfunction and subsequent adjustments of nuclear gene expression. Mitochondrial dysfunction is often found to correlate with alterations in nuclear gene expression. Further, there are other crucial factors that control nuclear gene expression, such as the ATP/ADP concentration: each mitochondrial promotor has been shown to have a unique sensitivity to the mitochondrial ATP level (Whelan and Zuckerbraun 2013).

Mitochondria have been called ‘fundamental arbiters of life and death of the cell’. It has been shown that pathways of intermediary metabolism involved in energy capture are important components and regulators of mitochondria-linked events of cell death and survival. These may involve interaction of signalling molecules with the mitochondrial membrane. Several chemotherapeutic agents have been shown to act on targets in the respiratory chain (Cerquetti et al. 2008).

Introduction

Figure 6. Gene expression in human mitochondria. (Taken from Zeviani et al. (2015)).

The mitochondrial permeability transition pore can be activated by factors which include a decreased membrane potential, with predominant role of ROS. The important mechanism of apoptosis involving mitochondrial membrane permeabilization and cyt c release has been described in the Section 1.1.8 and 1.1.9 when increased Ca²⁺ transmition plays a crucial role.

ROS are products of oxidative phosphorylation. Mitochondrial stress has been shown to result in excess production of ROS, and thay may contribute to cyt c release. ROS have also been found to be important second messengers for mitochondrial communication in both physiological and pathological circumstances. For example, expansion of ROS production can be an important second messenger in inducing factors such as nuclear erythroid-derived factors (Nrfs) that transfer to the nucleus and stimulate antioxidant response elements. ROS production induces conformation change of Keap1 protein, activating Nrf2 which transfers to the nucleus to stimulate antioxidant response elements, leading to enhanced mitochondrial biogenesis for inducing factors such as NRF-1. The

This dia gra m re fle cts a line a r vie w of the ge ne tic ma p of the he a vy (H) a nd light (L) s tra nds of huma n mtDNA. The long horizonta l a rrows indica te pre curs or polycis tronic tra ns cripts , while s hort ve rtica l line s indica te the loca tions of tRNA ge ne s a nd the ve rtica l a rrows indica te the proce s s ing s ite s whe re pre curs ors a re punctua te d by a ntis e ns e tRNAs or a re not punctua te d by tRNAs . Bla ck a nd white blocks re pre s e nt ma ture tra ns cripts for H- a nd L-s tra nds , re s pe ctive ly. As te ris ks de note the prote in coding tra ns cripts tha t re quire the a ddition of the poly (A) ta il to ge ne ra te a s top codon.

Introduction

genomes, and indirectly regulating the three mitochondrial-encoded COX subunit (Whelan and Zuckerbraun 2013). If oxygen species overwhelm the protective capacity of antioxidants, enhanced lipid peroxidation leads to increased permeability and progression to programmed cell death.