The structure of Artemia sp. haemoglobin. Cleavage of the native molecules into functional units by limited subtilisin digestion.
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ABSTRACT:
SUBMITTER: Moens L
PROVIDER: S-EPMC1144373 | biostudies-other | 1984 Nov
REPOSITORIES: biostudies-other
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Project description:BackgroundThe brine shrimp Artemia expresses four different types of haemoglobin subunits namely C1, C2, T1 and T2. Two of these four subunits dimerize in different combinations to produce the three isoforms of the heterodimeric Artemia haemoglobin: HbI (C1 and C2), HbII (C1 and T2) and HbIII (T1 and T2). Previous biochemical, biophysical and computational analyses demonstrate that the T and C polymers are rings of nine concatenated globin domains, which are covalently joined by interdomain linkers. Two such rings stacked coaxially give the functional molecule. This research aimed to construct a quaternary structural model of Artemia HbII that shows the interpolymer interface and domain-domain alignment, using the MS3D (mass spectrometry for three dimensional analysis) approach. This involved introducing chemical crosslinks between the two polymers, cleaving with trypsin and analyzing the resulting products by mass spectrometry. This was followed by computational analysis of the mass spectrometry data using the program SearchXlinks to identify putatively crosslinked peptides.ResultsSix putative EGS (ethylene glycol bis [succinimidylsuccinate]) crosslinked tryptic peptides were identified. All of them support a model in which the EF helices of all domains are in contact along the interpolymer surface, and Domain 1 of the T-polymer aligns with Domain 1 of the C-polymer. Any two adjacent interpolymer domain pairs contact through the early Helix H and early Helix A. The orientation of domains is different from the subunit proposed model proposed previously by this group. Crosslinking with GMBS (N- [gamma-maleimidobutyryloxy]succinimide ester) was also performed, and the results show good agreement with this model.ConclusionThe interpolymer EF-contact allows the hydrophobic E and F helices to be buried in the interface and therefore allow the complex to solubilize readily to facilitate efficient oxygen transport. Furthermore the EF-contact is a common contact in cooperative haemoglobins and thus the model is consistent with the cooperative behaviour of Artemia HbII.
| S-EPMC1865544 | biostudies-literature
Project description:Ozonide antimalarials, OZ277 (arterolane) and OZ439 (artefenomel), are synthetic peroxide-based antimalarials with potent activity against the deadliest malaria parasite, Plasmodium falciparum. Here we used a "multi-omics" workflow, in combination with activity-based protein profiling (ABPP), to demonstrate that peroxide antimalarials initially target the haemoglobin (Hb) digestion pathway to kill malaria parasites. Time-dependent metabolomic profiling of ozonide-treated P. falciparum infected red blood cells revealed a rapid depletion of short Hb-derived peptides followed by subsequent alterations in lipid and nucleotide metabolism, while untargeted peptidomics showed accumulation of longer Hb-derived peptides. Quantitative proteomics and ABPP assays demonstrated that Hb-digesting proteases were increased in abundance and activity following treatment, respectively. Ozonide-induced depletion of short Hb-derived peptides was less extensive in a drug-treated K13-mutant artemisinin resistant parasite line (Cam3.IIR539T) than in the drug-treated isogenic sensitive strain (Cam3.IIrev), further confirming the association between ozonide activity and Hb catabolism. To demonstrate that compromised Hb catabolism may be a primary mechanism involved in ozonide antimalarial activity, we showed that parasites forced to rely solely on Hb digestion for amino acids became hypersensitive to short ozonide exposures. Quantitative proteomics analysis also revealed parasite proteins involved in translation and the ubiquitin-proteasome system were enriched following drug treatment, suggestive of the parasite engaging a stress response to mitigate ozonide-induced damage. Taken together, these data point to a mechanism of action involving initial impairment of Hb catabolism, and indicate that the parasite regulates protein turnover to manage ozonide-induced damage.
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Project description:We report herein a two or three step synthesis of fluorinated π-conjugated oligomers through iterative C-H bond arylations. Palladium-catalyzed desulfitative arylation of heteroarenes allowed in a first step the synthesis of fluoroaryl-heteroarene units in high yields. Then, the next steps involve direct arylation with aryl bromides catalyzed by PdCl(C3H5)(dppb) to afford triad or tetrad heteroaromatic compounds via regioselective activation of C(sp(2))-H bonds.
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Project description:The eukaryotic convertase family plays an important role in posttranslational proteolytic processing and activation of many pro- and polypeptides that have at their cleavage sites the paired basic motif, RX(K/R)R. Recent studies have revealed that the cleavage site of insect pro-vitellogenins (pro-Vg) also contains this motif. To identify and characterize the insect pro-Vg processing enzyme, Vg convertase (VC), its cDNA was cloned from a vitellogenic female fat body cDNA library of the mosquito, Aedes aegypti. The 3735-bp-long VC cDNA has an open reading frame encoding a 115-kDa protein. In vitro transcription/translation of VC cDNA revealed that this 115-kDa protein becomes 140 kDa after co- and posttranslational modifications. The VC deduced amino acid sequence has high similarity to and a domain structure characteristic of furin-like convertases. Northern blot analysis showed that a single 4.2-kb transcript was expressed in the fat body during the first 18 hr of the Vg synthetic period. Coexpression of VC cDNA with mosquito Vg cDNA resulted in correct cleavage of pro-Vg. Thus, this newly identified convertase is, indeed, a functional fat body-specific enzyme for pro-Vg cleavage.
| S-EPMC39211 | biostudies-other
Project description:The X-ray crystallographic structure of the mature form of subtilisin E-S7 (SES7) at 1.90 Å resolution is reported here. Structural comparisons between the previously reported propeptide-subtilisin E complex (1SCJ) and our mature form subtilisin E-S7 (6O44) provide insight into active site adjustments involved in catalysis and specificity. To further investigate the protease substrate selectivity mechanism, we used SES7 to hydrolyze skim milk and analyzed the hydrolysates by LC-MS for peptide identification. The cleavage pattern suggests a high preference for proline at substrate P2 position. The results based on the peptide analysis are consistent with our structural observations, which is instrumental in future protein engineering by rational design. Furthermore, the ACE-inhibitor and NLN-inhibitor activity of the hydrolysates were determined to assess the utility of SES7 for further industrial applications; IC50-ACE = 67 ± 0.92 μg/mL and IC50-NLN = 263 ± 13 μg/mL.
| S-EPMC6541920 | biostudies-literature
Project description:To study enzyme-DNA interactions at single molecular level, both the attachment points and the immediate surroundings of surfaces must be carefully considered such that they do not compromise the structural information and biological properties of the sample under investigation. The present work demonstrates the feasibility of enzymatic digestion of single DNA molecules attached to nanoparticle-modified surfaces. With Nanogold linking DNA to the mica surface by electrostatic interactions, advantageous conditions with fewer effects on the length and topography of DNA are obtained, and an appropriate environment for the activities of DNA is created. We demonstrate that by using Dip-Pen Nanolithography, individual DNA molecules attached to modified mica surfaces can be efficiently digested by DNase I.
| S-EPMC2893842 | biostudies-literature
Project description:The nonenzymatic digestion of proteins by microwave D-cleavage is an effective technique for site-specific cleavage at aspartic acid (D). This specific cleavage C-terminal to D residues leads to inherently large peptides (15-25 amino acids) that are usually relatively highly charged (above +3) when ionized by electrospray ionization (ESI) due to the presence of several basic amino acids within their sequences. It is well-documented that highly charged peptide ions generated by ESI are well-suited for electron transfer dissociation (ETD), which produces c- and z-type fragment ions via gas-phase ion/ion reactions. In this paper, we describe the sequence analysis by ETD tandem mass spectrometry (MS/MS) of multiply charged peptides generated by microwave D-cleavage of several standard proteins. Results from ETD measurements are directly compared to CID MS/MS of the same multiply charged precursor ions. Our results demonstrate that the nonenzymatic microwave D-cleavage technique is a rapid (<6 min) and specific alternative to enzymatic cleavage with Lys-C or Asp-N to produce highly charged peptides that are amenable to informative ETD.
| S-EPMC2707827 | biostudies-literature
Project description:Since Darwin's time, waterbirds have been considered an important vector for the dispersal of continental aquatic invertebrates. Bird movements have facilitated the worldwide invasion of the American brine shrimp Artemia franciscana, transporting cysts (diapausing eggs), and favouring rapid range expansions from introduction sites. Here we address the impact of bird migratory flyways on the population genetic structure and phylogeography of A. franciscana in its native range in the Americas. We examined sequence variation for two mitochondrial gene fragments (COI and 16S for a subset of the data) in a large set of population samples representing the entire native range of A. franciscana. Furthermore, we performed Mantel tests and redundancy analyses (RDA) to test the role of flyways, geography and human introductions on the phylogeography and population genetic structure at a continental scale. A. franciscana mitochondrial DNA was very diverse, with two main clades, largely corresponding to Pacific and Atlantic populations, mirroring American bird flyways. There was a high degree of regional endemism, with populations subdivided into at least 12 divergent, geographically restricted and largely allopatric mitochondrial lineages, and high levels of population structure (? ST of 0.92), indicating low ongoing gene flow. We found evidence of human-mediated introductions in nine out of 39 populations analysed. Once these populations were removed, Mantel tests revealed a strong association between genetic variation and geographic distance (i.e., isolation-by-distance pattern). RDA showed that shared bird flyways explained around 20% of the variance in genetic distance between populations and this was highly significant, once geographic distance was controlled for. The variance explained increased to 30% when the factor human introduction was included in the model. Our findings suggest that bird-mediated transport of brine shrimp propagules does not result in substantial ongoing gene flow; instead, it had a significant historical role on the current species phylogeography, facilitating the colonisation of new aquatic environments as they become available along their main migratory flyways.
| S-EPMC3828609 | biostudies-literature
Project description:The haemocyanin from the freshwater gastropod Lymnaea stagnalis was digested with proteolytic enzymes under conditions where it existed as whole (native) molecules (mol.wt. approx. 9 X 10(6)), or as one-tenth molecules. Digestion of whole molecules yielded a fragment of mol.wt. approx. 110,000 believed to correspond to the 'collar' of the molecule, and an aggregate some 20--30 times the size of the original native molecule formed by end-to-end polymerization of the molecule after removal of the collar. Digestion of one-tenth molecules yielded a mixture of products that could be separated into three fractions by gel filtration. Analysis of these by sodium dodecylsulphate/polyacrylamide-gel electrophoresis revealed that they typically contained two or three components. The collar fragment was present as a component of the intermediate-molecular-weight fraction, and it dissociated on sodium dodecyl sulphate/polyacrylamide gels to give two bands corresponding to apparent mol.wts. 65,000 and 60,000. The c.d. spectra of the separated fractions were recorded and fitted with Gaussian curves by a computer procedure. The fractions each possessed distinct c.d. spectra, by which they could be identified: the collar-fragment c.d. and absorption spectra showed the most striking differences compared with those of the other fragments. The results were interpreted in terms of the postulated existence, within the haemocyanin molecule, of multi-domain structures, each comprising a single polypeptide chain of mol.wt. 200,000--300,000.
| S-EPMC1186668 | biostudies-other