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Thimet Oligopeptidase (also known as; TOP, EC 184.108.40.206 or EP24.15) Neurolysin (also known as; NLN, EC 220.127.116.11, EP 24.16 Oligopeptidase M (mitochondrial peptidase) or soluble angiotensin II-binding protein) are both members of the protease super family. Throughout this report Neurolysin will be referred to as, NLN and Thimet Oligopeptidase will be referred to as TOP. A protease is an enzyme (a protein catalyst; makes reactions happens at a quicker pace) which breaks down proteins. Proteins are made up of amino acids (like building blocks of the protein) that are linked by peptide bonds connecting the carboxyl terminus of one amino acid to the next terminus, thus forming a polypeptide chain. [i] These proteins can be broken down by proteolysis. This is a process where the enzymes cleave the peptides into smaller pieces thus triggering protein catabolism by hydrolysis (removal of a water molecule) of one or many peptide bonds that link amino acids together. [ii] This is done by a protein called ubiquitin which tags the protein and sends a signal to other ligases which attach to the ubiquitin molecules, thus degrading the protein and forming smaller polypeptides. However, this may be time-consuming so proteases are added to speed up the reaction. Proteases act as a catalyst, by speeding up the reaction and allowing cleavage (cutting the protein in specific areas and well-defined ways). Studies have been done (Reich et al., 1975) to show proteases are involved in many processes within the tissues and organs for example; maintenance, growth, development and repair processes. Peptides resistant to degradation by other cytosolic peptidases is hydrolysed by TOP, this reduces the pool of peptides available for antigen presentation. [iii] (Saric et al. 2001)
There are many groups of related proteases which were broken down into 6 super families by Rawlings and Barrett [iv] ; serine proteases, theonine proteases, cysteine proteases, aspartate proteases, glutamic acid proteases and metalloproteases. The serine protease family contains a serine amino acid in the active site of the enzyme. Thenonine proteases contain a threonine (Thr) residue within the active site of the enzyme; The prototype members within this group are the catalytic subunits of the proteasome. The aspartic proteases use an aspartatic acid residue for the catalysis of their peptide substrates and are inhibited by pepstatin. Glutamic-acid proteases were not described until 2004. 1 The metalloprotease family are mainly a zinc-dependant (some use cobalt) family which contain two subgroups; exoproteases (metalloexopeptidases) or endoproteases (metalloendopeptidases). Many members of the zinc metalloprotease family have a characteristic sequence within the active site motif, His-Glu-Xaa-Xaa-His (HEXXH). [v] This specific sequence forms part of the binding site which has thought to be for the metal cofactor zinc and the catalytic water molecule (Matthews et al. 1974) [vi] .The M3 family of zinc metalloendoproteases has 9 members altogether which share a various degree of sequence similarity to each other (Rawlings and Barrett 1995). Thimet oligopeptidase is a thiol-sensitive member of the M3 family of zinc metalloprotease, [vii] as well as Neurolysin which is another member of the M3 family of metalloproteases. The two enzymes with the highest sequence similarity in this are TOP and NLN with 60% sequence similarity. 2 As both TOP and NLN are metalloendoproteases they are both able to cleave the peptide bonds of the amino acids within the protein, and although they have such a high similarity they are still able to cut different sequences.
Thimet Oligopeptidase is a soluble highly homologous endopeptidase which is restricted to small peptide substrates of around 5-18 amino acids in length. [viii] Dando et al., 1993; Orlowski et al., 1983, 1989 helped determine the molecular weight of TOP resulting in the range between, 67,000 to 75,000 which composed of 645 amino acids. [ix] Pierotti et al determined a more specific molecular weight of around 72,985MW using polyacrylamide gel electrophoresis under denaturing and reducing conditions and by molecular sieving chromatography. [x] TOP is encoded by the THOP1 gene which is located on chromosome 19 on the short arm at position 13.3 (19p13.3) in humans. In the rat it is found on chromosome 7 at the position 11 on the long arm: 7q11. (http://rgd.mcw.edu/tools/genes/genes_view.cgi?id=68330) This gene starts at the 2,785,506 base pair and ends at the 2,813,599 base pair thus resulting in 28.094 bases long (plus strand orientation). [xi] The location of the THOP1 protein is within the subcellular location of the cytoplasm with the gene being found in high concentration within the nucleus. (Fontenele-Neto et al, 2001) The THOP1 gene is excluded from the Linkage Region for Late-Onset Alzheimer Disease. [xii]
TOP has many physiological roles one of which has thought to be involved in the metabolism of many small peptides such as; angiotension, opiods, bradykinin, somatostatin, and neurotension. # It is found predominantly in the neuroendocrine-gonadal axis where it plays a part in the process of bioactive peptides such as; Gonadotropin-releasing hormone (degrades GnRH which is released from the hypothalamus and in doing so modulates the LH levels), beta-neoendorphin, alpha-neoendorphin and dynorphin; the progression of spermatogenesis and the normal clearance of beta amyloid in brain cells. [xiii] TOP is thought to cleave peptide bonds on the C-terminus side of specific hydrophobic residues of around 5-18 amino acids in length and modulate the activity of many neuropeptides. (Crack et al, 1999) A unique characteristic about TOP would be its "fuzzy" substrate recognition that allows it to cleave the peptides with high specificity for certain sites but no obvious sequence similarity.
NLN is a homologue of the TOP endopeptidase which has a molecular weight of between 66,000 and 78,000 and around 704 amino acids long. NLN can be found on chromosome 5 at the position 12.3 of the long arm (5q12.3) in humans, whereas the genetic locus for the NLN gene within the rat maps to chromosome 2 at the position 13 of the long arm: 2q13. (http://rgd.mcw.edu/tools/genes/genes_view.cgi?id=621518) The gene location starts at the 65,018,023 base pair and ends at the 65,167,553 base pair thus making it 149,531 bases in size (plus strand orientation).
As well as TOP, NLN role is also involved in hydrolysing a number neuropeptides in vitro. In vivo NLN has thought to be involved in the metabolic inactivation of bioactive peptides such as the 13-residue neuropeptide, neurotensin. Neurotension is hydrolysed between residues Pro-10 and Tyr-11, thus creating a shorter fragment which becomes inactive. NLN is also involved in the destruction of ganglion and cortical cells. NLN is primarily located in the mitochondrial intermembrane space, where it is able to interact non-covalently with the inner membrane and also within the cytoplasm. (6) NLN is primarily located in the mitochondrial inter-membrane space however it may also be present in the cytoplasm, this is due to an alternative splicing site in the N-terminal. The splicing site determines whether the protein is spliced into the longer or shorter form of NLN. The longer form of NLN target the location of the mitochondrial inter-membrane space whereas the shorter form of NLN exists as cytosol in the cytoplasm.
Structure of TOP and NLN
The crystal structure of Neurolysin shows that it is predominately Î±-helical in nature and accounts for 53% of all residues, whereas, the Î²-secondary structure only accounts for just 5.9%. TOP is also predominately Î±-helical due to it having 24 Î±-helices and 9 short Î² strands. Within the N-terminal there are 13 residues which are disordered in the crystal structure. It is thought that an area of this flexible sequence may mediate the transport of NLN and TOP through the secretory pathways in specific cell types. [xiv] Neurolysin adopts an overall prolate ellipsoid shape.# Both TOP and NLN contain a deep narrow channel running the length of the molecule, thus dividing the enzyme into two large domains (domain I and domain II) The only thing connecting them is a few secondary structural elements at the floor. The active site can be found at the bottom of the channel at the base of domain II and is in the middle of the enzymes length. The active site only has a narrow pathway that allows access, this is due to it being buried by the channel walls. #1 Within the active site of TOP it contains an H-E-X-X-H zinc binding motif sequence [xv] , which is characteristic of many zinc metallopeptidases. 1#
Domain IFigure 1: these stereo ribbon diagrams show the crystal structure of both TOP and NLN
Legend: Figure A shows the crystal structure of TOP and domain I and II, figure B shows the crystal structure for NLN and domain I and II. The area in blue indicates the region in which they are similar. http://en.wikipedia.org/wiki/THOP1, http://en.wikipedia.org/wiki/NLN_(gene)
When TOP and NLN crystal structures were compared it showed that the active site found in TOP (base of a deep channel running the length of the elongated molecule) had an overall fold first seen in NLN. In a recent study by Kallol Ray et al., 2004 they discovered variations near one end of the channel. These variations allow TOP and NLN to form a variable three-dimensional shape which is determined by its folding/conformational change. TOP and NLN can cleave a variety of sequences, this may be due to a variety of potentially flexible elements. Although they have a high relation, few sequence differences between the two proteins in the substrate binding site were found. The four variable residues which were found in the substrate binding site may account for their differences in the substrate specificity. Further-more a loop segment found in TOP and NLN differ in degree of order and conformation from each other suggesting a role in the difference in cellular distribution. #2
Figure 2 shows the surface of NLN and the differences between TOP and NLN.An external file that holds a picture, illustration, etc. Object name is 51634-19f3_C4TT.jpg Object name is 51634-19f3_C4TT.jpg
Legend: 'A' shows the outside out the molecule, the blue colour indicates the difference in amino acids between TOP and NLN. 'B' shows the molecule cut in half at the substate binding channels floor and the sequence differences are labelled in the green boxes, red indicates TOP and black indicates NLN. 'C' indicates the sequence differences in the two channel walls and the orange circle shows the active site. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2373592/figure/f3/
NLN and TOP both cleave most bioactive peptides at the same peptide bond which may be expected as they have such a high similarity. 2# However, they do not cleave all bioactive peptides at the same point for example NLN cleaves neurotension between Pro-10 and Tyr-11 while TOP cleaves between Arg-8 and Arg-9. #2. TOP limits the extent of antigen presentation by MHC class I molecules due to degrading peptides released from proteasomes. 2# As stated above both TOP and NLN cleave either one or many sites of the bioactive peptides however in saying that the cleavage sequences vary widely. The site of hydrolysis unusually has no consistency with the amino acid preference at any position relative to this site. Prevalence of basic residues, glycines and prolines has thought to be the only common feature of these two substrates. Many differences occur between peptide cleavage as many peptides are cleaved at the same peptide bond, on the other hand a select few peptides are cleaved at different points. Much of the sequence difference (11%) between TOP and NLN is located at the floor of the channel and the walls. This difference accounts for 23% of the total molecular surface.
Figure 3: This figure show the sequence alignment of human TOP and NLN.
Legend: Sequence differences are highlighted in green. Structural elements of TOP are shown schematically above the TOP sequence. Accessible cysteines in TOP are indicated with red triangles, and a key residue difference that affects an active site loop is indicated by a red star. http://www.jbc.org/content/279/19/20480.full.pdf+html
Substrates in vitro and in vivo
As stated above, TOP cleaves the 13-residue peptide neurotensin at Arg-8-Arg-9, whereas neurolysin cleaves the peptide at between Pro-10 and Tyr-11. This sequence specificity may be due to TOP being strongly electronegative at the open end of the channel at the surface floor, whereas NLN has several basic patches at the corresponding surface area. Neurotension may bind with the two adjacent arginines due to the large acidic patch in TOP. This shifts them towards the open end of the channel thus optimizing the electrostatic interactions. However the opposite occurs in NLN as it has only some basic patches which cause neurotension to shift its binding site toward the closed end of the channel. This minimizes the electrostatic repulsion between the basic residues in the peptide and the enzyme.
TOP has a high distribution throughout the rat in many different subcellular locations and is generally uniformed in cells and tissues throughout the body. (Charles Pineau et al, 1999) It has been observed (Kallol Ray et al., 2004) that at the level of transcription the expression of TOP activity is regulated, however the activity of TOP is also regulated at the level of transcription. TOP has been known to occur in are; pituitary, many peripheral tissues, endocrine tissues, blood, brain cortex, hypothalamus, heart, intestine and lower levels in the spleen, lung, kidney, muscle, adrenal gland, and liver. The highest activity recorded is in the testes with a 3-5 fold higher level than that of the brain. (Charles Pineau et al, 1999) However, NLN distribution may vary in levels depending on the area. NLN is found in many different subcellular locations such as: a high activity in the liver and kidneys and lower levels in the cytosol and membrane fractions of the brain [xvi] . Many of these studies have looked into the distribution of TOP and NLN separately. This is due to the difficulty in developing selective in vivo inhibitors for both TOP and NLN. TOP and NLN have such a high similarity which makes it hard to determine distributions within specific cells and tissues for each of them. The high similarity also makes it hard to determine the function and process they are both involved in within the body. So far, they have found a better understanding of the localisation of TOP and NLN, the distribution of activity, protein and whether they are at a cellular or subcellular distribution.
Cellular Activity - the level of protein expression
TOP mRNA was previously found to be 30-fold higher in rat testis than in other tissues in the body. (Charles Pineau et al, 1999) There's a high volume of proteases found in the testis due to them maintaining the growth, development and repair process; as the testis undergoes restructuring and remodelling events which permanently occur, especially during spermatogenesis. (Charles Pineau et al, 1999) 10 Many different techniques were carried out to determine the exact distribution of TOP activity within the developing and adult gonads of human and rat testis. This was investigated in isolated cells and in situ by; immunohistochemistry (in situ), western and northern blot techniques (isolated cells).
Top's activity increases steadily the older the rat's get with a maximum activity in adulthood (TOP mRNA was not observed before 35 days of age in the testis). (Charles Pineau et al, 1999) Immunohistochemistry showed that TOP can be found in the Leydig cells and throughout the interstitial space. (Charles Pineauet al, 1999) Western blot analysis was used to confirm the immunohistochemistry results showing the distribution of the protein expressed. The Leydig cells displayed a different outcome as it was much lower than originally expected from the immunohistochemical data. (Charles Pineau et al, 1999) TOP was also detected in the post-meiotic germ cells within the seminiferous tubules, with the highest activity recorded during spermiogenesis in elongated spermatids and residual bodies. (Charles Pineau et al, 1999) TOP was not found in serminiferous tubules in immature testes however western blot analysis found protein in 9 day old testes homogenates. (Charles Pineau et al, 1999) In summary this experiment showed that both human and rats had the highest TOP mRNA expression in the post-meiotic germ cells and Leydig cells within the testes during spermiogenesis. Further research is going into the possible involvement of TOP in proteolytic events associated with the cell function of spermiogenesis and Leydig cells.
TOP may be present in many subcellular locations depending on the cell type with reports showing there are two main forms eg secreted and cytosolic forms, although it is not yet fully understood why it achieves certain localizations. The two forms of TOP are very similar if not identical with respect to sensitivity to specific inhibitors, immunological properties, and substrate specificity. TOP's activity can be found in either tissue or cell lines with around 80% of TOP's activity found in a solid form (cytosolic); the minority are either nuclear or associated with membranes. (Crack et al, 1999) Crack et al investigated the association of TOP with the extracellular surface of the AtT-20 cell plasma membrane (tumour derived, mouse pituitary cell line). (Crack et al, 1999) TOP is situated extracellularly either associated with the plasma membrane or located within the extracellular milieu, suggesting that the plasma membrane fraction could be the potential site of TOP action in the AtT-20 cells. (Crack et al, 1999) TOP is situated extracellularly to allow the metabolism of neuropeptides, this was further looked into by carrying out differential centrifugation then an equilibrium density centrifugation with a Percoll gradient. (Crack et al, 1999) The AtT-20 cells were suggested to be the active site for TOP as all the TOP enzymatic activity and immunoreactivity entered the Percoll gradient and was found to all occur with this enriched plasma membrane fraction. (Crack et al, 1999) this study shows that at the cell surface the actual enzymatic site is exposed and active. (Crack et al, 1999)
Cellular and subcellular distribution
The cellular and subcellular distribution of NLN was examined using electron microscope immunogold labelling. (Fontenele-Neto et al, 2001) They carried out a quantitative analysis on cerebral and cerebellar cortices and NTS which indicated various proportions between regions of labelled cells however within the same region, NLN stayed the same. (Fontenele-Neto et al, 2001) thus suggested that NLN's expression may vary due to the regional properties (neuronal activity) and not programmed expression within certain neurons (NLN is expressed by glial cells as well as neurons). (Fontenele-Neto et al, 2001) NLN's immunoreactivity was found throughout the nerve cell bodies, dendrites and in the axons and axon terminals, when located outside the nucleus. (Fontenele-Neto et al, 2001) Within the dendrites and axons a high portion of NLN immunoreactivity was associated with microtubules and neurofilaments. (Fontenele-Neto et al, 2001) Finding NLN within these cytoskeleton elements suggests that NLN is able to be transported to neuronal processes via the cytoskeleton. (Fontenele-Neto et al, 2001) When NLN was within the neuronal perikarya it was mainly observed with the endoplasmic reticulum which is associated with sites of enzyme synthesis. (Fontenele-Neto et al, 2001) NLN was also observed in the external or internal mitochondrial membranes. As mentioned before NLN will either be targeted to the mitochondrial intermembrane space or the cytoplasm due to the several splice varients. Fontenele-Neto et al, also determined NLN to be found in the luminal and cytoplasmic faces of the membrane for example, from the vesicles, TGN or the golgi. However, NLN being targeted to the luminal side of the vesicle is unclear as it lacks a signal peptide. (Fontenele-Neto et al, 2001)Furthermore a fraction of NLN was detected in the neuronal nuclei, which is slightly unexpected as NLN shows a nuclear localisation sequence. (Fontenele-Neto et al, 2001)