ABSTRACT
[This corrects the article DOI: 10.1021/acsomega.0c02813.].
ABSTRACT
Background: Perinatal depression (PND) is often under-treated and under-recognized. It has a negative impact on infant development and mother-child interactions. This study aims to estimate the prevalence of PND during pregnancy and in the postpartum period and the effect of sociodemographic factors, psychosocial stressors, and obstetric and neonatal factors on PND. Methods: 166 antenatal mothers attending tertiary center, who completed the 1st-trimester, were evaluated on baseline sociodemographic, psychosocial, obstetric, neonatal, and post neonatal factors by using a semi-structured questionnaire. Periodic prospective assessments were done using Hamilton depression rating scale (HAMD) at the end of the second and third trimesters and first and sixth weeks of the postpartum period. Results: Prevalence of PND was 21.7%, 32.2%, 35%, 30.4%, and 30.6%, at the end of the first trimester, during second, and third trimesters, and first and sixth week postpartum, respectively. Factors significantly associated with depressive symptoms included history of previous children with illness (P: 0.013, OR-5.16, CI-1.3-19.5) and preterm birth (P: 0.037, OR-3.73, CI-1.1- 13.2) at the time of recruitment; history of abuse (P: 0.044, OR-3.26, CI-1.1-10.8) and marital conflicts (P: 0.003, OR-3.2, CI-1.4-6.9) by the end of second trimester; history of miscarriages (P: 0.012, OR-2.58, CI-1.2-5.4) by the end of third trimester; lower SES (P: 0.001, OR-3.48, CI-1.64-7.37), unsatisfied living conditions (P: 0.004, OR-2.9, CI-1.4-6.04), alcohol use in husband (P: 0.049, OR-2.01, CI-1.1-4.11), history of depressive episodes (P: 0.049, OR-2.09, CI-1.1-4.46), history of high-risk pregnancy (P: 0.008, OR-2.7, CI-1.29-5.64), history of miscarriages (P: 0.049, OR-2.04, CI-1.1-4.2), stressful events in the postpartum period (P: 0.043, OR-2.58, CI-1.01-6.59), IUD (P: 0.002), preterm birth (P: 0.001), congenital malformations (P: 0.001), dissatisfaction with the sex of the child (P: 0.005, OR-3.75, CI-1.42-9.91), poor family support (P: 0.001), and low birth weight (P: 0.001, OR-16.78, CI-6.32-44.53) in the postpartum period. These analyses are purely exploratory. Conclusions: PND is highly prevalent from the early antenatal period onwards; this warrants periodic assessment of depression among high-risk mothers, using a validated tool, for early diagnosis and management.
ABSTRACT
Meteorin (Metrn) and Meteorin-like (Metrnl) are homologous secreted proteins involved in neural development and metabolic regulation. In this study, we have performed de novo structure prediction and analysis of both Metrn and Metrnl using Alphafold2 (AF2) and RoseTTAfold (RF). Based on the domain and structural homology analysis of the predicted structures, we have identified that these proteins are composed of two functional domains, a CUB domain and an NTR domain, connected by a hinge/loop region. We have identified the receptor binding regions of Metrn and Metrnl using the machine-learning tools ScanNet and Masif. These were further validated by docking Metrnl with its reported KIT receptor, thus establishing the role of each domain in the receptor interaction. Also, we have studied the effect of non-synonymous SNPs on the structure and function of these proteins using an array of bioinformatics tools and selected 16 missense variants in Metrn and 10 in Metrnl that can affect the protein stability. This is the first study to comprehensively characterize the functional domains of Metrn and Metrnl at their structural level and identify the functional domains, and protein binding regions. This study also highlights the interaction mechanism of the KIT receptor and Metrnl. The predicted deleterious SNPs will allow further understanding of the role of these variants in modulating the plasma levels of these proteins in disease conditions such as diabetes.Communicated by Ramaswamy H. Sarma.
ABSTRACT
Homologs of the protein Get3 have been identified in all domains yet remain to be fully characterized. In the eukaryotic cytoplasm, Get3 delivers tail-anchored (TA) integral membrane proteins, defined by a single transmembrane helix at their C terminus, to the endoplasmic reticulum. While most eukaryotes have a single Get3 gene, plants are notable for having multiple Get3 paralogs. Get3d is conserved across land plants and photosynthetic bacteria and includes a distinctive C-terminal α-crystallin domain. After tracing the evolutionary origin of Get3d, we solve the Arabidopsis thaliana Get3d crystal structure, identify its localization to the chloroplast, and provide evidence for a role in TA protein binding. The structure is identical to that of a cyanobacterial Get3 homolog, which is further refined here. Distinct features of Get3d include an incomplete active site, a "closed" conformation in the apo-state, and a hydrophobic chamber. Both homologs have ATPase activity and are capable of binding TA proteins, supporting a potential role in TA protein targeting. Get3d is first found with the development of photosynthesis and conserved across 1.2 billion years into the chloroplasts of higher plants across the evolution of photosynthesis suggesting a role in the homeostasis of photosynthetic machinery.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Photosynthesis , Adenosine Triphosphatases/metabolism , Embryophyta , Endoplasmic Reticulum/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolismABSTRACT
Autosomal recessive congenital ichthyosis (ARCI) is a diverse group of cornification diseases associated with severe clinical complications and decreased quality of life. Germline mutations in the TGM1 gene, which encodes the enzyme TGM1, are the predominant cause of ARCI. These TGM1 mutations trigger the abnormal epidermal differentiation and impaired cutaneous barrier function observed in patients with ARCI. Unfortunately, current ARCI therapies focus solely on symptomatic relief. Thus, there is a significant unmet need for therapeutic strategies aimed at correcting the TGM1 deficiency underlying ARCI. In this study, we investigated the ability of KB105, a gene therapy vector encoding full-length human TGM1, to deliver functional human TGM1 to keratinocytes. In vitro, KB105 efficiently infected TGM1-deficient human keratinocytes, produced TGM1 protein, and rescued transglutaminase enzyme function. In vivo studies demonstrated that both single and repeated topical KB105 administration induced TGM1 protein expression in the target epidermal layer without triggering fibrosis, necrosis, or acute inflammation. Toxicity and biodistribution assessments on repeat dosing indicated that KB105 was well-tolerated and restricted to the dose site. Overall, our results demonstrate that rescuing TGM1 deficiency in patients with ARCI through topical KB105 application represents a promising strategy for safely and noninvasively treating this debilitating disease.
Subject(s)
Genetic Vectors/administration & dosage , Herpesvirus 1, Human/genetics , Ichthyosis, Lamellar/therapy , Transglutaminases/genetics , Animals , Biopsy , Cells, Cultured , Enzyme Assays , Female , Genetic Therapy/methods , Genetic Vectors/genetics , Germ-Line Mutation , Humans , Ichthyosis, Lamellar/genetics , Ichthyosis, Lamellar/pathology , Keratinocytes , Male , Mice , Models, Animal , Primary Cell Culture , Quality of Life , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Skin/enzymology , Skin/pathology , Transglutaminases/metabolismABSTRACT
Penicillin V acylase (PVA, EC 3.5.1.11) hydrolyzes the side chain of phenoxymethylpenicillin (Pen V) and finds application in the manufacture of the pharmaceutical intermediate 6-aminopenicillanic acid (6-APA). Here, we report the scale-up of cultivation of Escherichia coli whole cells expressing a highly active PVA from Pectobacterium atrosepticum and their encapsulation in polyvinyl alcohol-poly(ethylene glycol) Lentikats hydrogels. A biocatalytic process for the hydrolysis of 2% (w/v) Pen V was set up in a 2 L reactor using the Lentikats-immobilized whole cells, with a customized setup to enable continuous downstream processing of the reaction products. The biocatalytic reaction afforded complete conversion of Pen V for 10 reaction cycles, with an overall 90% conversion up to 50 cycles. The bioprocess was further scaled up to the pilot-scale at 10 L, enabling complete conversion of Pen V to 6-APA for 10 cycles. The 6-APA and phenoxy acetic acid products were recovered from downstream processing with isolated yields of 85-90 and 87-92%, respectively. Immobilization in Lentikats beads improved the stability of the whole cells on storage, maintaining 90-100% activity and similar conversion efficiency after 3 months at 4 °C. The robust PVA biocatalyst can be employed in a continuous process to provide a sustainable route for bulk 6-APA production from Pen V.
ABSTRACT
Sugar will eventually be exported transporters (SWEETs), a novel family of sugar transporters found in both eukaryotes and prokaryotes, facilitate sugar flux across the cell membrane. Although these transporters were first discovered in plants, their homologs have been reported in different organisms. SWEETs have critical roles in various developmental processes, including phloem loading, nectar secretion, and pathogen nutrition. The structure of bacterial homologs, called SemiSWEETs, has been well studied thus far. Here, we provide an overview of SWEET protein structure and dynamic function by analyzing the solved crystal structures and predicted models that are available for a few SWEETs in a monocot plant (rice) and dicot plant (Arabidopsis thaliana). Despite the advancement in structure-related studies, the regulation of SWEETs remains unknown. In light of reported regulatory mechanisms of a few other sugar transporters, we propose the regulation of SWEETs at the post-translational level. We then enumerate the potential post-translational modification sites in SWEETs using computational tools. Overall, in this review, we critically analyze SWEET protein structure in plants to predict the post-translational regulation of SWEETs. Such findings have a direct bearing on plant nutrition and defense and targeting the regulation at these levels will be important in crop improvement.
Subject(s)
Arabidopsis , Membrane Transport Proteins , Oryza , Plant Proteins , Sugars/metabolism , Arabidopsis/metabolism , Biological Transport , Gene Expression Regulation, Plant , Membrane Transport Proteins/metabolism , Oryza/metabolism , Plant Proteins/metabolismABSTRACT
The prevalence of substrate cross-reactivity between AHL acylases and ß-lactam acylases provides a glimpse of probable links between quorum sensing and antibiotic resistance in bacteria. Both these enzyme classes belong to the N-terminal nucleophile (Ntn)-hydrolase superfamily. Penicillin V acylases alongside bile salt hydrolases constitute the cholylglycine hydrolase (CGH) group of the Ntn-hydrolase superfamily. Here we report the ability of two acylases, Slac1 and Slac2, from the marine bacterium Shewanella loihica-PV4 to hydrolyze AHLs. Three-dimensional structure of Slac1reveals the conservation of the Ntn hydrolase fold and CGH active site, making it a unique CGH exclusively active on AHLs. Slac1homologs phylogenetically cluster separate from reported CGHs and AHL acylases, thereby representing a functionally distinct sub-class of CGH that might have evolved as an adaptation to the marine environment. We hypothesize that Slac1 could provide the structural framework for understanding this subclass, and further our understanding of the evolutionary link between AHL acylases and ß-lactam acylases.
Subject(s)
Acyl-Butyrolactones/chemistry , Acyl-Butyrolactones/metabolism , Amidohydrolases/chemistry , Amidohydrolases/metabolism , Shewanella/enzymology , Amidohydrolases/genetics , Bile Acids and Salts/metabolism , Catalytic Domain , Enzyme Assays , Models, Molecular , Phylogeny , Protein Structure, Quaternary , Sequence Alignment , Shewanella/genetics , Substrate Specificity , beta-Lactams/metabolismABSTRACT
Enzymes are important tools for various applications. We have studied structural transitions and functional stability of a Kunitz trypsin inhibitor from Chickpea (CaTI2), a potent insect gut-protease inhibitor, under different stress conditions like non-neutral pH, elevated temperature and co-solvent concentrations. CaTI2 was cloned and expressed in an eukaryotic system P. pastoris and was investigated for conformational transitions using circular dichroism spectroscopy, differential scanning fluorimetry and activity assay. Native CaTI2 has a sheet dominant structure with 40% ß sheets and possess a single tryptophan residue situated in the hydrophobic core of the enzyme. The recombinant inhibitor maintained its maximum activity under alkaline pH with its secondary structure intact between pH 6-10. CaTI2 was observed to be thermally stable up to 55 °C with a Tm of 61.3 °C above which the protein unfolds. On treating with chemical denaturant (urea), the CaTI2 lost its inhibitory potential and native conformation beyond 2 M urea concentration. Moreover, the protein unfolded at lower temperatures as the concentration of denaturant increased, suggesting more complex structural changes. Further, the stability of the inhibitor was found to be directly proportional to the solvent polarity. The data, herein offers significant information of inhibitor stability and activity which could be exploited for its further development into an effective pesticide.
Subject(s)
Cicer/chemistry , Plant Proteins/chemistry , Trypsin Inhibitors/chemistry , Cicer/genetics , Circular Dichroism , Hydrophobic and Hydrophilic Interactions , Plant Proteins/biosynthesis , Plant Proteins/genetics , Protein Structure, Secondary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
Tau aggregation and accumulation is a key event in the pathogenesis of Alzheimer's disease. Inhibition of Tau aggregation is therefore a potential therapeutic strategy to ameliorate the disease. Phytochemicals are being highlighted as potential aggregation inhibitors. Epigallocatechin-3-gallate (EGCG) is an active phytochemical of green tea that has shown its potency against various diseases including aggregation inhibition of repeat Tau. The potency of EGCG in altering the PHF assembly of full-length human Tau has not been fully explored. By various biophysical and biochemical analyses like ThS fluorescence assay, MALDI-TOF analysis and Isothermal Titration Calorimetry, we demonstrate dual effect of EGCG on aggregation inhibition and disassembly of full-length Tau and their binding affinity. The IC50 for Tau aggregation by EGCG was found to be 64.2 µM.
Subject(s)
Catechin/analogs & derivatives , tau Proteins/metabolism , Catechin/chemistry , Catechin/pharmacology , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Molecular Docking Simulation , Molecular Dynamics Simulation , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Thermodynamics , tau Proteins/chemistryABSTRACT
One of the unique characteristic features of the domain archaea, are the lipids that form the hydrophobic core of their cell membrane. These membrane lipids are characterized by distinctive isoprenoid biochemistry and the building blocks are two core lipid structures, sn-2,3- diphytanyl glycerol diether (archaeol) and sn-2,3-dibiphytanyl diglycerol tetraether (caldarchaeol). Archaeol has two phytanyl chains (C20) in a bilayer structure connected to the glycerol moiety by an ether bond. The enzyme involved in this bilayer formation is Di-O-Geranylgeranyl Glyceryl Phosphate Synthase (DGGGPS), which is a member of a very versatile superfamily of enzymes known as UbiA superfamily. Multiple sequence analysis of the typical members of the UbiA superfamily indicates that the majority of conserved residues are located around the central cavity of these enzymes. Interestingly few of these conserved residues in the human homologs are centrally implicated in several human diseases, on basis of the major mutations reported against these diseases in the earlier clinical studies. It remains to be investigated about the role of these conserved residues in the biochemistry of these enzymes. The binding and active site of these enzymes found to be similar architecture but have different substrate affinities ranging from aromatic to linear compounds. So further investigation of UbiA superfamily may be translated to novel therapeutic and diagnostic application of these proteins in human disease management.
Subject(s)
Alkyl and Aryl Transferases , Archaeal Proteins , Cardiovascular Diseases , Membrane Lipids , Archaea/enzymology , Archaea/genetics , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Cardiovascular Diseases/enzymology , Cardiovascular Diseases/genetics , Glyceryl Ethers/metabolism , Humans , Membrane Lipids/biosynthesis , Membrane Lipids/geneticsABSTRACT
BACKGROUND: Insight is awareness of one's symptoms, illness, and need for treatment. In bipolar disorder, insight is better in bipolar depression and nonpsychotic depression when compared with mania and psychotic depression. Insight impacts on medication adherence. In our study, we measured and compared with various clinical parameters of insight. The aim of this study is to assess various dimensions of insight recovery prospectively in bipolar affective disorder patients with treatment and drug compliance. MATERIALS AND METHODS: Patient's insight was assessed using Mood Disorder Insight Scale (MDIS) at baseline, 1st, 3rd, and 6th months. Their insight was then compared with various sociodemographic profiles and correlated with number of mood episodes, family history of mental illness, and 6-month MDIS scores. RESULTS: Depression patients scored better in insight components (P = 0.001). The good compliance group attributed their symptoms to their illness than the poor compliance group (P = 0.013). The MDIS scores were gradually improving from baseline to 6 months (P ≤ 0.001). There was no relationship between insight and the number of episodes (P = 0.788). CONCLUSION: Depressive episode patients had better insight during the baseline, which improved during 6 months follow-up compared with manic patients. Among various components of insight, insight on the attribution of symptoms was a predictor of good compliance. Progression of insight was steady and proportionate to the duration of treatment in depressive episode patients.
ABSTRACT
Alzheimer's disease (AD) is a neurodegenerative disorder caused by protein misfolding, aggregation and accumulation in the brain. A large number of molecules are being screened against these pathogenic proteins but the focus for therapeutics is shifting towards the natural compounds as aggregation inhibitors, mainly due to their minimum adverse effects. Baicalein is a natural compound belonging to the class of flavonoids isolated from the Chinese herb Scutellaria baicalensis. Here we applied fluorescence, absorbance, microscopy, MALDI-TOF spectrophotometry and other biochemical techniques to investigate the interaction between Tau and Baicalein in vitro. We found the aggregation inhibitory properties of Baicalein for the repeat Tau. Overall, the potential of Baicalein in dissolving the preformed Tau oligomers as well as mature fibrils can be of utmost importance in therapeutics for Alzheimer's disease.
Subject(s)
Alzheimer Disease/metabolism , Flavanones/pharmacology , tau Proteins/metabolism , Cell Line, Tumor , Humans , Molecular Docking SimulationABSTRACT
A generalized method for sorting nanoparticles based on their cores does not exist; it is an immediate necessity, and an approach incorporating cost-effectiveness and biocompatibility is in demand. Therefore, an efficient method for the separation of various mixed core-compositions or dissimilar metallic nanoparticles to their pure forms at the nano-bio interface was developed. Various simple core-combinations of monodispersed nanoparticles with dual cores, including silver plus gold, iron oxide plus gold and platinum plus gold, to the complex three-set core-combinations of platinum plus gold plus silver and platinum plus iron plus gold were sorted using step-gradient centrifugation in a sucrose suspension. Viscosity mediated differential terminal velocities of the nanoparticles permitted diversified dragging at different gradients allowing separation. Stability, purity and properties of the nanoparticles during separation were evaluated based on visual confirmation and by employing advanced instrumentations. Moreover, theoretical studies validated our experimental observations, revealing the roles of various parameters, such as the viscosity of sucrose, the density of the particles and the velocity and duration of centrifugation, involved during the separation process. This remarkably rapid, cost-efficient and sustainable strategy can be adapted to separate other cores of nanoparticles for various biomedical research purposes, primarily to understand nanoparticle induced toxicity and particle fate and transformations in natural biotic environments.
ABSTRACT
The Lipid A component of the outer membrane of Gram-negative bacteria is an integral part of the permeability barrier known as LPS, which actively prevents the uptake of bactericidal compounds. It is clinically very significant, as it is known to elicit a strong immune response in the humans, through the TLR4 complex. The Lipid A species are synthesized through a highly conserved multistep biosynthetic pathway. The final step is catalyzed by acyltransferases of the HtrB/MsbB family, which are members of a superfamily of enzymes, present in all domains of life with important roles to play in various biological processes. The investigation of a putative dual functioning enzyme which can add both laurate and myristate residues to the (Kdo)2-lipid IVA (precursor of Lipid A) would give a snapshot into the versatility of substrates that these enzymes catalyze. In this study we have cloned and purified to homogeneity, such a putative dual functional acyltransferase from Chlorobium tepidum, and attempted to study the enzyme in more details in terms of its sequence and structural aspects, as it lacks conserved residues with other enzymes of the same family.
Subject(s)
Acyltransferases/chemistry , Bacterial Proteins/chemistry , Cell Membrane/enzymology , Chlorobium/enzymology , Acyltransferases/genetics , Acyltransferases/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Membrane/chemistry , Cell Membrane/genetics , Cell Membrane/metabolism , Chlorobium/chemistry , Chlorobium/genetics , Chlorobium/metabolism , Glycolipids/metabolism , Hydrophobic and Hydrophilic Interactions , Lipid A/analogs & derivatives , Lipid A/metabolism , Phylogeny , Sequence AlignmentABSTRACT
The twin-arginine translocase (Tat) pathway transports folded proteins across the plasma membrane and plays a critical role in protein transport in haloarchaea. Computational analysis and previous experimental evidence suggested that the Tat pathway transports almost the entire secretome in haloarchaea. The TatC, receptor component of this pathway shows greater variation in membrane topology in haloarchaea than in other organisms. The presence of a unique fourteen-transmembrane TatC homolog (TatCt) in haloarchaea, over and above the expected TatC topological variants, indicates a strong correlation between the additional homologs and the large number of substrates transported via the haloarchaeal Tat pathway. Various combinations of TatC homologs with different topologies-TatCo, TatCt, TatCn, and TatCx have been observed in haloarchaea. In this report, on the basis of these combinations we have segregated all haloarchaeal Tat substrates into two groups. The first group consists of substrates that are transported by TatCt alone, whereas the second group consists of substrates that are transported by the other TatC homologs (TatCo, TatCn, and TatCx). The various haloarchaea TatA components also shows the possible segregation towards the substrates. We have also identified the possible homologs for Tat substrate chaperones, which act as a quality-control mechanism for proper protein folding. Further sequence analysis implies that the two TatC domains of TatCt complement each other's functionally. Substrate analysis also revealed subtle differences between the substrates being transported by various homologs: further experimental analysis is therefore required for better understanding of the complexities of the haloarchaeal Tat pathway.
ABSTRACT
Kunitz-type trypsin inhibitors bind to the active pocket of trypsin causing its inhibition. Plant Kunitz-type inhibitors are thought to be important in defense, especially against insect pests. From sequence analysis of various Kunitz-type inhibitors from plants, we identified CaTI2 from chickpea as a unique variant lacking the functionally important arginine residue corresponding to the soybean trypsin inhibitor (STI) and having a distinct and unique inhibitory loop organization. To further explore the implications of these sequence variations, we obtained the crystal structure of recombinant CaTI2 at 2.8Å resolution. It is evident from the structure that the variations in the inhibitory loop facilitates non-substrate like binding of CaTI2 to trypsin, while the canonical inhibitor STI binds to trypsin in substrate like manner. Our results establish the unique mechanism of trypsin inhibition by CaTI2, which warrant further research into its substrate spectrum. Abbreviations BApNA Nα-Benzoyl-L-arginine 4-nitroanilide BPT bovine pancreatic trypsin CaTI2 Cicer arietinum L trypsin inhibitor 2 DrTI Delonix regia Trypsin inhibitor EcTI Enterolobium contortisiliquum trypsin inhibitor ETI Erythrina caffra trypsin inhibitor KTI Kunitz type inhibitor STI soybean trypsin inhibitor TKI Tamarindus indica Kunitz inhibitor Communicated By Ramaswamy H. Sarma.
Subject(s)
Cicer/chemistry , Models, Molecular , Plant Extracts/chemistry , Trypsin Inhibitor, Kunitz Soybean/chemistry , Trypsin Inhibitors/chemistry , Trypsin/chemistry , Amino Acid Sequence , Amino Acids , Animals , Binding Sites , Catalytic Domain , Cattle , Crystallography, X-Ray , Enzyme Activation , Kinetics , Molecular Docking Simulation , Molecular Dynamics Simulation , Plant Extracts/pharmacology , Protein Binding , Protein Conformation , Recombinant Proteins , Spectrum Analysis , Structure-Activity Relationship , Trypsin Inhibitor, Kunitz Soybean/pharmacology , Trypsin Inhibitors/pharmacologyABSTRACT
Acid ceramidase (N-acylsphingosine deacylase EC 3.5.1.23; AC) catalyzes the hydrolysis of ceramide into sphingosine (SPH) and free fatty acid. Zebrafish acid ceramidase (AC) has 60% homology with the human AC). Mutations in the human AC gene asah1 are known to cause Farber disease and spinal muscular atrophy with progressive myoclonic epilepsy. Zebrafish AC was overexpressed in Pichia pastoris by inserting asah1b gene into the genome. The majority of the overexpressed enzyme was secreted into the culture medium and purified to apparent homogeneity by stepwise chromatography. The recombinant protein was glycosylated precursor, that further undergoes limited autoproteolytic processing into two subunits (α and ß) which are visible in SDS-PAGE. The zebrafish AC is heterodimer associated with an inter-subunit disulfide bond. SDS-PAGE estimated the mass of native enzyme to be approximately 50â¯kDa & size exclusion chromatography estimated the mass of the active enzyme as approximately 100â¯kDa, suggesting the formation of a dimer of heterodimers. The protein was secreted as a mixture of processed and unprocessed forms in the culture media. A preliminary characterization of purified zebrafish AC was done by an enzyme assay. The zebrafish AC expressed in Pichia pastoris would be used for further structural and functional analysis.
Subject(s)
Acid Ceramidase/genetics , Acid Ceramidase/metabolism , Pichia/genetics , Zebrafish/genetics , Acid Ceramidase/isolation & purification , Animals , Biocatalysis , Cloning, Molecular , Gene Expression , Glycoproteins/metabolism , Glycosylation , ProteolysisABSTRACT
Tail-anchored (TA) proteins are a special class of membrane proteins that carry out vital functions in all living cells. Targeting mechanisms of TA proteins are investigated as the best example for post-translational protein targeting in yeast. Of the several mechanisms, Guided Entry of Tail-anchored protein (GET) pathway plays a major role in TA protein targeting. Many in silico and in vivo analyses are geared to identify TA proteins and their targeting mechanisms in different systems including Arabidopsis thaliana. Yet, crop plants that grow in specific and/or different conditions are not investigated for the presence of TA proteins and GET pathway. This study majorly investigates GET pathway in two crop plants, Oryza sativa subsp. Indica and Solanum tuberosum, through detailed in silico analysis. 508 and 912 TA proteins are identified in Oryza sativa subsp. Indica and Solanum tuberosum respectively and their localization with respect to endoplasmic reticulum (ER), mitochondria, and chloroplast has been delineated. Similarly, the associated GET proteins are identified (Get1, Get3 and Get4) and their structural inferences are elucidated using homology modelling. Get3 models are based on yeast Get3. The cytoplasmic Get3 from O. sativa is identified to be very similar to yeast Get3 with conserved P-loop and TA binding groove. Three cytoplasmic Get3s are identified for S. tuberosum. Taken together, this is the first study to identify TA proteins and GET components in Oryza sativa subsp. Indica and Solanum tuberosum, forming the basis for any further experimental characterization of TA targeting and GET pathway mechanisms in crop plants.
ABSTRACT
Legume Kunitz type trypsin inhibitor (KTI) family is one of the most versatile families of proteins. A typical KTI features a single peptide folded in ß-trefoil manner, with the molecular weight about 20-22kDa and two disulphide bonds. The members are known to inhibit a wide range of serpins proteases at the same time many of them possess unique features. Copaifera langsdorffii Trypsin inhibitor (CTI) has a ß-trefoil fold made up of two non-covalently bound polypeptide chains with only a single disulfide bridge. Delonix regia Trypsin inhibitor (DrTI) has one amino acid insertion between P1 and P2 of the reactive site distorting its conformation. Bauhinia bauhinioides Cruzipain inhibitor (BbCI) has a conservative ß-trefoil fold but lacks disulfide bonds. Such subtle differences in structures make Kunitz inhibitors different from other inhibitor families. Most of the studies on these inhibitors are focused towards their proposed role in defense from insect pests and wounding but their exact physiological role in nature is still uncharted. Thus, it would be very interesting to closely analyze the structural details of these inhibitors in order to ascertain their biological role and other fascinating applications.