Cysteines In Domain Research Articles

Intrauterine arsenic exposure induces glucose metabolism disorders in adult offspring by targeting TET2-mediated DNA hydroxymethylation reprogramming of HNF4α in developing livers, an effect alleviated by ascorbic acid

Exposure to arsenic during gestation has lasting health-related effects on the developing fetus, including an increase in the risk of metabolic disease later in life. Epigenetics is a potential mechanism involved in this process. Ten-eleven translocation 2 (TET2) has been widely considered as a transferase of 5-hydroxymethylcytosine (5hmC). Here, mice were exposed, via drinking water, to arsenic or arsenic combined with ascorbic acid (AA) during gestation. For adult offspring, intrauterine arsenic exposure exhibited disorders of glucose metabolism, which are associated with DNA hydroxymethylation reprogramming of hepatic nuclear factor 4 alpha (HNF4α). Further molecular structure analysis, by SEC-UV-DAD, SEC-ICP-MS, verified that arsenic binds to the cysteine domain of TET2. Mechanistically, arsenic reduces the stability of TET2 by binding to it, resulting in the decrease of 5hmC levels in Hnf4α and subsequently inhibiting its expression. This leads to the disorders of expression of its downstream key glucose metabolism genes. Supplementation with AA blocked the reduction of TET2 and normalized the 5hmC levels of Hnf4α, thus alleviating the glucose metabolism disorders. Our study provides targets and methods for the prevention of offspring glucose metabolism abnormalities caused by intrauterine arsenic exposure.

PAX3-FOXO1 uses its activation domain to recruit CBP/P300 and shape RNA Pol2 cluster distribution

Activation of oncogenic gene expression from long-range enhancers is initiated by the assembly of DNA-binding transcription factors (TF), leading to recruitment of co-activators such as CBP/p300 to modify the local genomic context and facilitate RNA-Polymerase 2 (Pol2) binding. Yet, most TF-to-coactivator recruitment relationships remain unmapped. Here, studying the oncogenic fusion TF PAX3-FOXO1 (P3F) from alveolar rhabdomyosarcoma (aRMS), we show that a single cysteine in the activation domain (AD) of P3F is important for a small alpha helical coil that recruits CBP/p300 to chromatin. P3F driven transcription requires both this single cysteine and CBP/p300. Mutants of the cysteine reduce aRMS cell proliferation and induce cellular differentiation. Furthermore, we discover a profound dependence on CBP/p300 for clustering of Pol2 loops that connect P3F to its target genes. In the absence of CBP/p300, Pol2 long range enhancer loops collapse, Pol2 accumulates in CpG islands and fails to exit the gene body. These results reveal a potential novel axis for therapeutic interference with P3F in aRMS and clarify the molecular relationship of P3F and CBP/p300 in sustaining active Pol2 clusters essential for oncogenic transcription.

Hydroxyethyl cellulose functionalised with maleimide groups as a new excipient with enhanced mucoadhesive properties

Hydroxyethylcellulose (HEC) is a non-ionic water-soluble polymer with poor mucoadhesive properties. The mucoadhesive properties of hydroxyethylcellulose can be improved by modifying it through conjugation with molecules containing maleimide groups. Maleimide groups interact with the thiol groups present in cysteine domains in the mucin via Michael addition reaction under physiological conditions to form a strong mucoadhesive bond. This will prolong the residence time of a dosage form containing this modified polymer and drug on mucosal surfaces. In this study HEC was modified by reaction with 4-bromophenyl maleimide in varying molar ratios and the successful synthesis was confirmed using 1H NMR and FTIR spectroscopies. The safety of the newly synthesised polymer derivatives was assessed with in vivo planaria assays and in vitro MTT assay utilising Caco-2 cell line. The synthesized maleimide-functionalised HEC solutions were sprayed onto blank tablets to develop a model dosage form. The physical properties and mucoadhesive behavior of these tablets were evaluated using a tensile test with sheep buccal mucosa. The maleimide-functionalised HEC exhibited superior mucoadhesive properties compared to unmodified HEC.

Palmitoylation of RGS20 affects Gαo-mediated signaling independent of its GAP activity

Regulator of protein signaling (RGS20) is a member of the RGS protein superfamily, which serve as key negative regulators of G protein-mediated signal transduction. Through their GTPase accelerating protein (GAP) activity, RGS proteins deactivate α-subunits of heterotrimeric G proteins. In addition, the majority of RGS proteins also have the ability to act through other, non-GAP related, functions. RGS20 is one of three members of the RZ subfamily, which all show selective GAP activity towards Gαz, however emerging data suggest that RGS20 can also regulate Gi/o-mediated signaling. While increased RGS20 expression is associated with the progression of multiple cancers, a large gap still exists relating to the mechanisms of RGS20 regulation and function. RGS20 contains a poly-cysteine string motif and a conserved cysteine in RGS domain, which are assumed to be palmitoylated. Palmitoylation, an important post-translational modification, plays an important role in cells by changing cellular functions of proteins. Consequently, the aim of this study was to confirm that RGS20 is palmitoylated and determine how palmitoylation affects its inhibition of Gαo-mediated signaling. We found a significant positive correlation between RGS20 palmitoylation and its association with active Gαo. We also showed that a conserved cysteine residue in the RGS domain is a critical site for its palmitoylation, with large impact on its association with Gαo. Palmitoylation on this site did not affect its GAP activity, however, it increased the inhibition of Gαo-mediated cAMP signaling. Altogether these data suggest that palmitoylation is a regulatory mechanism controlling RGS20 function, and that RGS20 can inhibit Gαo signaling through both GAP activity and non-GAP mechanisms.

N-glycosylation mediated folding and quality control in serine proteases of the hepsin family.

N-linked glycans are specifically attached to asparagine residues in a N-X-S/T motif of secretory pathway glycoproteins. N-glycosylation of newly synthesized glycoproteins directs their folding via the lectin chaperones calnexin and calreticulin that are associated with protein-folding enzymes and glycosidases of the endoplasmic reticulum (ER). Misfolded glycoproteins are retained in the ER by the same lectin chaperones. The work by Sun etal. (FEBS J 2023, 10.1111/febs.16757) in this issue focusses on hepsin, a serine protease on the surface of liver and other organs. The authors deduce that spatial positioning of N-glycans on one side of a conserved domain of hepsin, known as the scavenger receptor-rich cysteine domain, regulates calnexin selection for hepsin maturation and transport through the secretory pathway. If N-glycosylation is elsewhere on hepsin, then it is misfolded and has a prolonged accumulation with calnexin and BiP. This association coincides with the engagement of stress response pathways thatsense glycoprotein misfolding. The topological considerations of N-glycosylation dissected by Sun etal. may help unravel how key sites of N-glycosylation sites required for protein folding and transport have evolved to select the lectin chaperone calnexin pathway for folding and quality control.

S-acylation stabilizes ligand-induced receptor kinase complex formation during plant pattern-triggered immune signaling

Plant receptor kinases are key transducers of extracellular stimuli, such as the presence of beneficial or pathogenic microbes or secreted signaling molecules. Receptor kinases are regulated by numerous post-translational modifications.1,2,3 Here, using the immune receptor kinases FLS24 and EFR,5 we show that S-acylation at a cysteine conserved in all plant receptor kinases is crucial for function. S-acylation involves the addition of long-chain fatty acids to cysteine residues within proteins, altering their biochemical properties and behavior within the membrane environment.6 We observe S-acylation of FLS2 at C-terminal kinase domain cysteine residues within minutes following the perception of its ligand, flg22, in a BAK1 co-receptor and PUB12/13 ubiquitin ligase-dependent manner. We demonstrate that S-acylation is essential for FLS2-mediated immune signaling and resistance to bacterial infection. Similarly, mutating the corresponding conserved cysteine residue in EFR suppressed elf18-triggered signaling. Analysis of unstimulated and activated FLS2-containing complexes using microscopy, detergents, and native membrane DIBMA nanodiscs indicates that S-acylation stabilizes, and promotes retention of, activated receptor kinase complexes at the plasma membrane to increase signaling efficiency.

The Conserved Cysteine-Rich Secretory Protein MaCFEM85 Interacts with MsWAK16 to Activate Plant Defenses.

Metarhizium anisopliae is an entomopathogenic fungus which may enhance plant growth and resistance when acting as an endophyte in host plants. However, little is known about the protein interactions nor their activating mechanisms. Common in fungal extracellular membrane (CFEM) proteins have been identified as plant immune regulators that suppress or activate plant resistance responses. Here, we identified a CFEM domain-containing protein, MaCFEM85, which was mainly localized in the plasma membrane. Yeast two-hybrid (Y2H), glutathione-S-transferase (GST) pull-down, and bimolecular fluorescence complementation assays demonstrated that MaCFEM85 interacted with the extracellular domain of a Medicago sativa (alfalfa) membrane protein, MsWAK16. Gene expression analyses showed that MaCFEM85 and MsWAK16 were significantly upregulated in M. anisopliae and M. sativa, respectively, from 12 to 60 h after co-inoculation. Additional yeast two-hybrid assays and amino acid site-specific mutation indicated that the CFEM domain and 52th cysteine specifically were required for the interaction of MaCFEM85 with MsWAK16. Defense function assays showed that JA was up-regulated, but Botrytis cinerea lesion size and Myzus persicae reproduction were suppressed by transient expression of MaCFEM85 and MsWAK16 in the model host plant Nicotiana benthamiana. Collectively, these results provide novel insights into the molecular mechanisms underlying interactions of M. anisopliae with host plants.

Molecular and functional characterization of two IgM subclasses in large yellow croaker (Larimichthys crocea)

As the predominant immunoglobulin (Ig) isotype, IgM plays a crucial role in the acquired immunity of vertebrates. There is only one Igμ gene in mammals, except cattle, while the number of Igμ gene varies among teleost fish. In the current study, we found two functional Igμ genes (Igμ1 and Igμ2) and a pseudo Cμ gene (ψIgμ) in large yellow croaker (Larimichthys crocea). Both Igμ1 and Igμ2 genes possessed two transcript variants, which encoded the heavy chains of secreted (sIgM1 and sIgM2) and membrane-bound IgM1 and IgM2 (mIgM1 and mIgM2), respectively. Both the heavy chains of sIgM1 and sIgM2 consisted of a variable Ig domain, four constant Ig domains (CH1, CH2, CH3 and CH4) and a secretory tail, while those of mIgM1 and mIgM2 consisted of a variable Ig domain, three constant Ig domains (CH1, CH2 and CH3), a transmembrane domain and a short cytoplasmic tail. Cysteine residues that are necessary for the formation of intrachain and interchain disulfide bonds and tryptophan residues that are important for the folding of the Ig superfamily domain were well conserved in large yellow croaker IgM1 and IgM2. Interestingly, large yellow croaker IgM2 had an extra cysteine (C94) in the CH1 domain compared with IgM1, which may cause the structural difference between IgM1 and IgM2. A liquid chromatography–tandem mass spectrometry analysis revealed that both IgM1 and IgM2 were present at the protein level in large yellow croaker serum. Both the Igμ1 and Igμ2 genes were mainly expressed in systemic immune tissues, such as head kidney and spleen, but the expression level of Igμ2 was much lower than that of Igμ1. After Pseudomonas plecoglossicida infection, the expression levels of Igμ1 and Igμ2 in both the spleen and head kidney were significantly upregulated, with a higher upregulation of Igμ2 than that of Igμ1. These results suggested that Igμ1 and Igμ2 may play a differential role in the immune response of large yellow croaker against bacterial infection.

Effect of tryptophan mutation on the structure of LOV1 domain of phototropin1 protein of Ostreococcus tauri: A combined molecular dynamics simulation and biophysical approach

BackgroundLight, oxygen and voltage (LOV) proteins detect blue light by formation of a covalent ‘photoadduct’ between the flavin chromophore and the neighboring conserved cysteine residue. LOV proteins devoid of this conserved photoactive cysteine are unable to form this ‘photoadduct’ upon light illumination, but they can still elicit functional response via the formation of neutral flavin radical. Recently, tryptophan residue has been shown to be the primary electron donors to the flavin excited state. MethodsPhotoactive cysteine (Cys42) and tryptophan (Trp68) residues in the LOV1 domain of phototropin1 of Ostreococcus tauri (OtLOV1) was mutated to alanine and threonine respectively. Effect of these mutations have been studied using molecular dynamics simulation and spectroscopic techniques. ResultsMolecular dynamics simulation indicated that W68T did not affect the structure of OtLOV1 protein, but C42A leads to some structural changes. An increase in the fluorescence lifetime and quantum yield values was observed for the Trp68 mutant. ConclusionsAn increase in the fluorescence lifetime and quantum yield of Trp68 mutant compared to the wild type protein suggests that Trp68 residue participates in quenching of the flavin excited state followed by photoexcitation. General significanceEnhanced photo-physical properties of Trp68 OtLOV1 mutant might enable its use for the optogenetic and microscopic applications.

Significance of the p38MAPK-CRP2 axis in myofibroblastic phenotypic transition.

We have recently demonstrated that a LIM domain protein, cysteine and glycine-rich protein 2 (CSRP2 [CRP2]), plays a vital role in the functional expression of myofibroblasts and cancer-associated fibroblasts. CRP2 binds directly to myocardin-related transcription factors (MRTF [MRTF-A or MRTF-B]) and serum response factor (SRF) to stabilize the MRTF/SRF/CArG-box complex, leading to the expression of smooth muscle cell (SMC) genes such as α-smooth muscle actin (α-SMA) and collagens. These are the marker genes for myofibroblasts. Here, we show that the adhesion of cultured human skin fibroblasts (HSFs) to collagen reduces the myofibroblastic features. HSF adhesion to collagen suppresses the expression of CRP2 and CSRP2-binding protein (CSRP2BP [CRP2BP]) and reduces the expression of SMC genes. Although CRP2BP is known as an epigenetic factor, we find that CRP2BP also acts as an adaptor protein to enhance the function of CRP2 mentioned above. This CRP2BP function does not depend on its histone acetyltransferase activity. We also addressed the molecular mechanism of the reduced myofibroblastic features of HSFs on collagen. HSF adhesion to collagen inhibits the p38MAPK-mediated pathway, and reducing the p38MAPK activity decreases the expression of CRP2 and SMC genes. Thus, the activation of p38MAPK is critical for the myofibroblastic features. We also show evidence that CRP2 plays a role in the myofibroblastic transition of retinal pigment epithelial cells (RPEs). Like HSFs, such phenotypic modulation of RPEs depends on the p38MAPK pathway.Key words: CRP2, p38MAPK, MRTF, myofibroblasts, retinal pigment epithelial cells.

Efficient Production of Fc Fusion Proteins in the Cytoplasm of Escherichia coli: Dissecting and Mitigating Redox Heterogeneity

Cost-effective production of therapeutic proteins in microbial hosts is an indispensable tool towards accessible healthcare. Many of these heterologously expressed proteins, including all antibody formats, require disulfide bond formation to attain their native and functional state. A system for catalyzed disulfide bond formation (CyDisCo) has been developed allowing efficient production of recombinant proteins in the cytoplasm of one of the most used microbial expression systems, Escherichia coli. Here, we report high-yield production (up to 230 mg/L from 3 mL cultures) of in-demand therapeutics such as IgG1-based Fc fusion proteins in the E. coli cytoplasm. However, the production of this drug class using the CyDisCo system faces bottlenecks related to redox heterogeneity during oxidative folding. Our investigations identified and addressed one of the major causes of redox heterogeneity during CyDisCo-based production of Fc fusion proteins, i.e., disulfide bond formation in the IgG1 CH3 domain. Here, we communicate that mutating the cysteines in the CH3 domain of target Fc fusion proteins allows their production in a homogeneous redox state in the cytoplasm of E. coli without compromising on yields and thermal stability.

SEPHS1: Its evolution, function and roles in development and diseases

Selenophosphate synthetase (SEPHS) was originally discovered in prokaryotes as an enzyme that catalyzes selenophosphate synthesis using inorganic selenium and ATP as substrates. However, in contrast to prokaryotes, two paralogs, SEPHS1 and SEPHS2, occur in many eukaryotes. Prokaryotic SEPHS, also known as SelD, contains either cysteine (Cys) or selenocysteine (Sec) in the catalytic domain. In eukaryotes, only SEPHS2 carries out selenophosphate synthesis and contains Sec at the active site. However, SEPHS1 contains amino acids other than Sec or Cys at the catalytic position. Phylogenetic analysis of SEPHSs reveals that the ancestral SEPHS contains both selenophosphate synthesis and another unknown activity, and that SEPHS1 lost the selenophosphate synthesis activity. The three-dimensional structure of SEPHS1 suggests that its homodimer is unable to form selenophosphate, but retains ATPase activity to produce ADP and inorganic phosphate. The most prominent function of SEPHS1 is that it is implicated in the regulation of cellular redox homeostasis. Deficiency of SEPHS1 leads to the disturbance in the expression of genes involved in redox homeostasis. Different types of reactive oxygen species (ROS) are accumulated in response to SEPHS deficiency depending on cell or tissue types. The accumulation of ROS causes pleiotropic effects such as growth retardation, apoptosis, DNA damage, and embryonic lethality. SEPHS1 deficiency in mouse embryos affects retinoic signaling and other related signaling pathways depending on the embryonal stage until the embryo dies at E11.5. Dysregulated SEPHS1 is associated with the pathogenesis of various diseases including cancer, Crohn's disease, and osteoarthritis.

Oxidation of two cysteines within yeast Hsp70 impairs proteostasis while directly triggering an Hsf1-dependent cytoprotective response

Neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s diseases affect millions of Americans every year. One factor linked to the formation of aggregates associated with these diseases is damage sustained to proteins by oxidative stress. Management of protein misfolding by the ubiquitous Hsp70 chaperone family can be modulated by modification of two key cysteines in the ATPase domain by oxidizing or thiol-modifying compounds. To investigate the biological consequences of cysteine modification on the Hsp70 Ssa1 in budding yeast, we generated cysteine null (cysteine to serine) and oxidomimetic (cysteine to aspartic acid) mutant variants of both C264 and C303 and demonstrate reduced ATP binding, hydrolysis, and protein folding properties in both the oxidomimetic and hydrogen peroxide–treated Ssa1. In contrast, cysteine nullification rendered Ssa1 insensitive to oxidative inhibition. Additionally, we determined the oxidomimetic ssa1-2CD (C264D, C303D) allele was unable to function as the sole Ssa1 isoform in yeast cells and also exhibited dominant negative effects on cell growth and viability. Ssa1 binds to and represses Hsf1, the major transcription factor controlling the heat shock response, and we found the oxidomimetic Ssa1 failed to stably interact with Hsf1, resulting in constitutive activation of the heat shock response. Consistent with our in vitro findings, ssa1-2CD cells were compromised for de novo folding, post-stress protein refolding, and in regulated degradation of a model terminally misfolded protein. Together, these findings pinpoint Hsp70 as a key link between oxidative stress and proteostasis, information critical to understanding cytoprotective systems that prevent and manage cellular insults underlying complex disease states.

Palmitoylation of RGS20 Affects its Cellular Function

Regulator of G protein signaling 20 (RGS20) is a member of the RZ family of the RGS protein superfamily, which serve as key negative regulators of G protein‐mediated signal transduction. Previous studies showed that overexpression of RGS20 is correlated with progression of triple‐negative breast cancer (TNBC) in tumor tissues and contributes to breast cancer cell migration and invasion. However, there is still a large gap relating to the regulation and function of RGS20 due to its late discovery. RGS20 contains a poly cysteine string motif, which is assumed to be heavily palmitoylated. Palmitoylation, an important post‐translational protein modification, plays an important role in cells by changing cellular functions of proteins. Consequently, in this study we determined palmitoylation regulation of RGS20, which could provide more information for new potential treatment strategies for TNBC. We used click chemistry to show that RGS20 is palmitoylated in transfected HEK‐293T cells and MDA‐MB‐231 triple negative breast cancer cells. We also showed that constitutively activated Gαo, GαoQ205L, significantly increases palmitoylation levels of RGS20. Furthermore, palmitoylated RGS20 associates much more with GαoQ205L, while de‐palmitoylated RGS20 lost the association with GαoQ205L. Next, we detected how palmitoylation affects RGS20 GAP activity, as well as cAMP signaling downstream of Gαo. We mapped the palmitoylation site crucial for all of these effects to a conserved cysteine in the RGS domain. Mutating this cysteine residue resulted in a significant decrease in both palmitoylation signal and the association with GαoQ205L. Overall, we showed that palmitoylation affects functions of RGS20 in cells and that a conserved cysteine in the RGS domain is a critical site for its palmitoylation.

Mycobacterium tuberculosis requires SufT for Fe-S cluster maturation, metabolism, and survival in vivo.

Iron-sulfur (Fe-S) cluster proteins carry out essential cellular functions in diverse organisms, including the human pathogen Mycobacterium tuberculosis (Mtb). The mechanisms underlying Fe-S cluster biogenesis are poorly defined in Mtb. Here, we show that Mtb SufT (Rv1466), a DUF59 domain-containing essential protein, is required for the Fe-S cluster maturation. Mtb SufT homodimerizes and interacts with Fe-S cluster biogenesis proteins; SufS and SufU. SufT also interacts with the 4Fe-4S cluster containing proteins; aconitase and SufR. Importantly, a hyperactive cysteine in the DUF59 domain mediates interaction of SufT with SufS, SufU, aconitase, and SufR. We efficiently repressed the expression of SufT to generate a SufT knock-down strain in Mtb (SufT-KD) using CRISPR interference. Depleting SufT reduces aconitase's enzymatic activity under standard growth conditions and in response to oxidative stress and iron limitation. The SufT-KD strain exhibited defective growth and an altered pool of tricarboxylic acid cycle intermediates, amino acids, and sulfur metabolites. Using Seahorse Extracellular Flux analyzer, we demonstrated that SufT depletion diminishes glycolytic rate and oxidative phosphorylation in Mtb. The SufT-KD strain showed defective survival upon exposure to oxidative stress and nitric oxide. Lastly, SufT depletion reduced the survival of Mtb in macrophages and attenuated the ability of Mtb to persist in mice. Altogether, SufT assists in Fe-S cluster maturation and couples this process to bioenergetics of Mtb for survival under low and high demand for Fe-S clusters.

Mycobacterium tuberculosis DNA repair helicase UvrD1 is activated by redox-dependent dimerization via a 2B domain cysteine

Mycobacterium tuberculosis (Mtb) causes tuberculosis and, during infection, is exposed to reactive oxygen species and reactive nitrogen intermediates from the host immune response that can cause DNA damage. UvrD-like proteins are involved in DNA repair and replication and belong to the SF1 family of DNA helicases that use ATP hydrolysis to catalyze DNA unwinding. In Mtb, there are two UvrD-like enzymes, where UvrD1 is most closely related to other family members. Previous studies have suggested that UvrD1 is exclusively monomeric; however, it is well known that Escherichia coli UvrD and other UvrD family members exhibit monomer-dimer equilibria and unwind as dimers in the absence of accessory factors. Here, we reconcile these incongruent studies by showing that Mtb UvrD1 exists in monomer, dimer, and higher-order oligomeric forms, where dimerization is regulated by redox potential. We identify a 2B domain cysteine, conserved in many Actinobacteria, that underlies this effect. We also show that UvrD1 DNA-unwinding activity correlates specifically with the dimer population and is thus titrated directly via increasing positive (i.e., oxidative) redox potential. Consistent with the regulatory role of the 2B domain and the dimerization-based activation of DNA unwinding in UvrD family helicases, these results suggest that UvrD1 is activated under oxidizing conditions when it may be needed to respond to DNA damage during infection.

Mycobacterium tuberculosis DNA-repair helicase UvrD1 is activated by redox-dependent dimerization via a conserved 2b domain cysteine

Mycobacterium tuberculosis (Mtb) causes Tuberculosis and, during infection, is exposed to reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) from the host immune response that can cause DNA damage. UvrD-like proteins are involved in DNA repair and replication and belong to the SF1 family of DNA helicases that use ATP hydrolysis to catalyze DNA unwinding. In Mtb, there are two UvrD-like enzymes where UvrD1 is most closely related to other family members. Previous studies have suggested that UvrD1 is exclusively monomeric, however, it is well-known that E.

Gold nanoparticles from magnetite for the detection of amyloid proteins in neurodegenerative diseases

Introduction. Currently, neurodegenerative diseases (ND) are the fourth leading cause of death worldwide that pose a great challenge in the development of tools for early diagnosis. Thus, advances in science seek sensitive and selective detection systems and this manuscript will highlight the importance of nanotechnology.
 Material and methods. A literature review was conducted on the representative findings of NPs technologies in neurodegenerative diseases. Articles written in both English and Spanish were included. References between 2015-2021 were also taken into account.
 Results. One of the most representative techniques, AuNP was specifically implemented, together with a magnetic center composed of magnetite, which has as a specific ligand with a C-terminal cysteine domain present in the B-amyloid protein, which adhere directly to the surface of the NPs, characterizing the anomalous protein. Subsequently, by means of nanosensors capable of detecting and measuring different concentrations, these pathologies are identified at an early stage.
 Conclusions. Today, along with the advent of biotechnology, it has been possible to design techniques with NPs that allow the identification of specific mutations and provide diagnosis in individuals. In the investigative models of AuNP, it is possible to infer that the capabilities that make them representative focus on their magnetism and biofunctionality, by specifically binding to amyloid peptides and other ligands present in the protein, which are the major components of amyloid plaques used in these studies.

Regulation of the Expression, Oligomerisation and Signaling of the Inhibitory Receptor CLEC12A by Cysteine Residues in the Stalk Region.

CLEC12A is a myeloid inhibitory receptor that negatively regulates inflammation in mouse models of autoimmune and autoinflammatory arthritis. Reduced CLEC12A expression enhances myeloid cell activation and inflammation in CLEC12A knock-out mice with collagen antibody-induced or gout-like arthritis. Similarly to other C-type lectin receptors, CLEC12A harbours a stalk domain between its ligand binding and transmembrane domains. While it is presumed that the cysteines in the stalk domain have multimerisation properties, their role in CLEC12A expression and/or signaling remain unknown. We thus used site-directed mutagenesis to determine whether the stalk domain cysteines play a role in CLEC12A expression, internalisation, oligomerisation, and/or signaling. Mutation of C118 blocks CLEC12A transport through the secretory pathway diminishing its cell-surface expression. In contrast, mutating C130 does not affect CLEC12A cell-surface expression but increases its oligomerisation, inducing ligand-independent phosphorylation of the receptor. Moreover, we provide evidence that CLEC12A dimerisation is regulated in a redox-dependent manner. We also show that antibody-induced CLEC12A cross-linking induces flotillin oligomerisation in insoluble membrane domains in which CLEC12A signals. Taken together, these data indicate that the stalk cysteines in CLEC12A differentially modulate this inhibitory receptor’s expression, oligomerisation and signaling, suggestive of the regulation of CLEC12A in a redox-dependent manner during inflammation.

Expression and Refolding of the Plant Chitinase From Drosera capensis for Applications as a Sustainable and Integrated Pest Management.

Recently, the study of chitinases has become an important target of numerous research projects due to their potential for applications, such as biocontrol pest agents. Plant chitinases from carnivorous plants of the genus Drosera are most aggressive against a wide range of phytopathogens. However, low solubility or insolubility of the target protein hampered application of chitinases as biofungicides. To obtain plant chitinase from carnivorous plants of the genus Drosera in soluble form in E.coli expression strains, three different approaches including dialysis, rapid dilution, and refolding on Ni-NTA agarose to renaturation were tested. The developed « Rapid dilution » protocol with renaturation buffer supplemented by 10% glycerol and 2M arginine in combination with the redox pair of reduced/oxidized glutathione, increased the yield of active soluble protein to 9.5 mg per 1 g of wet biomass. A structure-based removal of free cysteines in the core domain based on homology modeling of the structure was carried out in order to improve the soluble of chitinase. One improved chitinase variant (C191A/C231S/C286T) was identified which shows improved expression and solubility in E. coli expression systems compared to wild type. Computational analyzes of the wild-type and the improved variant revealed overall higher fluctuations of the structure while maintaining a global protein stability. It was shown that free cysteines on the surface of the protein globule which are not involved in the formation of inner disulfide bonds contribute to the insolubility of chitinase from Drosera capensis. The functional characteristics showed that chitinase exhibits high activity against colloidal chitin (360 units/g) and high fungicidal properties of recombinant chitinases against Parastagonospora nodorum. Latter highlights the application of chitinase from D. capensis as a promising enzyme for the control of fungal pathogens in agriculture.