Biogenesis of d-amino acid containing peptides/proteins: where, when and how?

International audiencePeptides and proteins are chiral molecules with their structure determined by the composition and configuration of the amino acids constituting them. Natural amino acids (except glycine) display two chiral types (l- and d-enantiomers). For example, the presence of octopine, a derivative of l-arginine and d-alanine in octopus, or peptidyl poly-d-glutamic acid in a bacterial cell wall was demonstrated in the 1920s and 1930s, respectively. Nevertheless, an old dogma in biology was that proteins (in a strict sense) are composed of amino acids in the l-configuration exclusively, until a d-alanyl residue was reported in a frog skin opioid peptide in the early 1980s, and since, numerous d-amino acid containing peptides (DAACPs) have been discovered in multicellular organisms. Several hypotheses may be formulated to explain the origin of a d-residue in the peptide/protein chain. It may result from different mechanisms such as incorporation of a d-amino acid, non-enzymatic racemisation associated with ageing or diseases and enzymatic posttranslational modification. In the last case, the DAACPs are synthesised via a ribosome-dependent manner, and a normal codon for l-amino acid is present in the mRNA at the position where the d-residue is processed in the mature peptide by peptidyl aminoacyl l-d isomerisation, a peculiar and subtle posttranslational modification. In this review, the different pathways of biogenesis of DAACPs not only in bacteria but also in multicellular organisms are discussed, along with the description of the cellular specificity, the enzyme specificity and the substrate specificity of peptidyl aminoacyl l-d isomerisation

Experimental strategies for the analysis of d-amino acid containing peptides in crustaceans: A review.

International audienceDetection of d-amino acids in natural peptides has been, and remains a challenging task, as peptidyl isomerization is a peculiar and subtle posttranslational modification that does not induce any change in primary sequence or in physicochemical properties of the molecule such as molecular mass or pI. Therefore, the presence of a d-amino acid residue in a peptide chain is generally transparent to classical methods of peptide analysis (electrophoresis, chromatography, mass spectrometry, molecular biology). In this article, we will review the various experimental strategies and analytical techniques, which have been used to characterize and to study d-amino acid containing peptides in crustaceans

Transcriptome and peptidome characterisation of the main neuropeptides and peptidic hormones of a Euphausiid: the ice krill Euphausia crystallorophias

Background The Ice krill, Euphausia crystallorophias is one of the species at the base of the Southern Ocean food chain. Given their significant contribution to the biomass of the Southern Ocean, it is vitally important to gain a better understanding of their physiology and, in particular, anticipate their responses to climate change effects in the warming seas around Antarctica. Methodology/Principal Findings Illumina sequencing was used to produce a transcriptome of the ice krill. Analysis of the assembled contigs via two different methods, produced 36 new pre-pro-peptides, coding for 61 neuropeptides or peptide hormones belonging to the following families: Allatostatins (A, B et C), Bursicon (α and β), Crustacean Hyperglycemic Hormones (CHH and MIH/VIHs), Crustacean Cardioactive Peptide (CCAP), Corazonin, Diuretic Hormones (DH), the Eclosion Hormone (EH), Neuroparsin, Neuropeptide F (NPF), small Neuropeptide F (sNPF), Pigment Dispersing Hormone (PDH), Red Pigment Concentrating Hormone (RPCH) and finally Tachykinin. LC/MS/MS proteomics was also carried out on eyestalk extracts, which are the major site of neuropeptide synthesis in decapod crustaceans. Results confirmed the presence of six neuropeptides and six precursor-related peptides previously identified in the transcriptome analyses. Conclusions This study represents the first comprehensive analysis of neuropeptide hormones in a Eucarida non-decapod Malacostraca, several of which are described for the first time in a non-decapod crustacean. Additionally, there is a potential expansion of PDH and Neuropeptide F family members, which may reflect certain life history traits such as circadian rhythms associated with diurnal migrations and also the confirmation via mass spectrometry of several novel pre-pro-peptides, of unknown function. Knowledge of these essential hormones provides a vital framework for understanding the physiological response of this key Southern Ocean species to climate change and provides a valuable resource for studies into the molecular phylogeny of these organisms and the evolution of neuropeptide hormones

Antarctic krill (Euphausia superba) in a warming ocean: thermotolerance and deciphering Hsp70 responses

International audienceThe Antarctic krill, Euphausia superba, is a Southern Ocean endemic species of proven ecological importance to the region. In the context of predicted global warming, it is particularly important to understand how classic biomarkers of heat stress function in this species. In this respect, Hsp70s are acknowledged as good candidates. However, previous studies of expression kinetics have not been able to demonstrate significant upregulation of these genes in response to heat shocks at 3 °C and 6 °C for 3 and 6 h. The current work complements these previous results and broadens the prospects for the use of Hsp70s as a relevant marker of thermal shock in this krill species. New experiments demonstrate that induction of Hsp70 isoforms was not detected during exposure to heat shock, but increased expression was observed after several hours of recovery. To complete the analysis of the expression kinetics of the different isoforms, experiments were carried out over short time scales (1 and 2 h at 3 °C and 6 °C) as well as at higher temperatures (9 °C, 12 °C, and 15 °C for 3 h), without any significant response. A 6-week monitoring of animals at 3 °C showed that the time factor is decisive in the establishment of the response. CTmax experiments with incremental times of 1 °C per day or 1 °C every 3 days have shown a particularly high resilience of the animals. The demonstration of the abundance of Hsp70s present before thermal stress in various species of krill, as well as in specimens of E. superba of various origins, showed that the delay in the response in expression could be related to the high constitutive levels of Hsp70 available before the stress experiments. The alternative labelling of the two main isoforms of Hsp70 according to the origin of the animals allowed hypotheses to be put forward on the functioning of thermoregulation in Antarctic krill as well as ice krill

Alignment of the protein sequences of the pre-pro-peptides for Corazonin 1 and 2 from <i>E.</i><i>crystallorophias</i> (completes) and <i>E. superba</i> (partial).

<p>The mature peptides are highlighted in blue only for <i>E. crystallorophias.</i> The signal peptides are highlighted in green and the potential bibasic cleavage sites, in red. The PRP highlighted in yellow was characterised by mass spectroscopy.</p

Alphabetical list of peptide precursors, contig expression values and associated Blast matches.

<p>comp ID: sequences assembled with Trinity.<i>contig ID</i> (italic) sequences assembled with Newbler. Size (aa): deduced coding sequences. Size (pb): comp or <i>contig</i> sizes in pair bases. TPM  =  Transcripts Per Million. FPKM  =  Fragments Per Kilobase of exon per Million fragments mapped. Bold values are over the TPM or FPKM averages. Underlined peptides have been characterised by mass.</p

Complete protein sequences of the pre-pro-peptides of the Neuropeptides F1 and 2 from <i>E.</i><i>crystallorophias</i>.

<p>The mature peptides are highlighted in blue only for <i>E. crystallorophias.</i> The signal peptides are highlighted in green and the potential bibasic cleavage sites, in red. The PRP highlighted in yellow was characterised by mass spectroscopy.</p

Complete protein sequences of the pre-pro-peptides of the Pigment Dispersing Hormones (PDH) α and β of <i>E.</i><i>crystallorophias</i> and <i>E. superba</i>.

<p>The mature peptides are highlighted in blue only for <i>E. crystallorophias.</i> The signal peptides are highlighted in green and the potential bibasic cleavage sites, in red. The PRP highlighted in yellow was characterised by mass spectroscopy.</p

Complete protein sequence of the pre-pro-peptide containing the Tachykinins of <i>E. crystallorophias</i>.

<p>The 6 identical examples of Tachykinin are highlighted in blue. The signal peptide is highlighted in green and the potential bibasic cleavage sites, in red. The latter delimit the 4 potential PRPs. The PRP highlighted in yellow was characterised by mass spectrometry.</p

Complete and partial sequences from <i>E.</i><i>crystallorophias</i> of the pre-pro-peptides containing the Corticotrophin Releasing Factor-like Diuretic hormone (CRFLDH), the Crustacean Cardio Active Peptide (CCAP), the Crustacean Hyperglycaemic Hormone (CHH), the Eclosion Hormone (EH), the Red Pigment Dispersing Hormone (RPCH), the Allatostatin Bs and the SIFamide.

<p>The mature peptides are highlighted in blue. The potential bibasic cleavage site is highlighted in red. The CPRP (CHH PRP) is highlighted in yellow.</p