User:Alexander Rudecki/Sandbox 1: Difference between revisions

''Drosophila melanogaster'' glutaminyl cyclase (DromeQC but also known as CG10487 CG32412 or Dmel\CG32412) is a globular protein part of the α/β-hydrolase superfamily. DromeQC is an aminoacyltransferase (EC 2.3.2.5) that acts on N-terminal glutamine or glutamate residues, producing a stable cap resistant to protease degradation. The human orthologue of DromeQC (hQC) has been implicated in stabilizing amyloid Aβ peptides involved in neurodegenerative disorders such as Alzheimers<ref name="schilling">PMID: 18836460</ref>. It has been shown that DromeQC has a similar overall fold to hQC, as well as a conserved active site<ref name="main">PMID: 22897232</ref>. Thus DromeQC is an attractive candidate for transgenic models and mechanistic studies.

 

DromeQC is encoded by chromosome 3L, locus 64F4-64F5 in the ''D. melanogaster'' genome<ref name="genecard">DromeQC Gene Card. NCBI. [http://www.ncbi.nlm.nih.gov/gene?cmd=Retrieve&dopt=Graphics&list_uids=38663]</ref>. It is transcribed into a 1622 nucleotide transcript, containing 5' (36 nucleotides) and 3' (83 nucleotides) untranslated regions <ref name="genecard"/>. The translated protein contains 340 residues corresponding to a M=38,028 Da<ref>DromeQC. UniProt. [http://www.uniprot.org/uniprot/Q9VRQ9]</ref>. It contains a 27 residue signal sequence, suggesting its involvement in the secretory pathway <ref name="schilling"/>.

DromeQC belongs to the α/β-hydrolase fold superfamily; this superfamily exhibits a β-sheet core (5-8 strands) connected to α helices forming an α/β/α sandwhich<ref name=“family”>PMID: 23193256</ref>. As of present, the ESTHER database (ESTerases and α/β-Hydrolase Enzymes and Relatives) contains 168 protein families<ref>Lenfant, N., Hotelier, T., Velluet, E., Bourne, Y., Marchot, P., and A Chatonnet. (2013) ESTHER, the database of the alpha/beta-hydrolase fold superfamily of proteins: tools to explore diversity of functions. Nucleic Acids Research 41: D423-9. [http://bioweb.ensam.inra.fr/ESTHER/general?what=index]</ref>. Most of the proteins in this superfamily function via a conserved catalytic triad - a nucleophile, acid and base - with the residues present on the loops of the active site<ref name="family"/>. Further information on catalysis is found in the 'Catalytic Mechanism' section on this page.  

It is interesting that DromeQC prevents protein degradation from aminopeptidases, yet theses two enzymes share a common fold and active site residues<ref>PMID: 15028118</ref>. This suggests that the enzymes act in a similar manner, with contrasting effects on substrate.  

<Structure load='4F9U' size='350' frame='true' align='right' caption='A 3D ribbon model of DromeQC as determined by X-ray crystallography' scene='Insert optional scene name here'/>

DromeQC is a <scene name='58/580851/Two_chains/2'>homodimer</scene> consisting of an <span style="font-size:150%"><font color='blue'>A Chain</font></span> and a <span style="font-size:150%"><font color='limegreen'>B Chain</font></span>. This homodimer can also be viewed in <scene name='58/580851/Trace/1'>trace</scene> and <scene name='58/580851/Space_fill/1'>space filling</scene> representations. The backbone of DromeQC can be easily seen as an <scene name='58/580851/N_to_c_rainbow/1'>N to C rainbow</scene>. Here the polypeptide progression is depicted by the following template:

Within each subunit is 1 cysteine bond (C113→C136) linking the β3 strand to the α3 helix. These cysteine residues are situated close to the active site, and are conserved in the human orthologue suggesting a pivotal role in catalysis<ref name="schilling"/>. However, when cysteines were replaced with alanines via site-directed mutagenesis, no kinetic differences were observed<ref name="main"/>. In contrast, this mutation did affect structural differences as determined by thermal unfolding experiments<ref name="main"/>. These results correspond to the structural stabilization of this disulfide bond in hQC, and lack of its effect on kinetic activity<ref>Ruiz-Carrillo, D., Koch, B., Parthier, C., Wermann, M., Dambe, T., Buchholz, M., Ludwig, H., Heiser, U., Rahfeld, J., Stubbs, M. T., Schilling, S., and H. Demuth. (2011) Structures of glycosylated mammalian glutaminyl cyclases reveal conformational variability near the active center. Biochemistry. 50: 6280-6288. [http://dx.doi.org/10.1021/bi200249h DOI: 10.1021/bi200249h]</ref>.

 

[[Image:DromeQCActiveSite.png|thumb|300px|left|Figure 3. A comparison between the active sites of Drosophila melanogaster DromeQC crystalized either with a PBD150 inhibitor (right, 4F90) or without (left, 4FWU). Protein loops surrounding the active site are denoted in blue, and a key catalytic Zn²⁺ is shown as a grey sphere, chelated by three residues shown in light blue. The PBD150 inhibitor (red) involve interactions with W296 (yellow), F292 (green), W176 (beige) and D271 (pink).]]

The active site of DromeQC is located on four loops that lack secondary structure (Figure 3). Using these loops as a scaffold, a catalytic zinc ion is chelated via D131 OD2, E171 OE2, and H297 NE2. Thus, under the absence of substrate or inhibitor, Zn²⁺ exhibits trivalency (Figure 3). However, when DromeQC was crystalized in presence of a PBD150 inhibitor, Zn²⁺ was additionally chelated by the PBD150 imidazole moiety<ref name="main"/>. It is plausible that the amide oxygen of glutamine of peptide substrates chelate the zinc ion in a similar fashion, leading to position, polarization, and stabilization for cyclization.

 

Binding ofomeQCActiveSite.png|thumb|700px|center|Figure 3. A comparison between the active sites of Drosophila melanogaster DromeQC crystalized either with a PBD150 inhibitor (right, 4F90) or without (left, 4FWU). Protein loops surrounding the active site are denoted in blue, and a key catalytic Zn²⁺ is shown as a grey sphere, chelated by three residues shown in light blue. The PBD150 inhibitor (red) involve interactions with W296 (yellow), F292 (green), W176 (beige) and D271 (pink).]]

 

[[Image:Cyclase_reaction.png|thumb|500px|right|Figure 4. Cyclization of a terminal glutamine residue via DromeQC.]]

The N-terminus of many proteins (ie gonadotropin releasing hormone and thyrotropin-releasing hormone) contain a pyroglutamic acid (pGlu) residue<ref>PMID: 196172</ref>. A pGlu ‘cap’ protects these proteins against degradation by aminopeptidases, and influences the conformation of the hormone or its associated receptor, leading to their activation<ref name="schilling"/>. Cyclization also leads to decreased basicity in the peptide. Though cyclization of Gln-tRNA to pGlu-tRNA has been shown to occur in papaya latex,<ref>PMID: 4881333</ref> N terminal pGlu formation must be post translational due to an essential methionine that initiates translation in all organisms.   

 

 

 

::-acyltransferase (2.3)

::-aminoacyltransferase (2.3.2)

::-acts on glutaminyl/glutamyl residues (2.3.2.5)

 

The cyclization reaction occurs via a nucleophilic attack of the α-amine on the γ carbon in the glutamine side chain. The enzymatic mechanism for DromeQC is still undetermined, but it seems plausible that it follows that of its human orthologue. In hQC, the N-terminus of the peptide substrate is inserted into the active site pocket, where the γ amide oxygen chelates the catalytic zinc ion<ref name="mechanism">PMID: 18470930</ref>. This ion-dipole interaction causes carbonyl polarization, making it a better electrophile. To facilitate the reaction, a conserved glutamate (Glu201) acts as both a general base and acid. Glu201 abstracts a proton from the α-amino group, causing it to nucleophilically attack the γ amide oxygen. This produces a tetrahedral intermediate with a charged oxygen that is stabilized by Zn²⁺. Glu201 then protonates the γ amide nitrogen, and an amine group is expelled as the carbonyl reforms. Also essential to this mechanism is a conserved aspartate (Asp248) that coordinates/stabilizes the leaving amine group.

 

 

DromeQC is known to localize in the brain and peripheral nerves of ''D. melanogaster''<ref name="schilling"/>. This fact was unveiled by the discovery of adipokinetic hormone; this protein has an N-terminal pGlu, supporting a post translational modification via DromeQC<ref>PMID: 2117437</ref>. Adipokinetic hormone was found to localize in neurone and nerve endings by immunohistochemistry, suggesting that it functions as a locally released modulator in tissues such as heart and skeletal muscle<ref>PMID: 6342796</ref>. Thus, It has been suggested that DromeQC is part of the secretory pathway, being targeted to secretory vesicles where hormone maturation takes place<ref name="schilling"/>. This claim is supported by a 27 residue signal sequence contained at the N-terminus of DromeQC<ref name="schilling"/>. Further support stems from immunohistochemical evidence that DromeQC and its modified substrate are excreted from the cell, where they can be found in the extracellular medium<ref name="schilling"/>.