Biology/Life Sciences
Immunoproteomics: Approach to Diagnostic and Vaccine Development
The study of large protein sets (proteomics) involved in the immunological reaction is known as immunoproteomics. The methodology of immunoproteomics plays a major role in identifying possible vaccine candidates that could protect against pathogenic infection. The study of immunogenic proteins that are expressed during the outset of infection is the focus of the cross-talk between proteomics and immune protection antigens utilizing serum. Peptide presentation by MHC provides the new ‘window’ into changes that occur in the cell. Thus there is strong intense pressure on the pathogen that has been mutated in such an unusual manner that it can bypass the MHC peptide presentation by the MHC molecule. The pathogen's ability to evade the immune system is strongly restricted by the two unique distinct properties of MHC molecules i.e. polygenic and polymorphic properties. MHC-I restriction epitope identification has traditionally been accomplished using genetic motif prediction. The study of immune system proteins and their interactions is the main emphasis of the specialist field of immunoproteomics within proteomics. Methodologies include mass spectrometry (MS) SRM assay MALDI-TOF Chromatography ELISA 2DG PAGE and bioinformatics tools. Challenges are the complexity of the immune system protein abundance and dynamics sample variability post-translational modifications (PTMs) and data integration. Current advancements are enhanced mass spectrometry techniques single-cell proteomics artificial intelligence and machine learning advanced protein labeling techniques integration with other omics technologies and functional proteomics. However the recently emerging field of immunoproteomics has more promising possibilities in the field of peptide-based vaccines and virus-like particle vaccines. The importance of immunoproteomics technologies and methodologies as well as their use in the field of vaccinomics are the main topics of this review. Here we have discussed immunoproteomics in relation to a step towards the future of vaccination.
Exploring the Regulatory Interaction of Differentially Expressed Proteins in Cleft Palate Induced by Retinoic Acid
This study aimed to identify novel proteins involved in retinoic acid (RA)-induced embryonic cleft palate development.
The palate tissues of the control and RA-treated E14.5 were dissected and subjected to iTRAQ-based proteomic analysis.
Differential expression analysis identified 196 significantly upregulated and 149 downregulated considerably proteins in RA-induced palate tissues. Comprehensive Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed the significant involvement of cytoplasmic translation ribosome biogenesis glycolysis/gluconeogenesis and glutathione metabolism pathways in cleft palate pathogenesis triggered by RA. In particular ribosome-related pathways were highly enriched while glycolysis was disrupted. Protein-protein interaction analysis facilitated by the STRING database revealed a tightly interconnected network of differentially expressed proteins. Further analysis using the cytoHubba plugin in Cytoscape identified ten hub proteins including Eif4a1 Gapdh Eno1 Imp3 Rps20 Rps27a Eef2 Hsp90ab1 Rpl19 and Rps16 indicating their potential roles in RA-induced cleft palate development and thus positioning them as potential biomarkers for cleft palate.
These findings provide valuable insights into the proteomic changes associated with RA-induced cleft palate and shed light on key pathways and proteins that can contribute significantly to the pathogenesis of this congenital condition.
Neuropeptide Network of Polycystic Ovary Syndrome – A Review
Polycystic Ovary Syndrome (PCOS) the ubiquitous reproductive disorder has been documented as highly prevalent (6-9%) in India. 10% of women globally are predicted to have the disease. The highly mutable endocrinopathy with differential clinical criteria for each diagnosis of PCOS can mask the severity of the syndrome by influencing the incidence and occurrence of PCOS.
When there is a solid theoretical hypothesis between the neuroendocrine origin and ovarian origin of PCOS recent evidence supports the neuroendocrine derivation of the pathology. It is considered of neuroendocrine basis – as it controls the ovarian axis and acts as a delicate target because it possesses receptors for various gonadal hormones neurotransmitters & neuropeptides. Can these neuroendocrine alterations variations in central brain circuits and neuropeptide dysregulation be the tie that would link the pathophysiology of the disorder the occurrence of all the 1˚ and 2˚ symptoms like polycystic ovaries hyperandrogenism obesity insulin resistance etc. in PCOS?
This review anticipates providing a comprehensive overview of how neuropeptides such as Kisspeptin Neurokinin B Dynorphin A β-Endorphin Nesfatin Neuropeptide Y Phoenixin Leptin Ghrelin Orexin and Neudesin influence PCOS the understanding of which may help to establish potential drug candidates against precise targets in these central circuits.
MicroRNA-605-3p Inhibited the Growth and Chemoresistance of Osteosarcoma Cells via Negatively Modulating RAF1
Osteosarcoma (OS) is the leading cancer-associated mortality in childhood and adolescence. Increasing evidence has demonstrated the key function of microRNAs (miRNAs) in OS development and chemoresistance. Among them miRNA-605-3p acted as an important tumor suppressor and was frequently down-regulated in multiple cancers. However the function of miR-650-3p in OS has not been reported.
The aim of this work is to explore the novel role of miR-605-3p in osteosarcoma and its possible involvement in OS chemotherapy resistance.
The expression levels of miR-605-3p in OS tissues and cells were assessed by reverse transcription quantitative PCR (RT-qPCR). The relevance of miR-605-3p with the prognosis of OS patients was determined by the Kaplan-Meier analysis. Additionally the influence of miR-605-3p on OS cell growth was analyzed using the cell counting kit-8 colony formation assay and flow cytometry. The mRNA and protein expression of RAF1 were detected by RT-qPCR and western blot. The binding of miR-605-3p with the 3’-UTR of RAF1 was confirmed by dual-luciferase reporter assay.
Our results showed that miR-605-3p was markedly decreased in OS tissues and cells. A lower level of miR-605-3p was strongly correlated with lymph node metastasis and poor 5-year overall survival rate of OS patients. In vitro assay found that miR-605-3p suppressed OS cell proliferation and promoted cell apoptosis. Mechanistically the proto-oncogene RAF1 was seen as a target of miR-605-3p and strongly suppressed by miR-605-3p in OS cells. Restoration of RAF1 markedly eliminated the inhibitory effect of miR-605-3p on OS progression suggesting RAF1 as a key mediator of miR-605-3p. Consistent with the decreased level of RAF1 miR-605-3p suppressed the activation of both MEK and ERK in OS cells which are the targets of RAF1. Moreover lower levels of miR-605-3p were found in chemoresistant OS patients and down-regulated miR-605-3p increased the resistance of OS cells to therapeutic agents.
Our data revealed that miR-605-3p serves as a tumor suppressor gene by regulating RAF1 and increasing the chemosensitivity of OS cells which provided the novel working mechanism of miR-605-3p in OS. Engineering stable nanovesicles that could efficiently deliver miR-605-3p with therapeutic activity into tumors could be a promising therapeutic approach for the treatment of OS.
A Functional Human Glycogen Debranching Enzyme Encoded by a Synthetic Gene: Its Implications for Glycogen Storage Disease Type III Management
Glycogen Storage Disease type III (GSD III) is a metabolic disorder resulting from a deficiency of the Glycogen Debranching Enzyme (GDE) a large monomeric protein (approximately 170 kDa) with cytoplasmic localization and two distinct enzymatic activities: 4-α-glucantransferase and amylo-α-16-glucosidase. Mutations in the Agl gene with consequent deficiency in GDE lead to the accumulation of abnormal/toxic glycogen with shorter chains (phosphorylase limit dextrin PLD) in skeletal and/or heart muscle and/or in the liver. Currently there is no targeted therapy and available treatments are symptomatic relying on specific diets.
Enzyme Replacement Therapy (ERT) might represent a potential therapeutic strategy for GSD III. Moreover the single-gene nature of GSD III the subcellular localization of GDE and the type of affected tissues represent ideal conditions for exploring gene therapy approaches. Toward this direction we designed a synthetic codon-optimized cDNA encoding the human GDE.
This gene yielded high amounts of soluble enzymatically active protein in Escherichia coli. Moreover when transfected in Human Embryonic Kidney cells (HEK-293) it successfully encoded a functional GDE.
These results suggest that our gene or protein might complement the missing function in GSD III patients opening the door to further exploration of therapeutic approaches for this disease.
miR-1204 Positioning in 8q24.21 Involved in the Tumorigenesis of Colorectal Cancer by Targeting MASPIN
Colorectal cancer remains to be the third leading cause of cancer mortality rates. Despite the diverse effects of the miRNA cluster located in PVT1 of 8q24.21 across various tumors the specific biological function in colorectal cancer has not been clarified.
The amplification of the miR-1204 cluster was analyzed with the cBioPortal database while the expression and survival analysis of the miRNAs in the cluster were obtained from several GEO databases of colorectal cancer. To investigate the functional role of miR-1204 in colorectal cancer overexpression and silencing experiments were performed by miR-1204 mimic and inhibitor transfection in colorectal cancer cell lines respectively. Then the effects of miR-1204 on cell proliferation were assessed through CCK-8 colony formation and Edu assay. In addition cell migration was evaluated using wound healing and Transwell assay. Moreover candidate genes identified through RNA sequencing and predicted databases were identified and validated using PCR and western blot. A Dual-luciferase reporter experiment was conducted to identify MASPIN as the target gene of miR-1204.
In colorectal cancer the miR-1204 cluster exhibited high amplification and the expression levels of several cluster miRNAs were also significantly increased. Furthermore miR-1204 was found to be significantly associated with disease-specific survival according to the analysis of GSE17536. Functional experiments demonstrated that transfection of miR-1204 mimic or inhibitor could enhance or decrease cancer cell proliferation and migration. MASPIN was identified as a target of miR-1204. Additionally the overexpression of MASPIN partially rescued the effect of miR-1204 mimics on tumorigenic abilities in LOVO cells.
miR-1204 positioning in 8q24.21 promotes the proliferation and migration of colorectal cancer cells by targeting MASPIN.
The GA-Hecate Peptide inhibits the ZIKV Replicative Cycle in Different Steps and can Inhibit the Flavivirus NS2B-NS3 Protease after Cell Infection
Peptide drugs are advantageous because they are subject to rational design and exhibit highly diverse structures and broad biological activities. The NS2B-NS3 protein is a particularly promising flavivirus therapeutic target with extensive research on the development of inhibitors as therapeutic candidates and was used as a model in this work to determine the mechanism by which GA-Hecate inhibits ZIKV replication.
The present study aimed to evaluate the potential of GA-Hecate a new antiviral developed by our group against the Brazilian Zika virus and to evaluate the mechanism of action of this compound on the flavivirus NS2B-NS3 protein.
Solid-phase peptide Synthesis High-Performance Liquid Chromatography and Mass Spectrometry were used to obtain purify and characterize the synthesized compound. Real-time and enzymatic assays were used to determine the antiviral potential of GA-Hecate against ZIKV.
The RT-qPCR results showed that GA-Hecate decreased the number of ZIKV RNA copies in the virucidal pre-treatment and post-entry assays with 5- to 6-fold fewer RNA copies at the higher nontoxic concentration in Vero cells (HNTC: 10 μM) than in the control cells. Enzymatic and kinetic assays indicated that GA-Hecate acts as a competitive ZIKV NS2B-NS3 protease inhibitor with an IC50 of 32 nM and has activity against the yellow fever virus protease.
The results highlight the antiviral potential of the GA-Hecate bioconjugate and open the door for the development of new antivirals.
The Role of TGFBR3 in the Development of Lung Cancer
The Transforming Growth Factor-β (TGF-β) mediates embryonic development maintains cellular homeostasis regulates immune function and is involved in a wide range of other biological processes. TGF-β superfamily signaling pathways play an important role in cancer development and can promote or inhibit tumorigenesis. Type III TGF-β receptor (TGFBR3) is a co-receptor in the TGF-β signaling pathway which often occurs with reduced or complete loss of expression in many cancer patients and can act as a tumor suppressor gene. The reduction or deletion of TGFBR3 is more pronounced compared to other elements in the TGF-β signaling pathway. In recent years lung cancer is one of the major malignant tumors that endanger human health and its prognosis is poor. Recent studies have reported that TGFBR3 expression decreases to varying degrees in different types of lung cancer both at the tissue level and at the cellular level. The invasion metastasis angiogenesis and apoptosis of lung cancer cells are closely related to the expression of TGFBR3 which strengthens the inhibitory function of TGFBR3 in the evolution of lung cancer. This article reviews the mechanism of TGFBR3 in lung cancer and the influencing factors associated with TGFBR3. Clarifying the physiological function of TGFBR3 and its molecular mechanism in lung cancer is conducive to the diagnosis and treatment of lung cancer.
The Agonistic Activity of the Human Epidermal Growth Factor is Reduced by the D46G Substitution
Resistance to anti-tumor agents targeting the epidermal growth factor receptor (EGFR) reduces treatment response and requires the development of novel EGFR antagonists. Mutant epidermal growth factor (EGF) forms with reduced agonistic activity could be promising agents in cancer treatment.
EGF D46G affinity to EGFR domain III was assessed with affinity chromatography. EGF D46G acute toxicity in Af albino mice at 320 and 3200 μg/kg subcutaneous doses was evaluated. EGF D46G activity in human epidermoid carcinoma cells at 10 ng/mL concentration in serum-free medium and in subcutaneous Ehrlich ascites carcinoma mice model at 320 μg/kg dose was studied.
The D46G substitution decreases the thermal stability of EGF complexes with EGFR domain III by decreasing the ability of the C-terminus to be released from the intermolecular β-sheet. However with remaining binding sites for EGFR domain I EGF D46G effectively competes with other EGF-like growth factors for binding to EGFR and does not demonstrate toxic effects in mice. EGF D46G inhibits the proliferation of human epidermoid carcinoma cells compared to native EGF. A single subcutaneous administration of EGF D46G along with Ehrlich carcinoma cells injection inhibits the proliferation of these cells and delays tumor formation for up to seven days.
EGF D46G can be defined as a partial EGFR agonist as this mutant form demonstrates reduced agonistic activity compared to native EGF. The study emphasizes the role of the EGF C-terminus in establishing interactions with EGFR domain III which are necessary for EGFR activation and subsequent proliferation of cells.
Anionic Host Defence Peptides from the Plant Kingdom: Their Anticancer Activity and Mechanisms of Action
It is becoming increasingly clear that plants ranging across the plant kingdom produce anionic host defence peptides (AHDPs) with potent activity against a wide variety of human cancers cells. In general this activity involves membrane partitioning by AHDPs which leads to membranolysis and / or internalization to attack intracellular targets such as DNA. Several models have been proposed to describe these events including: the toroidal pore and Shai-Matsuzaki- Huang mechanisms but in general the mechanisms underpinning the membrane interactions and anticancer activity of these peptides are poorly understood. Plant AHDPs with anticancer activity can be conveniently discussed with reference to two groups: cyclotides which possess cyclic molecules stabilized by cysteine knot motifs and other ADHPs that adopt extended and -helical conformations. Here we review research into the anticancer action of these two groups of peptides along with current understanding of the mechanisms underpinning this action.
The Amazing World of Peptide Engineering: the Example of Antimicrobial Peptides from Frogs and Their Analogues
This review discusses the importance and properties of antimicrobial peptides from frogs and their synthetic analogues as potential therapeutic alternatives in fighting not only bacterial infections but also protozoans involved with the major neglected diseases which afflict human populations (e.g. Chagas disease African sleeping sickness Leishmaniasis and malaria). Here we emphasize their multifunctional properties such as promising broad-spectrum drugs that target protozoan parasites too.
Induced Folding Under Membrane Mimetic and Acidic Conditions Implies Undiscovered Biological Roles of Prokaryotic Ubiquitin-Like Protein Pup
Ubiquitin-like proteins play important roles in diverse biological processes. In Mycobacterium tuberculosis Pup (prokaryotic ubiquitin-like protein) a functional homologue of eukaryotic ubiquitin interacts with the proteasome ATPase subunit Mpa to recognize and unfold substrates and then translocate them into the proteasome core for degradation. Previous studies revealed that Pup an intrinsically disordered protein (IDP) adopts a helical structure upon binding to the N-terminal coiled-coil domain of Mpa at its disordered C-terminal region. In the present study using circular dichroism (CD) surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR) we show that membrane mimetic and acidic conditions also induce Pup to adopt helical conformations. Moreover at low pH Pup via both of its N- and C-terminal regions binds to Mpa on sites from the N-terminal region in addition to the C-terminal region of the coiled-coil domain. Our results imply Pup may play undiscovered roles in some biological processes e.g. those involve in membrane.
C-terminal Lysine-Linked Magainin 2 with Increased Activity Against Multidrug-Resistant Bacteria
Due to the growing problem of antibiotic-resistant microorganisms the development of novel antimicrobial agents is a very important challenge. Dimerization of cationic antimicrobial peptides (cAMPs) is a potential strategy for enhancing antimicrobial activity. Here we studied the effects of magainin 2 (MG2) dimerization on its structure and biological activity. Lysine and glutamic acid were used to synthesize the C- and N-terminal dimers of MG2 respectively in order to evaluate the impact of linker position used to obtain the dimers. Both MG2 and its dimeric versions showed a random coil structure in aqueous solution. However in the presence of a structure-inducing solvent or a membrane mimetic all peptides acquired helical structure. N-terminal dimerization did not affect the biological activity of the peptide. On the other hand the C-terminal dimer (MG2)2K showed antimicrobial activity 8–16 times higher than that of MG2 and the time required to kill Escherichia coli was lower. The enhanced antimicrobial activity was related to membrane permeabilization. (MG2)2K was also more active against multidrug-resistant bacteria of clinical origin. Overall the results presented here demonstrate that C-terminal lysine-linked dimerization improve the activity of MG2 and (MG2)2K can be considered as a potential antimicrobial agent.
Ribosome-inactivating Proteins from Root Tubers and Seeds of Trichosan-thes kirilowii and Other Trichosanthes Species
Ribosome-inactivating proteins have been isolated from Trichosanthes kirilowii root tubers and seeds including trichosanthin karasurin and T 33 from root tubers and trichosanthrip trichokirin alpha-kirilowin beta-kirilowin and trichoanguin from seeds. The aforementioned proteins show structural and functional similarities. Among them trichosanthin is the best known and most intensely studied. Trichosanthin manifests anticancer activity in vitro and in tumor bearing mice against a variety of cancers/cancer cell lines. It also exhibits anti-HIV-1 and anti-HSV-1 activities. Trichosanthin has been found to be useful for treatment of cesarean scar pregnancies and ectopic pregnancy and for preventing acute rejection of major histocompatibility complex-mismatched mouse skin allograft. Trichosanthin selectively lesions some neurons and thus can be used in neuroscience research.
Wasp Venom Toxins as a Potential Therapeutic Agent
It is high time now to discover novel drugs due to the increasing rate of drug resistance by the pathogen organisms and target cells as well as the dependence or tolerance of the body towards the drug. As it is obvious that significant numbers of the modern day pharmaceuticals are derived from natural products it is equally astonishing to accept that venoms of various origins have therapeutic potentials. Wasp venoms are also a rich source of therapeutically important toxins which includes short cationic peptides kinins polyamines and polyDNA viruses to name a few indentified. Wasp venom cationic peptides namely mastoparan and its analogs show a very important potency as an antimicrobial and anticancer agents of the future. They have proven to be the better candidates due to their lesser toxic effects and higher selectivity upon chemical modification and charge optimization. They also have superiority over the conventional chemical drugs as the target cells very rarely develop resistance against them because these peptides primarily imparts its effect through biophysical interaction with the target cell membrane which is dependent upon the net charge of the peptide its hydrophobicity and anionicity and fluidity of the target cell membranes. Besides the other components of wasp venom such as kinins polyamines and polyDNA viruses show various pharmacological promise in the treatment of pain inflammatory disease and neurodegenerative diseases such as epilepsy and aversion.
Activities of Venom Proteins and Peptides with Possible Therapeutic Applications from Bees and WASPS
The variety of proteins and peptides isolated from honey bee venom and wasp venom includes melittin adiapin apamine bradykinin cardiopep mast cell degranulating peptide mastoparan phospholipase A2 and secapin. Some of the activities they demonstrate may find therapeutic applications.
Insights into Antimicrobial Peptides from Spiders and Scorpions
The venoms of spiders and scorpions contain a variety of chemical compounds. Antimicrobial peptides (AMPs) from these organisms were first discovered in the 1990s. As of May 2015 there were 42 spider's and 63 scorpion's AMPs in the Antimicrobial Peptide Database (http://aps.unmc.edu/AP). These peptides have demonstrated broad or narrow-spectrum activities against bacteria fungi viruses and parasites. In addition they can be toxic to cancer cells insects and erythrocytes. To provide insight into such an activity spectrum this article discusses the discovery classification structure and activity relationships bioinformatics analysis and potential applications of spider and scorpion AMPs. Our analysis reveals that in the case of linear peptides spiders use both glycine-rich and helical peptide models for defense whereas scorpions use two distinct helical peptide models with different amino acid compositions to exert the observed antimicrobial activities and hemolytic toxicity. Our structural bioinformatics study improves the knowledge in the field and can be used to design more selective peptides to combat tumors parasites and viruses.
High-Level Secretory Expression and Purification of Recombinant Human Interleukin 1 Beta in Pichia pastoris
As an important pro-inflammatory cytokine interleukin-1beta (IL-1β) participates in a variety of physiological and pathological responses. In order to obtain higher yielded recombinant human interleukin-1 beta (rhIL-1β) we cloned hIL-1β cDNA sequences based on the coding sequence of human mature IL-1. After recombinant pPICZA/hIL-1β was separated and sequenced we transformed recombinant pPICZA/hIL-1β into Pichia pastoris GS115 SMD1168 and X-33 strain via electroporation. The results showed that recombinant pPICZA/ hIL-1β had the highest expression level in X-33 Pichia pastoris. Subsequently rhIL-1β was purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and identified by Western blot. Then the fermentation process was optimized to increase product yield. Under the fermentation conditions of the absorption value of fermentation liquor before induction of 600 oxygen concentration of 20% methanol concentration of 0.25% with pH 5.0 the yield of rhIL-1β reached to 250 mg/L after 72 h induction at 26°C. After aqueous two-phase extraction combined with chromatography the purity of rhIL-1β was 95% and the yield was up to 85%. The biological activity of rhIL-1β was detected by MTT assay and the result showed that rhIL-1 significantly inhibited the growth and proliferation of B16 melanoma cells.