RESUMO
Plant lipids are important as alternative sources of carbon and energy when sugars or starch are limited. Here, we applied combined heat and darkness or extended darkness to a panel of â¼300 Arabidopsis (Arabidopsis thaliana) accessions to study lipid remodeling under carbon starvation. Natural allelic variation at 3-KETOACYL-COENZYME A SYNTHASE4 (KCS4), a gene encoding an enzyme involved in very long chain fatty acid (VLCFA) synthesis, underlies the differential accumulation of polyunsaturated triacylglycerols (puTAGs) under stress. Ectopic expression of KCS4 in yeast and plants proved that KCS4 is a functional enzyme localized in the endoplasmic reticulum with specificity for C22 and C24 saturated acyl-CoA. Allelic mutants and transient overexpression in planta revealed the differential role of KCS4 alleles in VLCFA synthesis and leaf wax coverage, puTAG accumulation, and biomass. Moreover, the region harboring KCS4 is under high selective pressure and allelic variation at KCS4 correlates with environmental parameters from the locales of Arabidopsis accessions. Our results provide evidence that KCS4 plays a decisive role in the subsequent fate of fatty acids released from chloroplast membrane lipids under carbon starvation. This work sheds light on both plant response mechanisms and the evolutionary events shaping the lipidome under carbon starvation.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Humanos , Arabidopsis/metabolismo , Coenzima A/genética , Coenzima A/metabolismo , Escuridão , Amigos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ácidos Graxos/metabolismo , Triglicerídeos/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
The ongoing research on biomaterials that support bone regeneration led to the quest for materials or material modifications that can actively influence the activity or balance of bone tissue cells. The bone biocompatibility of porous chitosan scaffolds was modified in the present study by the addition of calcium phosphates or hemocyanin. The first strategy comprised the incorporation of calcium phosphates into chitosan to create a biomimetic chitosan-mineral phase composite. The second strategy comprised dip-coating of chitosan scaffolds with hemocyanin extracted from crayfish hemolymph. The cytocompatibility was assessed in a mono-culture of human bone marrow stromal cells (hBMSCs) and their differentiation to osteoblasts; in a mono-culture of human monocytes (hMs) and their maturation to osteoclasts; and in a co-culture of hBMSC/osteoblasts-hM/osteoclasts. Mineral incorporation caused an increase in scaffold bioactivity, as shown by reduced calcium concentration in the cell culture medium, delayed differentiation of hBMSCs, and reduced osteoclastic maturation of hMs in mono-culture. Dip-coating with hemocyanin led to increased proliferation of hBMSCs and equivalent osteoclast maturation in mono-culture, while in co-culture, both an inhibitory effect of mineral incorporation on osteoblastogenesis and stimulatory effects of hemocyanin were observed. It was concluded that highly bioactive scaffolds (containing mineral phases) restrain osteoblast and osteoclast development, while hemocyanin coating significantly supports osteoblastogenesis. These influences on the osteoblasts/osteoclasts activity ratio may support scaffold-driven bone healing in the future.
Assuntos
Fosfatos de Cálcio/química , Quitosana/química , Técnicas de Cocultura/métodos , Hemocianinas/química , Hemocianinas/farmacologia , Osteoblastos/citologia , Osteoclastos/citologia , Células Cultivadas , Durapatita/química , Humanos , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacosRESUMO
Some crustaceans possess exoskeletons that are reinforced with calcium carbonate. In the crayfish Cherax quadricarinatus, the molar tooth, which is part of the mandibular exoskeleton, contains an unusual crystalline enamel-like apatite layer. As this layer resembles vertebrate enamel in composition and function, it offers an interesting example of convergent evolution. Unlike other parts of the crayfish exoskeleton, which is periodically shed and regenerated during the molt cycle, molar mineral deposition takes place during the pre-molt stage. The molar mineral composition transforms continuously from fluorapatite through amorphous calcium phosphate to amorphous calcium carbonate and is mounted on chitin. The process of crayfish molar formation is entirely extracellular and presumably controlled by proteins, lipids, polysaccharides, low-molecular weight molecules and calcium salts. We have identified a novel molar protein termed Cq-M15 from C. quadricarinatus and cloned its transcript from the molar-forming epithelium. Its transcript and differential expression were confirmed by a next-generation sequencing library. The predicted acidic pI of Cq-M15 suggests its possible involvement in mineral arrangement. Cq-M15 is expressed in several exoskeletal tissues at pre-molt and its silencing is lethal. Like other arthropod cuticular proteins, Cq-M15 possesses a chitin-binding Rebers-Riddiford domain, with a recombinant version of the protein found to bind chitin. Cq-M15 was also found to interact with calcium ions in a concentration-dependent manner. This latter property might make Cq-M15 useful for bone and dental regenerative efforts. We suggest that, in the molar tooth, this protein might be involved in calcium phosphate and/or carbonate precipitation.
Assuntos
Exoesqueleto/química , Proteínas de Artrópodes/química , Astacoidea/anatomia & histologia , Quitina/química , Exoesqueleto/metabolismo , Animais , Apatitas/química , Apatitas/metabolismo , Proteínas de Artrópodes/genética , Astacoidea/crescimento & desenvolvimento , Carbonato de Cálcio/química , Carbonato de Cálcio/metabolismo , Fosfatos de Cálcio/química , Fosfatos de Cálcio/metabolismoRESUMO
Across the animal kingdom, the involvement of insulin-like peptide (ILP) signaling in sex-related differentiation processes is attracting increasing attention. Recently, a gender-specific ILP was identified as the androgenic sex hormone in Crustacea. However, moieties modulating the actions of this androgenic insulin-like growth factor were yet to be revealed. Through molecular screening of an androgenic gland (AG) cDNA library prepared from the crayfish Cherax quadricarinatus, we have identified a novel insulin-like growth factor-binding protein (IGFBP) termed Cq-IGFBP. Based on bioinformatics analyses, the deduced Cq-IGFBP was shown to share high sequence homology with IGFBP family members from both invertebrates and vertebrates. The protein also includes a sequence determinant proven crucial for ligand binding, which according to three-dimensional modeling is assigned to the exposed outer surface of the protein. Recombinant Cq-IGFBP (rCq-IGFBP) protein was produced and, using a "pulldown" methodology, was shown to specifically interact with the insulin-like AG hormone of the crayfish (Cq-IAG). Particularly, using both mass spectral analysis and an immunological tool, rCq-IGFBP was shown to bind the Cq-IAG prohormone. Furthermore, a peptide corresponding to residues 23-38 of the Cq-IAG A-chain was found sufficient for in vitro recognition by rCq-IGFBP. Cq-IGFBP is the first IGFBP family member shown to specifically interact with a gender-specific ILP. Unlike their ILP ligands, IGFBPs are highly conserved across evolution, from ancient arthropods, like crustaceans, to humans. Such conservation places ILP signaling at the center of sex-related phenomena in early animal development.
Assuntos
Androgênios/fisiologia , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/fisiologia , Insulina/fisiologia , Sequência de Aminoácidos , Animais , Astacoidea , Sequência de Bases , Primers do DNA , DNA Complementar , Feminino , Masculino , Reação em Cadeia da PolimeraseRESUMO
The SLC20A2 transporter supplies phosphate ions (Pi) for diverse biological functions in vertebrates, yet has not been studied in crustaceans. Unlike vertebrates, whose skeletons are mineralized mainly by calcium phosphate, only minute amounts of Pi are found in the CaCO3-mineralized exoskeletons of invertebrates. In this study, a crustacean SLC20A2 transporter was discovered and Pi transport to exoskeletal elements was studied with respect to the role of Pi in invertebrate exoskeleton biomineralization, revealing an evolutionarily conserved mechanism for Pi transport in both vertebrates and invertebrates. Freshwater crayfish, including the study animal Cherax quadricarinatus, require repeated molt cycles for their growth. During the molt cycle, crayfish form transient exoskeletal mineral storage organs named gastroliths, which mostly contain amorphous calcium carbonate (ACC), an unstable polymorph long-thought to be stabilized by Pi. RNA interference experiments via CqSLC20A2 dsRNA injections reduced Pi content in C. quadricarinatus gastroliths, resulting in increased calcium carbonate (CaCO3) crystallinity and grain size. The discovery of a SLC20A2 transporter in crustaceans and the demonstration that knocking down its mRNA reduced Pi content in exoskeletal elements offers the first direct proof of a long-hypothesized mechanism by which Pi affects CaCO3 biomineralization in the crustacean exoskeleton. This research thus demonstrated the distinct role of Pi as an amorphous mineral polymorph stabilizer in vivo, suggesting further avenues for amorphous biomaterial studies. STATEMENT OF SIGNIFICANCE: ⢠Crustaceans exoskeletons are hardened mainly by CaCO3, with Pi in minute amounts ⢠Pi was hypothesized to stabilize exoskeletal amorphous mineral forms in vivo ⢠For the first time, transport protein for Pi was discovered in crayfish ⢠Transport knock-down resulted in exoskeletal CaCO3 crystallization and reduced Pi.
Assuntos
Biomineralização , Carbonato de Cálcio , Animais , Carbonato de Cálcio/química , Minerais/metabolismo , Astacoidea/química , Astacoidea/metabolismo , Interferência de RNARESUMO
Vitellogenesis, a key process in oviparous animals, is characterized by enhanced synthesis of the lipoprotein vitellogenin, which serves as the major yolk-protein precursor. In most oviparous animals, and specifically in crustaceans, vitellogenin is mainly synthesized in the hepatopancreas, secreted to the hemolymph, and taken up into the ovary by receptor-mediated endocytosis. In the present study, localization of the vitellogenin receptor and its interaction with vitellogenin were investigated in the freshwater prawn Macrobrachium rosenbergii. The receptor was immuno-histochemically localized to the cell periphery and around yolk vesicles. A receptor blot assay revealed that the vitellogenin receptor interacts with most known vitellogenin subunits, the most prominent being the 79 kDa subunit. The receptor was, moreover, able to interact with trypsin-digested vitellogenin peptides. By combining a novel peptide-array approach with tandem mass spectrometry, eleven vitellogenin-derived peptides that interacted with the receptor were identified. A 3D model of vitellogenin indicated that four of the identified peptides are N-terminally localized. One of the peptides is homologous to the receptor-recognized site of vertebrate vitellogenin, and assumes a conserved ß-sheet structure. These findings suggest that this specific ß-sheet region in the vitellogenin N-terminal lipoprotein domain is the receptor-interacting site, with the rest of the protein serving to enhance affinity for the receptor. The conservation of the receptor recognition site in invertebrate and vertebrate vitellogenin might have vast implications for oviparous species reproduction, development, immunity, and pest management.
Assuntos
Peptídeos/química , Vitelogeninas/química , Sequência de Aminoácidos , Animais , Proteínas do Ovo/química , Proteínas do Ovo/metabolismo , Evolução Molecular , Ligantes , Dados de Sequência Molecular , Palaemonidae/metabolismo , Peptídeos/metabolismo , Análise Serial de Proteínas , Estrutura Secundária de Proteína , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Vitelogeninas/metabolismoRESUMO
Gastroliths are transient extracellular calcium deposits formed by the crayfish Cherax quadricarinatus von Martens on both sides of the stomach wall during pre-molt. Gastroliths are made of a rigid chitinous organic matrix, constructed as sclerotized chitin-protein microfibrils within which calcium carbonate is deposited. Although gastroliths share many characteristics with the exoskeleton, they are simpler in structure and relatively homogeneous in composition, making them an excellent cuticle-like model for the study of cuticular proteins. In searching for molt-related proteins involved in gastrolith formation, two integrated approaches were employed, namely the isolation and mass spectrometric analysis of proteins from the gastrolith matrix, and 454-sequencing of mRNAs from both the gastrolith-forming and sub-cuticular epithelia. SDS-PAGE separation of gastrolith proteins revealed a set of bands at apparent molecular masses of 75-85 kDa; mass spectrometry data matched peptide sequences from the deduced amino acid sequences of seven hemocyanin transcripts. This assignment was then examined by immunoblot analysis using anti-hemocyanin antibodies, also used to determine the spatial distribution of the proteins in situ. Apart from contributing to oxygen transport, crustacean hemocyanins were previously suggested to be involved in several aspects of the molt cycle, including hardening of the new post-molt exoskeleton via phenoloxidation. The phenoloxidase activity of gastrolith hemocyanins was demonstrated. It was also noted that hemocyanin transcript expression during pre-molt was specific to the hepatopancreas. Our results thus reflect a set of functionally versatile proteins, expressed in a remote metabolic tissue and dispersed via the hemolymph to perform different roles in various organs and structures.
Assuntos
Astacoidea/enzimologia , Cálcio/metabolismo , Quitina/metabolismo , Hemocianinas/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Estômago/enzimologia , Animais , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hemocianinas/genética , Hemolinfa/metabolismo , Especificidade de Órgãos/genética , Peptídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espectrometria de Massas em TandemRESUMO
Crustacean male sexual differentiation is governed by the androgenic gland (AG) and specifically by the secreted insulin-like AG hormone (IAG), thus far identified in several decapod species including the Australian red claw crayfish Cherax quadricarinatus (termed Cq-IAG). While a few insulin-like AG genes have been identified in crustaceans, other AG-specific genes have not been documented until now. In the present study, we describe the recent identification of a non-IAG AG-specific transcript obtained from the C. quadricarinatus AG cDNA library. This transcript, termed C. quadricarinatus membrane-anchored AG-specific factor (Cq-MAG), was fully sequenced and found to encode a putative product of 189 amino acids including a signal anchoring peptide. Expression of a recombinant GFP fusion protein lacking the signal anchor encoding sequence dramatically affected recombinant protein localization pattern. While the expression of the deleterious fusion protein was observed throughout most of the cell, the native GFP::Cq-MAG fusion protein was observed mainly surrounding the periphery of the nucleus, demonstrating an endoplasmic reticulum (ER)-like localization pattern. Moreover, co-expression of the wild-type Cq-MAG (fused to GFP) and the Cq-IAG hormone revealed that these peptides indeed co-localize. This study is the first to report a protein specifically associated with the insulin-like AG hormone in addition to the finding of another AG-specific transcript in crustaceans. Previous knowledge suggests that insulin/insulin-like factor secretion involves tissue-specific transcripts and membrane-anchored proteins. In this regard, Cq-MAG's tissue specificity, anchoring properties and intracellular co-localization with Cq-IAG suggest that it may play a role in the processing and secretion of this insulin-like AG hormone.
Assuntos
Androgênios/metabolismo , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Astacoidea/crescimento & desenvolvimento , Astacoidea/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Astacoidea/química , Astacoidea/metabolismo , Sequência de Bases , Insulina/metabolismo , Masculino , Dados de Sequência Molecular , Diferenciação Sexual , Transcrição GênicaRESUMO
In Crustacea, an early evolutionary group (â¼50â000 species) inhabiting most ecological niches, sex differentiation is regulated by a male-specific androgenic gland (AG). The identification of AG-specific insulin-like factors (IAGs) and genomic sex markers offers an opportunity for a deeper understanding of the sexual differentiation mechanism in crustaceans and other arthropods. Here, we report, to our knowledge, the first full and functional sex reversal of male freshwater prawns (Macrobrachium rosenbergii) through the silencing of a single IAG-encoding gene. These "neofemales" produced all-male progeny, as proven by sex-specific genomic markers. This finding offers an insight regarding the biology and evolution of sex differentiation regulation, with a novel perspective for the evolution of insulin-like peptides. Our results demonstrate how temporal intervention with a key regulating gene induces a determinative, extreme phenotypic shift. Our results also carry tremendous ecological and commercial implications. Invasive and pest crustacean species represent genuine concerns worldwide without an apparent solution. Such efforts might, therefore, benefit from sexual manipulations, as has been successfully realized with other arthropods. Commercially, such manipulation would be significant in sexually dimorphic cultured species, allowing the use of nonbreeding, monosex populations while dramatically increasing yield and possibly minimizing the invasion of exotic cultured species into the environment.
Assuntos
Inativação Gênica/fisiologia , Hormônios Esteroides Gonadais/genética , Palaemonidae/fisiologia , Diferenciação Sexual/fisiologia , Animais , Feminino , Hormônios Esteroides Gonadais/fisiologia , Masculino , Ovário/embriologia , Palaemonidae/genética , Diferenciação Sexual/genética , Testículo/embriologia , Fatores de TempoRESUMO
In crustaceans, molting is known to be under the control of neuropeptide hormones synthesized and secreted from the eyestalk ganglia. While the role of molt-inhibiting hormone (MIH) in regulating molting has been described in several species using classical methods, an in vivo specific MIH targeted manipulation has not been described yet. In the present study, an MIH cDNA was isolated and sequenced from the eyestalk ganglia of the Australian freshwater red claw crayfish Cherax quadricarinatus (Cq) by 5' and 3' RACE. We analyzed the putative Cq-MIH based on sequence homology, a three dimensional structure model and transcript's tissue specificity. We further examined the involvement of Cq-MIH in the control of molt in the crayfish through RNAi by in vivo injections of Cq-MIH double-stranded RNA, which resulted in, similarly to eyestalk ablation, acceleration of molt cycles. This acceleration was reflected by a significant reduction (up to 32%) in molt interval and an increased rate in molt mineralization index (MMI), which correlated with the induction of ecdysteroid hormones compared to control. Altogether, this study provides a proof of function for the involvement of the Cq-MIH gene in molt regulation in the crayfish.
Assuntos
Astacoidea/fisiologia , Hormônios de Invertebrado/genética , Muda/fisiologia , Animais , Astacoidea/genética , Muda/genética , Interferência de RNA/fisiologiaRESUMO
Gastroliths, the calcium storage organs of crustaceans, consist of chitin-protein-mineral complexes in which the mineral component is stabilized amorphous calcium carbonate. To date, only three proteins, GAP 65, gastrolith matrix protein (GAMP), and orchestin, have been identified in gastroliths. Here, we report a novel protein, GAP 10, isolated from the gastrolith of the crayfish Cherax quadricarinatus and specifically expressed in its gastrolith disc. The encoding gene was cloned by partial sequencing of the protein extracted from the gastrolith matrix. Based on an assembled microarray cDNA chip, GAP 10 transcripts were found to be highly (12-fold) up-regulated in premolt gastrolith disc and significantly down-regulated in the hypodermis at the same molt stage. The deduced protein sequence of GAP 10 lacks chitin-binding domains and does not show homology to known proteins in the GenBank data base. It does, however, have an amino acid composition that has similarity to proteins extracted from invertebrate and ascidian-calcified extracellular matrices. The GAP 10 sequence contains a predicted signal peptide and predicted phosphorylation sites. In addition, the protein is phosphorylated and exhibits calcium-binding ability. Repeated daily injections of GAP 10 double strand RNA to premolt C. quadricarinatus resulted in a prolonged premolt stage and in the development of gastroliths with irregularly rough surfaces. These findings suggest that GAP 10 may be involved in the assembly of the gastrolith chitin-protein-mineral complex, particularly in the deposition of amorphous calcium carbonate.
Assuntos
Astacoidea/metabolismo , Proteínas de Ligação ao Cálcio/biossíntese , Cálcio/metabolismo , Proteínas da Matriz Extracelular/biossíntese , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/fisiologia , Estruturas Animais/metabolismo , Animais , Astacoidea/genética , Sequência de Bases , Proteínas de Ligação ao Cálcio/genética , Clonagem Molecular , Matriz Extracelular/genética , Proteínas da Matriz Extracelular/genética , Perfilação da Expressão Gênica , Masculino , Dados de Sequência Molecular , Muda/fisiologia , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Despite the proclamation of Lowenstam and Weiner that crustaceans are the "champions of mineral mobilization and deposition of the animal kingdom," relatively few proteins from the two main calcification sites in these animals, i.e., the exoskeleton and the transient calcium storage organs, have been identified, sequenced, and their roles elucidated. Here, a 65-kDa protein (GAP 65) from the gastrolith of the crayfish, Cherax quadricarinatus, is fully characterized and its function in the mineralization of amorphous calcium carbonate (ACC) of the extracellular matrix is demonstrated. GAP 65 is a negatively charged glycoprotein that possesses three predicted domains: a chitin-binding domain 2, a low-density lipoprotein receptor class A domain, and a polysaccharide deacetylase domain. Expression of GAP 65 was localized to columnar epithelial cells of the gastrolith disk during premolt. In vivo administration of GAP 65 dsRNA resulted in a significant reduction of GAP 65 transcript levels in the gastrolith disk. Such gene silencing also caused dramatic structural and morphological deformities in the chitinous-ACC extracellular matrix structure. ACC deposited in these gastroliths appeared to be sparsely packed with large elongated cavities compared with the normal gastrolith, where ACC is densely compacted. ACC spherules deposited in these gastroliths are significantly larger than normal. GAP 65, moreover, inhibited calcium carbonate crystallization in vitro and stabilized synthetic ACC. Thus, GAP 65 is the first protein shown to have dual function, involved both in extracellular matrix formation and in mineral deposition during biomineralization.
Assuntos
Carbonato de Cálcio/química , DNA/fisiologia , Matriz Extracelular/metabolismo , Minerais/química , Sequência de Aminoácidos , Animais , Astacoidea , Quitina/química , DNA/genética , Proteínas da Matriz Extracelular/química , Perfilação da Expressão Gênica , Inativação Gênica , Microscopia Eletrônica de Varredura , Modelos Biológicos , Dados de Sequência Molecular , Interferência de RNA , RNA Mensageiro/metabolismoRESUMO
During their life, crustaceans undergo several molts, which if theoretically compared to the human body would be equivalent to replacing all bones at a single event. Such a dramatic repetitive event is coupled to unique molecular mechanisms of mineralization so far mostly unknown. Unlike human bone mineralized with calcium phosphate, the crustacean exoskeleton is mineralized mainly by calcium carbonate. Crustacean growth thus necessitates well-timed mobilization of bicarbonate to specific extracellular sites of biomineralization at distinct molt cycle stages. Here, by looking at the crayfish Cherax quadricarinatus at different molting stages, we suggest that the mechanisms of bicarbonate ion transport for mineralization in crustaceans involve the SLC4 family of transporters and that these proteins play a key role in the tight coupling between molt cycle events and mineral deposition. This discovery of putative bicarbonate transporters in a pancrustacean with functional genomic evidence from genes encoding the SLC4 family-mostly known for their role in pH control-is discussed in the context of the evolution of calcium carbonate biomineralization.
Assuntos
Astacoidea/fisiologia , Biomineralização/genética , Muda/genética , Simportadores de Sódio-Bicarbonato/genética , Animais , Transporte Biológico , Biologia Computacional , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Modelos Biológicos , Fenótipo , Filogenia , Simportadores de Sódio-Bicarbonato/metabolismoRESUMO
Stable amorphous calcium carbonate (ACC) is a unique material produced naturally exclusively as a biomineral. It was demonstrated that proteins extracted from biogenic stable ACC induce and stabilize synthetic ACC in vitro. Polyphosphate molecules were similarly shown to induce amorphous calcium carbonate formation in vitro. Accordingly, we tested the hypothesis that biogenic ACC induction and stabilization is mediated by the phosphorylated residues of phosphoproteins. We show that extracellular organic matrix extracted from gastroliths of the red claw crayfish Cherax quadricarinatus induce stable ACC formation in vitro. The proteinaceous fraction of this organic matrix is highly phosphorylated and is incorporated into the ACC mineral phase during precipitation. We have identified the major phosphoproteins of the organic matrix and showed that they have high calcium binding capacity. Based on the above, in vitro precipitation experiments with single phosphoamino acids were performed, indicating that phosphoserine or phosphothreonine alone can induce the formation of highly stable ACC. The results indicate that phosphoproteins may play a major role in the control of ACC formation and stabilization and that their phosphoamino acid moieties are key components in this process.
Assuntos
Carbonato de Cálcio/metabolismo , Fosfoaminoácidos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Animais , Astacoidea/química , Astacoidea/metabolismo , Carbonato de Cálcio/química , Masculino , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Fosfoaminoácidos/química , Análise Espectral RamanRESUMO
Crustacean chitin-hemocyanin-calcium mineral complexes were designed as bone biomimetics, with emphasis on their ability to bind or release calcium ions. Chitin scaffolds were prepared by dissolving chitin flakes in LiCl/dimethylacetamide, followed by gel formation and freeze-drying. Some of these scaffolds were modified by incorporation of CaCO3 . In some of the chitin-CaCO3 scaffolds, macroporosity was introduced by HCl treatment. Hemocyanin from the crayfish Cherax quadricarinatus was used to further modify the chitin scaffolds by dip coating. Cytocompatibility, cellular adherence and proliferation of human mesenchymal stem cells (hMSCs) were evaluated in terms of cell number as reflected in lactate dehydrogenase activity. The chitin, chitin-CaCO3 , and porous chitin-CaCO3 scaffolds were all found to facilitate cell attachment. Hemocyanin dip-coating of these scaffolds led to increased initial cell adhesion, enhanced proliferation, and osteogenic differentiation. Since the hemocyanin loading of the scaffolds was impaired by sterilization by gamma-irradiation (as required for biomedical applications), the hemocyanin loading was performed on previously sterilized scaffolds. All scaffolds facilitated osteogenic differentiation of osteoblasts, with the highest cell ALP-activity being found on hemocyanin-modified porous chitin-CaCO3 scaffolds. Thus, chitin-hemocyanin scaffolds enhanced the initial stages of bone cell development and could serve as promising biomaterials for bone regeneration.
Assuntos
Astacoidea , Substitutos Ósseos/química , Quitina/química , Hemocianinas/química , Células-Tronco Mesenquimais/citologia , Osteogênese , Animais , Astacoidea/metabolismo , Substitutos Ósseos/farmacologia , Células Cultivadas , Quitina/farmacologia , Hemocianinas/farmacologia , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacosRESUMO
The pancrustacean theory groups crustaceans and hexapods (once thought to comprise separate clades within the Arthropoda) into a single clade. A key feature common to all pancrustaceans is their chitinous exoskeleton, with a major contribution by cuticular proteins. Among these, are the CPAP3's, a family of cuticular proteins, first identified in the hexapod Drosophila melanogaster and characterized by an N-terminal signaling peptide and three chitin-binding domains. In this study, CPAP3 proteins were mined from a transcriptomic library of a decapod crustacean, the crayfish Cherax quadricarinatus. Phylogenetic analysis of other CPAP3 proteins from hexapods and other crustaceans showed a high degree of conservation. Characterization of the crayfish proteins, designated CqCPAP3's, suggested a major role for CPAP3'sin cuticle formation. Loss-of-function experiments using RNAi supported such a notion by demonstrating crucial roles for several CqCPAP3 proteins during molting. A putative mode of action for the CqCPAP3 proteins -theoretically binding three chitin strands- was suggested by the structural data obtained from a representative recombinant CqCPAP3. The similarities between the CqCPAP3 proteins and their hexapod homologues further demonstrated common genetic and proteinaceous features of cuticle formation in pancrustaceans, thereby reinforcing the linkage between these two highly important phylogenetic groups.
Assuntos
Proteínas de Artrópodes/química , Astacoidea/genética , Quitina/química , Insetos/genética , Filogenia , Transcriptoma , Exoesqueleto/química , Exoesqueleto/metabolismo , Animais , Proteínas de Artrópodes/antagonistas & inibidores , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Astacoidea/classificação , Astacoidea/metabolismo , Biomineralização/genética , Quitina/biossíntese , Quitina/genética , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Insetos/classificação , Insetos/metabolismo , Muda , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The phosphorylation profile of ciliary proteins under basal conditions and after stimulation by extracellular ATP was investigated in intact tissue and in isolated cilia from porcine airway epithelium using anti-phosphoserine and anti-phosphothreonine specific antibodies. In intact tissue, several polypeptides were serine phosphorylated in the absence of any treatment (control conditions). After stimulation by extracellular ATP, changes in the phosphorylation pattern were detected on seven ciliary polypeptides. Serine phosphorylation was enhanced for three polypeptides (27, 37, and 44 kD), while serine phosphorylation was reduced for four polypeptides (35, 69, 100, and 130 kD). Raising intracellular Ca2+ with ionomycin induced identical changes in the protein phosphorylation profile. Inhibition of the NO pathway by inhibiting either NO synthase (NOS), guanylyl cyclase (GC), or cGMP-dependent protein kinase (PKG) abolished the changes in phosphorylation induced by ATP. The presence of PKG within the axoneme was demonstrated using a specific antibody. In addition, in isolated permeabilized cilia, submicromolar concentrations of cGMP induced protein phosphorylation. Taken together, these results suggest that the axoneme is an integral part of the intracellular NO pathway. The surprising observation that ciliary activation is accompanied by sustained dephosphorylation of ciliary proteins via NO pathway was not detected in isolated cilia, suggesting that the protein phosphatases were either lost or deactivated during the isolation procedure. This work reveals that any pharmacological manipulation that abolished phosphorylation and dephosphorylation also abolished the enhancement of ciliary beating. Thus, part or all of the phosphorylated polypeptides are likely directly involved in axonemal regulation of ciliary beating.
Assuntos
Cálcio/metabolismo , Movimento Celular/fisiologia , Cílios/fisiologia , Óxido Nítrico/metabolismo , Mucosa Respiratória/fisiologia , Transdução de Sinais/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Células Cultivadas , Cílios/ultraestrutura , GMP Cíclico/metabolismo , Líquido Intracelular/metabolismo , Fosforilação , Mucosa Respiratória/citologia , SuínosRESUMO
Chitin is a major component of arthropod cuticles, where it forms a three-dimensional network that constitutes the scaffold upon which cuticles form. The chitin fibers that form this network are closely associated with specific structural proteins, while the cuticular matrix contains many additional structural, enzymatic and other proteins. We study the crayfish gastrolith as a simple model for the assembly of calcified cuticular structures, with particular focus on the proteins involved in this process. The present study integrates a gastrolith-forming epithelium transcriptomic library with data from mass spectrometry analysis of proteins extracted from the gastrolith matrix to obtain a near-complete picture of gastrolith protein content. Using native protein separation we identified 24 matrix proteins, of which 14 are novel. Further analysis led to discovery of three putative protein complexes, all containing GAP 65 the most abundant gastrolith structural protein. Using immunological methods we further studied the role of GAP 65 in the gastrolith matrix and forming epithelium, as well as in the newly identified protein complexes. We propose that gastrolith matrix construction is a sequential process in which protein complexes are dynamically assembled and disassembled around GAP 65, thus changing their functional properties to perform each step in the construction process. BIOLOGICAL SIGNIFICANCE: The scientific interest on which this study is based arises from three main features of gastroliths: (1) Gastroliths possess partial analogy to cuticles both in structural and molecular properties, and may be regarded, with the appropriate reservations (see Introduction), as simple models for cuticle assembly. At the same time, gastroliths are terminally assembled during a well-defined period, which can be controlled in the laboratory, making them significantly easier to study than cuticles. (2) Gastroliths, like the crayfish exoskeleton, contain stable amorphous calcium carbonate (ACC) rather than crystalline calcite. The biological mechanism for the stabilization of a naturally unstable, but at the same time biologically highly available, calcium carbonate polymorph is of great interest from the pharmaceutical point of view. (3) The gastrolith organic matrix is based on a highly structured chitin network that interacts with a variety of substances. This biologically manipulated, biodegradable structure is in itself of biotechnological and pharmaceutical potential. A growing body of evidence indicates that proteins play central roles in all above aspects of gastrolith construction. This study offers the first comprehensive screening of gastrolith proteins, and we believe that the analysis presented in this work can not only help reveal basic biological questions regarding assembly of mineralized and non-mineralized cuticular structures, but may also serve as basis for applied research in the fields of agriculture (e.g. cuticle-based pest management), health (e.g. bioavailable calcium supplements and biodegradable drug carriers) and materials science (e.g. non-toxic scaffolds for water purification).
Assuntos
Astacoidea/metabolismo , Quitina/metabolismo , Conexinas/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Proteoma/metabolismo , AnimaisRESUMO
The molar tooth of the crayfish Cherax quadricarinatus is part of the mandible, and is covered by a layer of apatite (calcium phosphate). This tooth sheds and is regenerated during each molting cycle together with the rest of the exoskeleton. We discovered that molar calcification occurs at the pre-molt stage, unlike calcification of the rest of the new exoskeleton. We further identified a novel molar protein from C. quadricarinatus and cloned its transcript from the molar-forming epithelium. We termed this protein Cq-M13. The temporal level of transcription of Cq-M13 in an NGS library of molar-forming epithelium at different molt stages coincides with the assembly and mineralization pattern of the molar tooth. The predicted protein was found to be related to the pro-resilin family of cuticular proteins. Functionally, in vivo silencing of the transcript caused molt cycle delay and a recombinant version of the protein was found to bind chitin and exhibited elastic properties.
Assuntos
Proteínas de Artrópodes/metabolismo , Astacoidea/metabolismo , Quitina/metabolismo , Muda/fisiologia , Dente/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/genética , Astacoidea/crescimento & desenvolvimento , Clonagem Molecular , Elasticidade , Epitélio/metabolismo , Expressão Gênica , Dados de Sequência Molecular , Muda/genética , Filogenia , Ligação Proteica , Dente/crescimento & desenvolvimento , Calcificação de Dente/genética , Calcificação de Dente/fisiologiaRESUMO
Like many metazoans, the freshwater prawn Macrobrachium rosenbergii begins its post-embryonic life with a set of morphologically distinct planktonic larval stages, followed by a benthic post-larval stage during which the maturing organism differs from the larvae both ecologically and physiologically. Understanding of the molecular basis underlying morphogenesis in crustaceans is limited to the observation that methyl farnesoate, the non-epoxidated form of the insect juvenile hormone, acts as the active crustacean juvenoid. Molt steroids were also linked to morphogenesis and several other molecular pathways, such as Hedgehog and Wnt, are known to underlie morphogenesis in all metazoans examined and, as such, are thought to do the same in crustaceans. Using next generation sequencing, we deep-sequenced the transcriptomes of several larval and post-larval stages. De novo assembly, followed by bioinformatics analysis, revealed that many novel transcripts are over-expressed in either larvae- or post-larvae-stage prawn, shedding light on the molecular basis underlying M. rosenbergii metamorphosis. Fast larval molting rates and periodic morphological changes were reflected in over-expression of transcripts annotated to the cell cycle, DNA replication and morphogenic pathways (i.e., Hedgehog and Wnt). Further characterization of transcripts assigned to morphogenic pathways by real-time RT-PCR reconfirmed their over-expression in larvae, albeit with a more complex expression pattern when examined in the individual developmental stages. The expression level of an orthologue of cytochrome P450, 15A1, known to epoxidize methyl farnesoate in insects, was increased in the late larval and early post-larval stages, in accordance with the role of methyl farnesoate in crustacean metamorphosis. This study exemplifies the applicability of a high-throughput sequencing approach for studying complex traits, including metamorphosis, providing new insight into this unexplored area of crustacean research.