-
Journal of Pharmacy & Bioallied Sciences Apr 2024contains andrograpanin, which is both anti-inflammatory and anti-infective. comprises over 150-200 species from the family . exerts various properties, including...
contains andrograpanin, which is both anti-inflammatory and anti-infective. comprises over 150-200 species from the family . exerts various properties, including anti-inflammatory property. Herbal mouthwash was made using and extract. The anti-inflammatory effect was evaluated using an albumin denaturation assay and egg albumin denaturation. The percentage of protein denaturation that is inhibited by the formulation of and indicates that it has strong anti-inflammatory effect. According to the findings, as concentration is raised, the formulation's anti-inflammatory activity rises. The formulation's percentage inhibition values are also equivalent to those of a typical anti-inflammatory medicine, indicating that it may be effective as a natural anti-inflammatory agent.
PubMed: 38882775
DOI: 10.4103/jpbs.jpbs_581_23 -
BioRxiv : the Preprint Server For... May 2024display technologies, exemplified by phage and yeast display, have emerged as powerful platforms for antibody discovery and engineering. However, the identification of...
display technologies, exemplified by phage and yeast display, have emerged as powerful platforms for antibody discovery and engineering. However, the identification of antibodies that disrupt target functions beyond binding remains a challenge. In particular, there are very few strategies that support identification and engineering of either protein-based irreversible binders or inhibitory enzyme binders. Expanding the range of chemistries in antibody libraries has the potential to lead to efficient discovery of function-disrupting antibodies. In this work, we describe a yeast display-based platform for the discovery of chemically diversified antibodies. We constructed a billion-member antibody library that supports the presentation of a range of chemistries within antibody variable domains via noncanonical amino acid (ncAA) incorporation and subsequent bioorthogonal click chemistry conjugations. Use of a polyspecific orthogonal translation system enables introduction of chemical groups with various properties, including photo-reactive, proximity-reactive, and click chemistry-enabled functional groups for library screening. We established conjugation conditions that facilitate modification of the full library, demonstrating the feasibility of sorting the full billion-member library in "protein-small molecule hybrid" format in future work. Here, we conducted initial library screens after introducing -(2-bromoethyl)tyrosine (OBeY), a weakly electrophilic ncAA capable of undergoing proximity-induced crosslinking to a target. Enrichments against donkey IgG and protein tyrosine phosphatase 1B (PTP1B) each led to the identification of several OBeY-substituted clones that bind to the targets of interest. Flow cytometry analysis on the yeast surface confirmed higher retention of binding for OBeY-substituted clones compared to clones substituted with ncAAs lacking electrophilic side chains after denaturation. However, subsequent crosslinking experiments in solution with ncAA-substituted clones yielded inconclusive results, suggesting that weakly reactive OBeY side chain is not sufficient to drive robust crosslinking in the clones isolated here. Nonetheless, this work establishes a multi-modal, chemically expanded antibody library and demonstrates the feasibility of conducting discovery campaigns in chemically expanded format. This versatile platform offers new opportunities for identifying and characterizing antibodies with properties beyond what is accessible with the canonical amino acids, potentially enabling discovery of new classes of reagents, diagnostics, and even therapeutic leads.
PubMed: 38853888
DOI: 10.1101/2024.05.29.596443 -
Current Health Sciences Journal 2024Inflammation and the injuries produced by free radicals are interconnected and influence each other. The underlying mechanisms of inflammation are partially attributed...
Inflammation and the injuries produced by free radicals are interconnected and influence each other. The underlying mechanisms of inflammation are partially attributed to the release of free radicals by immune cells, prooxidants that can also cause protein alteration. This study was performed in order to assess the potential anti-inflammatory effect of two bee venom samples harvested from Apis mellifera. Free radical scavenging capacity was investigated using DPPH and ABTS.+ tests and protective effect on proteins through the inhibitory activity on thermal denaturation of albumin.
PubMed: 38846469
DOI: 10.12865/CHSJ.50.01.11 -
RSC Advances May 2024An operationally simple one-pot three-component and convenient synthesis method for a series of diverse purine analogues of...
An operationally simple one-pot three-component and convenient synthesis method for a series of diverse purine analogues of 5-amino-7-(substituted)--(4-sulfamoylphenyl)-4,7-dihydro-[1,2,4]-triazolo[1,5-][1,3,5]triazine-2-carboxamide derivatives generated the reaction of 2-hydrazinyl--(4-sulfamoylphenyl)-2-thioxoacetamide, cyanoguanidine and a variety of aldehydes was achieved under green conditions. This experiment was conducted to evaluate the anti-inflammatory effect of the newly synthesized compounds using indomethacin as a reference medication; all compounds were tested for anti-inflammatory activity using the inhibition of albumin denaturation, RBC hemolysis technique and COX inhibition assay. The results showed that all evaluated compounds exhibited significant anti-inflammatory efficacy leading to excellently effective RBC membrane stabilization, inhibition of protein denaturation, and inhibition of COX enzymes when compared to those of indomethacin. At concentrations of 50, 100, 200, and 300 μg ml, these compounds decreased COX-1 and COX-2 activities more than indomethacin and have IC values in the range of 40.04-87.29 μg ml for COX-1 and 27.76-42.3 μg ml for COX-2 while indomethacin showed IC = 91.57 for COX-1 and 42.66 μg ml for COX-2. The anti-inflammatory findings show the need for more investigation to define the properties underlying the evaluated compounds' anti-inflammatory abilities. The enzyme cyclooxygenase-2 (COX 2) (PDB ID: 5IKT) was docked with ten synthetic substances. With docking scores () of -8.82, -7.82, and -7.76 kcal mol, 7-furan triazolo-triazine (4), 7-(2-hydroxy phenyl) triazolo-triazine (11), and 7-(4-dimethylamino phenyl) triazolo-triazine (12) had the greatest binding affinities, respectively. Therefore, these substances have COX-2 (PDB ID: 5IKT) inhibitory capabilities and hence may be investigated for COX 2 targeting development. Furthermore, both the top-ranked compounds (4 and 11) and the standard indomethacin were subjected to DFT analysis. The HOMO - LUMO energy difference (Δ) of the mentioned compounds was found to be less than that of indomethacin.
PubMed: 38832248
DOI: 10.1039/d4ra02970d -
Frontiers in Pharmacology 2024Cancer targeted therapy is essential to minimize damage to normal cells and improve treatment outcomes. The elevated activity of Cystathionine beta-synthase (CBS), an...
Benserazide, a cystathionine beta-synthase (CBS) inhibitor, potentially enhances the anticancer effects of paclitaxel via inhibiting the S-sulfhydration of SIRT1 and the HIF1-α/VEGF pathway.
Cancer targeted therapy is essential to minimize damage to normal cells and improve treatment outcomes. The elevated activity of Cystathionine beta-synthase (CBS), an enzyme responsible for producing endogenous hydrogen sulfide (HS), plays a significant role in promoting tumor growth, invasiveness, and metastatic potential. Consequently, the selective inhibition of CBS could represent a promising therapeutic strategy for cancer. Currently, there is much interest in combining paclitaxel with other drugs for cancer treatment. This study aimed to investigate the efficacy of combining benserazide, a CBS inhibitor, with paclitaxel in treating tumors. Firstly, we demonstrated CBS is indeed involved in the progression of multiple cancers. Then it was observed that the total binding free energy between the protein and the small molecule is -98.241 kJ/mol. The release of HS in the group treated with 100 μM benserazide was reduced by approximately 90% compared to the negative control, and the thermal denaturation curve of the complex protein shifted to the right, suggesting that benserazide binds to and blocks the CBS protein. Next, it was found that compared to paclitaxel monotherapy, the combination of benserazide with paclitaxel demonstrated stronger antitumor activity in KYSE450, A549, and HCT8 cells, accompanied by reduced cell viability, cell migration and invasion, as well as diminished angiogenic and lymphangiogenic capabilities. studies showed that the combined administration of benserazide and paclitaxel significantly reduced the volume and weight of axillary lymph nodes in comparison to the control group and single administration group. Further mechanistic studies revealed that the combination of benserazide and paclitaxel significantly suppressed the S-sulfhydration of SIRT1 protein, thereby inhibiting the expression of SIRT1 protein and activating SIRT1 downstream Notch1/Hes1 signaling pathway in KYSE450, A549, and HCT8 cells. Meanwhile, we observed that benserazide combined with paclitaxel induced a more significant downregulation of HIF-1α, VEGF-A, VEGF-C, and VEGF-D proteins expression levels in KYSE450, A549, and HCT8 cells compared to paclitaxel alone. These findings indicated that benserazide enhances the anticancer effects of paclitaxel via inhibiting the S-sulfhydration of SIRT1 and down-regulating HIF-1α/VEGF signaling pathway. This study suggests that benserazide may have potential as a chemosensitizer in cancer treatment.
PubMed: 38828455
DOI: 10.3389/fphar.2024.1404532 -
Biochemical and Biophysical Research... Sep 2024Rab3A is a member of the Rab GTPase family involved in synaptic vesicle trafficking. Recent evidence has demonstrated that Rab3A is phosphorylated by leucine-rich repeat...
Rab3A is a member of the Rab GTPase family involved in synaptic vesicle trafficking. Recent evidence has demonstrated that Rab3A is phosphorylated by leucine-rich repeat kinase 2 (LRRK2) that is implicated in both familial and sporadic forms of Parkinson's disease (PD), and an abnormal increase in Rab3A phosphorylation has been proposed as a cause of PD. Despite the potential importance of Rab3A in PD pathogenesis, its structural information is limited and the effects of bound nucleotides on its biophysical and biochemical properties remain unclear. Here, we show that GDP-bound Rab3A is preferentially phosphorylated by LRRK2 compared with GTP-bound Rab3A. The secondary structure of Rab3A, measured by circular dichroism (CD) spectroscopy, revealed that Rab3A is resistant to heat-induced denaturation at pH 7.4 or 9.0 regardless of the nucleotides bound. In contrast, Rab3A underwent heat-induced denaturation at pH 5.0 at a lower temperature in its GDP-bound form than in its GTP-bound form. The unfolding temperature of Rab3A was studied by differential scanning fluorimetry, which showed a significantly higher unfolding temperature in GTP-bound Rab3A than in GDP-bound Rab3A, with the highest at pH 7.4. These results suggest that Rab3A has unusual thermal stability under physiologically relevant conditions and that bound nucleotides influence both thermal stability and phosphorylation by LRRK2.
Topics: Phosphorylation; Guanosine Triphosphate; Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Protein Structure, Secondary; rab3A GTP-Binding Protein; Guanosine Diphosphate; Protein Stability
PubMed: 38824807
DOI: 10.1016/j.bbrc.2024.150199 -
Acta Neuropathologica Communications May 2024Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies....
Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of αsyn at serine 129 (PSER129) was considered rare in the healthy human brain but is enriched in pathological αsyn aggregates and is used as a specific marker for disease inclusions. However, recent observations challenge this assumption by demonstrating that PSER129 results from neuronal activity and can be readily detected in the non-diseased mammalian brain. Here, we investigated experimental conditions under which two distinct PSER129 pools, namely endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the mammalian brain. Results showed that in the wild-type (WT) mouse brain, perfusion fixation conditions greatly influenced the detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30-min and 1-h postmortem interval). Exposure to anesthetics (e.g., Ketamine or xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase calf alkaline phosphatase (CIAP) selectively dephosphorylated endogenous-PSER129 while αsyn preformed fibril (PFF)-seeded aggregates and genuine disease aggregates (Lewy pathology and Papp-Lantos bodies in Parkinson's disease and multiple systems atrophy brain, respectively) were resistant to CIAP-mediated dephosphorylation. The phosphatase resistance of aggregates was abolished by sample denaturation, and CIAP-resistant PSER129 was closely associated with proteinase K (PK)-resistant αsyn (i.e., a marker of aggregation). CIAP pretreatment allowed for highly specific detection of seeded αsyn aggregates in a mouse model that accumulates non-aggregated-PSER129. We conclude that αsyn aggregates are impervious to phosphatases, and CIAP pretreatment increases detection specificity for aggregated-PSER129, particularly in well-preserved biological samples (e.g., perfusion fixed or flash-frozen mammalian tissues) where there is a high probability of interference from endogenous-PSER129. Our findings have important implications for the mechanism of PSER129-accumulation in the synucleinopathy brain and provide a simple experimental method to differentiate endogenous-from aggregated PSER129.
Topics: Animals; Humans; Male; Mice; Alkaline Phosphatase; alpha-Synuclein; Brain; Mice, Inbred C57BL; Mice, Transgenic; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Aggregates; Protein Aggregation, Pathological; Synucleinopathies
PubMed: 38822421
DOI: 10.1186/s40478-024-01785-0 -
Heliyon May 2024Physico-chemical properties of fish flesh are reliable predictors of fillet quality and nutritional value. In our study, the age-related variations of the chemical...
Physico-chemical properties of fish flesh are reliable predictors of fillet quality and nutritional value. In our study, the age-related variations of the chemical composition, pH, water activity (aw), water holding capacity (WHC), color and texture analysis, protein thermal stability, myofibrillar fragmentation index (MFI), glycogen content, protein oxidation and protein profiles were investigated in (rainbow trout) fillet. The results revealed that protein denaturation temperatures (T1 and T2) decreased by 2 % and 11.6 % depending on fish age. T1 and T2 values in the same groups were raised 71 % at 11 months' fish and this increase was 58 % at 23 months' fish. An age-related reduction by 66.6 % and 31.25 % was noticed for protein oxidation markers sulfhydryl groups and disulfide bonds. MFI value increased by 86.6 % connected with age. The characteristics of fish meat quality are complex and are influenced by various factors that affect the degree of freshness of the product and its acceptance in the market. Taking into account the different demands of the consumer, this study has shown that age at slaughter has an impact on final product quality and that the recommended age at slaughter, taking into account market weight, positively affects meat quality.
PubMed: 38813226
DOI: 10.1016/j.heliyon.2024.e31146 -
MBio May 2024Pathogenic bacteria rely on secreted virulence factors to cause disease in susceptible hosts. However, in Gram-positive bacteria, the mechanisms underlying secreted...
UNLABELLED
Pathogenic bacteria rely on secreted virulence factors to cause disease in susceptible hosts. However, in Gram-positive bacteria, the mechanisms underlying secreted protein activation and regulation post-membrane translocation remain largely unknown. Using proteomics, we identified several proteins that are dependent on the secreted chaperone PrsA2. We followed with phenotypic, biochemical, and biophysical assays and computational analyses to examine the regulation of a detected key secreted virulence factor, listeriolysin O (LLO), and its interaction with PrsA2 from the bacterial pathogen (). Critical to virulence is internalization by host cells and the subsequent action of the cholesterol-dependent pore-forming toxin, LLO, which enables bacterial escape from the host cell phagosome. Since is a Gram-positive organism, the space between the cell membrane and wall is solvent exposed. Therefore, we hypothesized that the drop from neutral to acidic pH as the pathogen is internalized into a phagosome is critical to regulating the interaction of PrsA2 with LLO. Here, we demonstrate that PrsA2 directly interacts with LLO in a pH-dependent manner. We show that PrsA2 protects and sequesters LLO under neutral pH conditions where LLO can be observed to aggregate. In addition, we identify molecular features of PrsA2 that are required for interaction and ultimately the folding and activity of LLO. Moreover, protein-complex modeling suggests that PrsA2 interacts with LLO via its cholesterol-binding domain. These findings highlight a mechanism by which a Gram-positive secretion chaperone regulates the secretion, stability, and folding of a pore-forming toxin under conditions relevant to host cell infection.
IMPORTANCE
is a ubiquitous food-borne pathogen that can cause severe disease to vulnerable populations. During infection, relies on a wide repertoire of secreted virulence factors including the LLO that enables the bacterium to invade the host and spread from cell to cell. After membrane translocation, secreted factors must become active in the challenging bacterial cell membrane-wall interface. However, the mechanisms required for secreted protein folding and function are largely unknown. encodes a chaperone, PrsA2, that is critical for the activity of secreted factors. Here, we show that PrsA2 directly associates and protects the major virulence factor, LLO, under conditions corresponding to the host cytosol, where LLO undergoes irreversible denaturation. Additionally, we identify molecular features of PrsA2 that enable its interaction with LLO. Together, our results suggest that and perhaps other Gram-positive bacteria utilize secreted chaperones to regulate the activity of pore-forming toxins during infection.
PubMed: 38809022
DOI: 10.1128/mbio.00743-24 -
Journal of Pharmaceutical Sciences May 2024Protein denaturation and aggregation resulting from the effects of interfacial stress, often enhanced by flow and shear stress, pose significant challenges in the...
Protein denaturation and aggregation resulting from the effects of interfacial stress, often enhanced by flow and shear stress, pose significant challenges in the production of therapeutic proteins and monoclonal antibodies. The influence of flow on protein stability is closely intertwined with interfacial effects. In this study, we have developed a microfluidic device capable of exposing low volume (< 320 µL) protein solutions to highly uniform shear. To disentangle the synergistic impact of flow and interfaces on protein aggregation, we fabricated two devices composed of different materials, namely poly(methyl methacrylate) (PMMA) and stainless steel. Upon application of shear, we observed formation of protein particles in the micron-size range. Notably, The number of particles generated in the steel devices was ∼ 3.5 fold lower than in the PMMA device, hinting at an interface-mediated effect. With increasing the protein concentration from 1 to 50 mg/mL we observed a saturation in the amount of aggregates, further confirming the key role of solid-liquid interfaces in inducing particle formation. Introduction of non-ionic surfactants prevented protein aggregation, even at the highest tested protein concentration and low surfactant concentrations of 0.05 mg/mL. Overall, our findings corroborate the synergistic impact of shear and interface effects on protein aggregation. The device developed in this study offers a small-scale platform for assessing the stability of antibody formulations throughout various stages of the development and manufacturing process.
PubMed: 38801973
DOI: 10.1016/j.xphs.2024.05.024