scholarly journals Amyloidogenic proteins in the SARS-CoV and SARS-CoV-2 proteomes

2021 ◽  
Author(s):  
Taniya Bhardwaj ◽  
Kundlik Gadhave ◽  
Shivani Krishna Kapuganti ◽  
Prateek Kumar ◽  
Zacharias Faidon Brotzakis ◽  
...  

The phenomenon of protein aggregation is widespread and associated with a wide range of human diseases. Our knowledge on the aggregation behaviour of viral proteins, however, is still rather limited. Here, we investigated the distribution of aggregation-prone regions in the the SARS-CoV and SARS-CoV-2 proteomes. An initial analysis using a panel of sequence-based predictors suggested the presence of multiple aggregation-prone regions in these proteomes, and revealed an enhanced aggregation propensity in some SARS-CoV-2 proteins. We then studied the in vitro aggregation of predicted aggregation-prone regions in the of SARS-CoV-2 proteome, including the signal sequence peptide and fusion peptide 1 of the spike protein, a peptide from the NSP6 protein (NSP6-p), the ORF10 protein, and the NSP11 protein. Our re-sults show that these peptides and proteins form aggregates via a nucleation-dependent mecha-nism. Moreover, we demonstrated that the aggregates of NSP11 are toxic to mammalian cell cultures. These findings provide evidence about the aggregation of proteins in the SARS-CoV-2 proteome.

Author(s):  
Xin Zhang ◽  
Miao Li ◽  
Layla El Moussawi ◽  
Sally Saab ◽  
Shasha Zhang ◽  
...  

Humoral immune responses in animals are often tightly controlled by regulated proteolysis. This proteolysis is exerted by extracellular protease cascades, whose activation culminates in the proteolytic cleavage of key immune proteins and enzymes. A model for such immune system regulation is the melanization reaction in insects, where the activation of prophenoxidase (proPO) leads to the rapid formation of eumelanin on the surface of foreign entities such as parasites, bacteria and fungi. ProPO activation is tightly regulated by a network of so-called clip domain serine proteases, their proteolytically inactive homologs, and their serpin inhibitors. In Anopheles gambiae, the major malaria vector in sub-Saharan Africa, manipulation of this protease network affects resistance to a wide range of microorganisms, as well as host survival. However, thus far, our understanding of the molecular make-up and regulation of the protease network in mosquitoes is limited. Here, we report the function of the clip domain serine protease CLIPB10 in this network, using a combination of genetic and biochemical assays. CLIPB10 knockdown partially reversed melanotic tumor formation induced by Serpin 2 silencing in the absence of infection. CLIPB10 was also partially required for the melanization of ookinete stages of the rodent malaria parasite Plasmodium berghei in a refractory mosquito genetic background. Recombinant serpin 2 protein, a key inhibitor of the proPO activation cascade in An. gambiae, formed a SDS-stable protein complex with activated recombinant CLIPB10, and efficiently inhibited CLIPB10 activity in vitro at a stoichiometry of 1.89:1. Recombinant activated CLIPB10 increased PO activity in Manduca sexta hemolymph ex vivo, and directly activated purified M. sexta proPO in vitro. Taken together, these data identify CLIPB10 as the second protease with prophenoloxidase-activating function in An. gambiae, in addition to the previously described CLIPB9, suggesting functional redundancy in the protease network that controls melanization. In addition, our data suggest that tissue melanization and humoral melanization of parasites are at least partially mediated by the same proteases.


2016 ◽  
Vol 397 (9) ◽  
pp. 927-937 ◽  
Author(s):  
Edoardo Del Poggetto ◽  
Ludovica Gori ◽  
Fabrizio Chiti

Abstract Profilin-1 is a small protein involved in actin-mediated cytoskeleton rearrangement. Recently, mutations of profilin-1 have been associated with familial amyotrophic lateral sclerosis. It was previously reported that pathogenic mutations of profilin-1 increase the aggregation propensity of this protein, leaving its function unaffected. However, it is not clear if the mutations act by decreasing the conformational stability or by promoting structural perturbations of the folded state of this protein. In this work we have purified three novel profilin-1 mutants that were recently discovered and have investigated their conformational stability, structural features and aggregation behaviour in vitro. Analysis of the data obtained with the three novel variants, and a global statistical analysis with all profilin-1 mutants so far characterised, indicate significant correlations between aggregation propensity and structural perturbations of the folded state, rather than its conformational stability, in this group of mutants.


Recombinant DNA techniques have been used to express the proteins of influenza virus individually. Target cells expressing single viral proteins were then used to identify the molecules recognized by cytotoxic Tlymphocytes (CTLS). Results have shown that, contrary to expectation, the majority of the proteins recognized by class I major histocompatibility complex-restricted CTLS are not transmembrane glycoproteins. Experiments with deletion mutants of the nucleoprotein (NP) gene showed that transport of epitopes to the membrane for recognition by CTLS was independent of a definable signal sequence. In addition, the epitopes recognized were contained within short linear sequences of amino acids, and rapid degradation of large NP fragments within the target cell did not prevent recognition by CTLS. These results led to the suggestion that the epitopes recognized by class-I-restricted CTLS resulted from degradation of viral proteins. If so, the epitopes should, like those for class-II-restricted T cells, be replaceable in vitro with short synthetic peptides. Five different epitopes of NP have now been demonstrated that can be defined with short peptides in vitro . Each peptide is recognized with a specific class I molecule (D b , K k , K d and HLA B37). This has been extended to the influenza matrix protein, and a peptide epitope defined that is recognized by human CTLS in association with HLA -A2. T he question arose as to whether a similar phenomenon would be found with viral proteins which are naturally inserted in the target cell membrane. A mutant haemagglutinin has been produced that lacks a hydrophobic signal sequence. This protein is expressed as a short-lived, unglycosylated, intracellular protein. However, target cells expressing this molecule were recognized efficiently by CTLS raised to the wild-type haemagglutinin and vice versa. These and more recent results with nonviral glycoproteins are consistent with the existence of a mechanism for degrading viral (and perhaps host) proteins and exposing them at the cell surface for recognition by cytotoxic T cells in association with class I molecules of the major histocompatibility complex.


2021 ◽  
Vol 49 (18) ◽  
pp. 10618-10629
Author(s):  
Marta Cela ◽  
Anne Théobald-Dietrich ◽  
Joëlle Rudinger-Thirion ◽  
Philippe Wolff ◽  
Renaud Geslain ◽  
...  

Abstract Malaria is a life-threatening and devastating parasitic disease. Our previous work showed that parasite development requires the import of exogenous transfer RNAs (tRNAs), which represents a novel and unique form of host–pathogen interaction, as well as a potentially druggable target. This import is mediated by tRip (tRNA import protein), a membrane protein located on the parasite surface. tRip displays an extracellular domain homologous to the well-characterized OB-fold tRNA-binding domain, a structural motif known to indiscriminately interact with tRNAs. We used MIST (Microarray Identification of Shifted tRNAs), a previously established in vitro approach, to systematically assess the specificity of complexes between native Homo sapiens tRNAs and recombinant Plasmodium falciparum tRip. We demonstrate that tRip unexpectedly binds to host tRNAs with a wide range of affinities, suggesting that only a small subset of human tRNAs is preferentially imported into the parasite. In particular, we show with in vitro transcribed constructs that tRip does not bind specific tRNAs solely based on their primary sequence, hinting that post-transcriptional modifications modulate the formation of our host/parasite molecular complex. Finally, we discuss the potential utilization of the most efficient tRip ligands for the translation of the parasite's genetic information.


2020 ◽  
Vol 88 (4) ◽  
Author(s):  
Abhijeet Nayak ◽  
Wolfgang Schüler ◽  
Stefan Seidel ◽  
Ivan Gomez ◽  
Andreas Meinke ◽  
...  

ABSTRACT The development of vaccines for prevention of diseases caused by pathogenic species can encounter major obstacles if high sequence diversity is observed between individual strains. Therefore, development might be restricted either to conserved antigens, which are often rare, or to multivalent vaccines, which renders the production more costly and cumbersome. In light of this complexity, we applied a structure-based surface shaping approach for the development of a Lyme borreliosis (LB) vaccine suitable for the United States and Europe. The surface of the C-terminal fragment of outer surface protein A (OspA) was divided into distinct regions, based primarily on binding sites of monoclonal antibodies (MAbs). In order to target the six clinically most relevant OspA serotypes (ST) in a single protein, exposed amino acids of the individual regions were exchanged to corresponding amino acids of a chosen OspA serotype. Six chimeric proteins were constructed, and, based on their immunogenicity, four of these chimeras were tested in mouse challenge models. Significant protection could be demonstrated for all four proteins following challenge with infected ticks (OspA ST1, OspA ST2, and OspA ST4) or with in vitro-grown spirochetes (OspA ST1 and OspA ST5). Two of the chimeric proteins were linked to form a fusion protein, which provided significant protection against in vitro-grown spirochetes (OspA ST1) and infected ticks (OspA ST2). This article presents the proof-of-concept study for a multivalent OspA vaccine targeting a wide range of pathogenic LB Borrelia species with a single recombinant antigen for prevention of Lyme borreliosis.


1991 ◽  
Vol 30 (01) ◽  
pp. 35-39 ◽  
Author(s):  
H. S. Durak ◽  
M. Kitapgi ◽  
B. E. Caner ◽  
R. Senekowitsch ◽  
M. T. Ercan

Vitamin K4 was labelled with 99mTc with an efficiency higher than 97%. The compound was stable up to 24 h at room temperature, and its biodistribution in NMRI mice indicated its in vivo stability. Blood radioactivity levels were high over a wide range. 10% of the injected activity remained in blood after 24 h. Excretion was mostly via kidneys. Only the liver and kidneys concentrated appreciable amounts of radioactivity. Testis/soft tissue ratios were 1.4 and 1.57 at 6 and 24 h, respectively. Testis/blood ratios were lower than 1. In vitro studies with mouse blood indicated that 33.9 ±9.6% of the radioactivity was associated with RBCs; it was washed out almost completely with saline. Protein binding was 28.7 ±6.3% as determined by TCA precipitation. Blood clearance of 99mTc-l<4 in normal subjects showed a slow decrease of radioactivity, reaching a plateau after 16 h at 20% of the injected activity. In scintigraphic images in men the testes could be well visualized. The right/left testis ratio was 1.08 ±0.13. Testis/soft tissue and testis/blood activity ratios were highest at 3 h. These ratios were higher than those obtained with pertechnetate at 20 min post injection.99mTc-l<4 appears to be a promising radiopharmaceutical for the scintigraphic visualization of testes.


1997 ◽  
Vol 77 (04) ◽  
pp. 725-729 ◽  
Author(s):  
Mario Colucci ◽  
Silvia Scopece ◽  
Antonio V Gelato ◽  
Donato Dimonte ◽  
Nicola Semeraro

SummaryUsing an in vitro model of clot lysis, the individual response to a pharmacological concentration of recombinant tissue plasminogen activator (rt-PA) and the influence on this response of the physiological variations of blood parameters known to interfere with the fibrinolytic/thrombolytic process were investigated in 103 healthy donors. 125I-fibrin labelled blood clots were submersed in autologous plasma, supplemented with 500 ng/ml of rt-PA or solvent, and the degree of lysis was determined after 3 h of incubation at 37° C. Baseline plasma levels of t-PA, plasminogen activator inhibitor 1 (PAI-1), plasminogen, α2-anti-plasmin, fibrinogen, lipoprotein (a), thrombomodulin and von Willebrand factor as well as platelet and leukocyte count and clot retraction were also determined in each donor. rt-PA-induced clot lysis varied over a wide range (28-75%) and was significantly related to endogenous t-PA, PAI-1, plasminogen (p <0.001) and age (p <0.01). Multivariate analysis indicated that both PAI-1 antigen and plasminogen independently predicted low response to rt-PA. Surprisingly, however, not only PAI-1 but also plasminogen was negatively correlated with rt-PA-ginduced clot lysis. The observation that neutralization of PAI-1 by specific antibodies, both in plasma and within the clot, did not potentiate clot lysis indicates that the inhibitor, including the platelet-derived form, is insufficient to attenuate the thrombolytic activity of a pharmacological concentration of rt-PA and that its elevation, similarly to the elevation of plasminogen, is not the cause of clot resistance but rather a coincident finding. It is concluded that the in vitro response of blood clots to rt-PA is poorly influenced by the physiological variations of the examined parameters and that factors other than those evaluated in this study interfere with clot dissolution by rt-PA. In vitro clot lysis test might help to identify patients who may be resistant to thrombolytic therapy.


Growth regulators, phytohormones, both natural and artificial, are the main means to control plant ontogenesis. They are involved in regulating the processes of cell differentiation and cell divisions, the formation of tissues and organs, the changes in the rate of growth and development, the duration of the certain stages of ontogenesis. The main classes of phytohormones used in plant biotechnology, in particular, in the induction of haploid structures, are auxins and cytokinins. The mechanism of action of phytohormones on a cell is rather complicated and may have a different character. Understanding the characteristics of the action of phytohormones is complicated by the fact that the system of hormonal regulation of plant life is multicomponent. This is manifested in the fact that the same physiological process is most often influenced not by one, but by several phytohormones, covering a wide range of aspects of cell metabolism. In connection with the foregoing, the purpose of our work was to test a set of nutrient media with different basic composition and different proportions of phytohormones to determine the patterns of their influence on the processes of haploid structure induction in rape anther culture using accessions, developed at the Institute of Oilseed Crops NAAS. The material used was two accessions of winter rapeseed (No. 1 and No. 2) and one sample of spring rapeseed, provided by the Rapeseed Breeding laboratory of the Institute of Oilseed Crops. Incised inflorescences were kept against the background of low temperature of 6–8 ° C for several days, and then, under aseptic conditions, anthers with unripe pollen grains were isolated and planted on nutrient media differing in both basic mineral composition and content of phytohormones. MS (Murashige & Skoog 1962) and B5 (Gamborg et al 1968) media were used as basic media. Phytohormones were added to the basic media in various combinations – BA, 2,4-D, NAA at the concentrations of 0.1-0.6 mg/l. In each treatment up to 300 anthers were cultivated. Differences between treatments were evaluated using standard t-test. Studies have shown that in the anther culture of rapeseed on the tested nutrient media, morphogenic structures of different types (embryoids and callus) were originated. Synthetic auxin 2,4-D, regardless of the composition of the basic medium, caused the formation of structures of both types, though with a low frequency. Phytohormone BA of the cytokinin type had a similar effect. In this case, the frequency of structures was slightly higher, and the developed structures were represented mainly by embryoids. The joint action of cytokinin and auxin was the most favorable for the initiation of morphogenic structures. Such combination of phytohormones caused the formation of these structures with a frequency of 24.5-14.7% in the studied genotypes of winter rape. A similar effect of phytohormones on the induction and development of morphogenic structures was also observed in spring rape. In this case, a single basic MS medium was used. The experiment included treatments where phytohormones were absent (control), as well as various combinations of auxin and cytokinin. In the control treatment, the formation of new structures was not noted. In treatments with phytohormones, in addition to the medium with the combination of auxin and cytokinin, the medium in which only cytokinin was present was also rather effective. The treatment in which the action of auxin 2,4-D was combined with the action of another auxin, NAA, turned out to be practically ineffective. Thus, it was found that for the induction of morphogenic structures from microspores in rape anther culture of the tested genotypes, the combination of cytokinin with auxin, or the use of only single cytokinin BA without other phytohormones, had the most positive effect.


Author(s):  
Roohi Mohi-ud-din ◽  
Reyaz Hassan Mir ◽  
Prince Ahad Mir ◽  
Saeema Farooq ◽  
Syed Naiem Raza ◽  
...  

Background: Genus Berberis (family Berberidaceae), which contains about 650 species and 17 genera worldwide, has been used in folklore and various traditional medicine systems. Berberis Linn. is the most established group among genera with around 450-500 species across the world. This comprehensive review will not only help researchers for further evaluation but also provide substantial information for future exploitation of species to develop novel herbal formulations. Objective: The present review is focussed to summarize and collect the updated review of information of Genus Berberis species reported to date regarding their ethnomedicinal information, chemical constituents, traditional/folklore use, and reported pharmacological activities on more than 40 species of Berberis. Conclusion: A comprehensive survey of the literature reveals that various species of the genus possess various phytoconstituents mainly alkaloids, flavonoid based compounds isolated from different parts of a plant with a wide range of pharmacological activities. So far, many pharmacological activities like anti-cancer, anti-hyperlipidemic, hepatoprotective, immunomodulatory, anti-inflammatory both in vitro & in vivo and clinical study of different extracts/isolated compounds of different species of Berberis have been reported, proving their importance as a medicinal plant and claiming their traditional use.


2019 ◽  
Vol 20 (12) ◽  
pp. 1227-1243
Author(s):  
Hina Qamar ◽  
Sumbul Rehman ◽  
D.K. Chauhan

Cancer is the second leading cause of morbidity and mortality worldwide. Although chemotherapy and radiotherapy enhance the survival rate of cancerous patients but they have several acute toxic effects. Therefore, there is a need to search for new anticancer agents having better efficacy and lesser side effects. In this regard, herbal treatment is found to be a safe method for treating and preventing cancer. Here, an attempt has been made to screen some less explored medicinal plants like Ammania baccifera, Asclepias curassavica, Azadarichta indica, Butea monosperma, Croton tiglium, Hedera nepalensis, Jatropha curcas, Momordica charantia, Moringa oleifera, Psidium guajava, etc. having potent anticancer activity with minimum cytotoxic value (IC50 >3μM) and lesser or negligible toxicity. They are rich in active phytochemicals with a wide range of drug targets. In this study, these medicinal plants were evaluated for dose-dependent cytotoxicological studies via in vitro MTT assay and in vivo tumor models along with some more plants which are reported to have IC50 value in the range of 0.019-0.528 mg/ml. The findings indicate that these plants inhibit tumor growth by their antiproliferative, pro-apoptotic, anti-metastatic and anti-angiogenic molecular targets. They are widely used because of their easy availability, affordable price and having no or sometimes minimal side effects. This review provides a baseline for the discovery of anticancer drugs from medicinal plants having minimum cytotoxic value with minimal side effects and establishment of their analogues for the welfare of mankind.


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