Soluble Monomeric Form Research Articles

CONFORMATIONAL PARTICULARITIES OF A(25-35) PEPTIDE DETERMINED BY INFRARED SPECTROSCOPY

The Alzheimer’s amyloid-β (25-35) peptide retains the toxicity and ability to aggregate of the amyloid-(1-40) peptide. The study of the conformational properties of Aβ (25-35) peptide in its soluble monomeric form can play an essential role in determining the mechanism of oligomerization. In this study, we probed the secondary structure of a soluble Aβ (25-35) peptide in water solution at physiological conditions (pH 7.4). We have used Fourier Transformed Infrared Spectroscopy (FTIR) to examine the secondary structure of this peptide. The amide I bands of A (25-35) obtained from transmission-FTIR spectra consists of one main band at 1658 cm-1, at both concentrations used (200 M and 1 mM). This band show us that A (25-35) in aqueous solution was mostly organized into a -helical structure (48%). The contribution of the unordered structure was found to be about 12%. The proportion of -sheet and -turn structures are slightly lower, 12-15% and 13-14%, respectively for both concentrations. FTIR spectra of the peptide in water solution (100 µM) after incubation for 12h showed Aβ peptide conformation is critically dependent on the environmental conditions used.

Lessons learned from the failure of solanezumab as a prospective treatment strategy for Alzheimer’s disease

ABSTRACT Introduction In the last decade, the efforts conducted for discovering Alzheimer’s Disease (AD) treatments targeting the best-known pathogenic factors [amyloid–β (Aβ), tau protein, and neuroinflammation] were mostly unsuccessful. Given that a systemic failure of Aβ clearance was supposed to primarily contribute to AD development and progression, disease-modifying therapies with anti-Aβ monoclonal antibodies (e.g. solanezumab, bapineuzumab, gantenerumab, aducanumab, lecanemab and donanemab) are ongoing in randomized clinical trials (RCTs) with contrasting results. Areas covered The present Drug Discovery Case History analyzes the failures of RCTs of solanezumab on AD. Furthermore, the authors review the pharmacokinetics, pharmacodynamics, and tolerability effect of solanezumab from preclinical studies with its analogous m266 in mice. Finally, they describe the RCTs with cognitive, cerebrospinal fluid and neuroimaging findings in mild-to-moderate AD (EXPEDITION studies) and in secondary prevention studies (A4 and DIAN-TU). Expert opinion Solanezumab was one of the first anti-Aβ monoclonal antibodies to be tested in preclinical and clinical AD showing to reduce brain Aβ level by acting on soluble monomeric form of Aβ peptide without significant results on deposits. Unfortunately, this compound showed to accelerate cognitive decline in both asymptomatic and symptomatic trial participants, and this failure of solanezumab further questioned the Aβ cascade hypothesis of AD.

Cytotoxic Staphylococcus aureus PSMα3 inhibits the aggregation of human insulin in vitro.

Phenol-soluble modulins (PSMs) are extracellular short amphipathic peptides secreted by the bacteria Staphylococcus aureus (S. aureus). They play an essential role in the bacterial lifecycle, biofilm formation, and stabilisation. From the PSM family, PSMα3 has been of special interest recently due to its cytotoxicity and highly stable α-helical conformation, which also remains in its amyloid fibrils. In particular, PSMα3 fibrils were shown to be composed of self-associating "sheets" of α-helices oriented perpendicular to the fibril axis, mimicking the architecture of canonical cross-β fibrils. Therefore, they were called cross-α-fibrils. PSMα3 was synthesised and verified for identity with wild-type sequences (S. aureus). Then, using several experimental techniques, we evaluated its propensity for in vitro aggregation. According to our findings, synthetic PSMα3 (which lacks the N-terminal formyl groups found in bacteria) does not form amyloid fibrils and maintains α-helical conformation in a soluble monomeric form for several days of incubation. We also evaluated the influence of PSMα3 on human insulin fibrillation in vitro, using a variety of experimental approaches in combination with computational molecular studies. First, it was shown that PSMα3 drastically inhibits the fibrillation of human insulin. The anti-fibrillation effect of PSMα3 was concentration-dependent and required a concentration ratio of PSMα3: insulin equal to or above 1 : 100. Molecular modelling revealed that PSMα3 most likely inhibits the production of insulin primary nuclei by competing for residues involved in its dimerization.

Membrane protein chaperone and sodium chloride modulate the kinetics and morphology of amyloid beta aggregation.

Protein aggregation is a biological phenomenon caused by the accumulation of misfolded proteins. Amyloid beta (Aβ) peptides are derived from the cleavage of a larger membrane protein molecule and accumulate to form plaques extracellularly. According to the amyloid hypothesis, accumulation of Aβ aggregates in the brain is primarily responsible for the pathogenesis of Alzheimer's disease (AD). Therefore, the disassembly of Aβ aggregates may provide opportunities for alleviating or treating AD. Here, we show that the novel protein targeting machinery from chloroplast, chloroplast signal recognition particle 43 (cpSRP43), is an ATP-independent membrane protein chaperone that can both prevent and reverse Aβ aggregation effectively. Using of thioflavin T dye, we obtained the aggregation kinetics of Aβ aggregation and determined that the chaperone prevents Aβ aggregation in a concentration-dependent manner. Size exclusion chromatography and sedimentation assays showed that 10-fold excess of cpSRP43 can keep Aβ in the soluble monomeric form. Electron microscopy showed that the fibril structure was disrupted in the presence of this chaperone. Importantly, cpSRP43 utilizes the binding energy to actively remodel the preformed Aβ aggregates without assistance by a co-chaperone and ATP, emphasizing its unique function among protein chaperones. Moreover, when sodium chloride concentration is higher than 25 mm, the Aβ aggregation rate increases drastically to form tightly associated aggregates and generate more oligomers. Our results demonstrate that the presence of cpSRP43 and low NaCl levels inhibit or retard Aβ peptide aggregation, potentially opening new avenues to strategically develop an effective treatment for AD.

Solution-state NMR assignment and secondary structure propensity of the full length and minimalistic-truncated prefibrillar monomeric form of biofilm forming functional amyloid FapC from Pseudomonas aeruginosa.

Functional bacterial amyloids provide structural scaffolding to bacterial biofilms. In contrast to the pathological amyloids, they have a role in vivo and are tightly regulated. Their presence is essential to the integrity of the bacterial communities surviving in biofilms and may cause serious health complications. Targeting amyloids in biofilms could be a novel approach to prevent chronic infections. However, structural information is very scarce on them in both soluble monomeric and insoluble fibrillar forms, hindering our molecular understanding and strategies to fight biofilm related diseases. Here, we present solution-state NMR assignment of 250 amino acid long biofilm-forming functional-amyloid FapC from Pseudomonas aeruginosa. We studied full-length (FL) and shorter minimalistic-truncated (L2R3C) FapC constructs without the signal-sequence that is required for secretion. 91% and 100% backbone NH resonance assignments for FL and L2R3C constructs, respectively, indicate that soluble monomeric FapC is predominantly disordered, with sizeable secondary structural propensities mostly as PP2 helices, but also as α-helices and β-sheets highlighting hotspots for fibrillation initiation interface. A shorter construct showing almost identical NMR chemical shifts highlights the promise of utilizing it for more demanding solid-state NMR studies that require methods to alleviate signal redundancy due to almost identical repeat units. This study provides key NMR resonance assignments for future structural studies of soluble, pre-fibrillar and fibrillar forms of FapC.

Plant Defense Elicitation by the Hydrophobin Cerato-Ulmin and Correlation with Its Structural Features.

Cerato-ulmin (CU) is a 75-amino-acid-long protein that belongs to the hydrophobin family. It self-assembles at hydrophobic-hydrophilic interfaces, forming films that reverse the wettability properties of the bound surface: a capability that may confer selective advantages to the fungus in colonizing and infecting elm trees. Here, we show for the first time that CU can elicit a defense reaction (induction of phytoalexin synthesis and ROS production) in non-host plants (Arabidopsis) and exerts its eliciting capacity more efficiently when in its soluble monomeric form. We identified two hydrophobic clusters on the protein's loops endowed with dynamical and physical properties compatible with the possibility of reversibly interconverting between a disordered conformation and a β-strand-rich conformation when interacting with hydrophilic or hydrophobic surfaces. We propose that the plasticity of those loops may be part of the molecular mechanism that governs the protein defense elicitation capability.

Multi-Modal Imaging to Assess the Follicular Delivery of Zinc Pyrithione.

Zinc pyrithione (ZnPT) is a widely used antifungal, usually applied as a microparticle suspension to facilitate delivery into the hair follicles. It then dissociates into a soluble monomeric form that is bioactive against yeast and other microorganisms. In this study, we use multiphoton microscopy (MPM) and fluorescence lifetime imaging microscopy (FLIM) to characterise ZnPT formulations and map the delivery of particles into follicles within human skin. To simulate real-world conditions, it was applied using a massage or no-massage technique, while simultaneously assessing the dissolution using Zinpyr-1, a zinc labile fluorescent probe. ZnPT particles can be detected in a range of shampoo formulations using both MPM and FLIM, though FLIM is optimal for detection as it allows spectral and lifetime discrimination leading to increased selectivity and sensitivity. In aqueous suspensions, the ZnPT 7.2 µm particles could be detected up to 500 µm in the follicle. The ZnPT particles in formulations were finer (1.0–3.3 µm), resulting in rapid dissolution on the skin surface and within follicles, evidenced by a reduced particle signal at 24 h but enhanced Zinpyr-1 intensity in the follicular and surface epithelium. This study shows how MPM-FLIM multimodal imaging can be used as a useful tool to assess ZnPT delivery to skin and its subsequent dissolution.

The central inflammasome adaptor protein ASC activates the inflammasome after transition from a soluble to an insoluble state

Apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD) (ASC) is a 22 kDa protein that functions as the central adaptor for inflammasome assembly. ASC forms insoluble specks in monocytes undergoing pyroptosis, and the polymerization of ASC provides a template of CARDs that leads to proximity-mediated autoactivation of caspase-1 in canonical inflammasomes. However, specks are insoluble protein complexes, and solubility is typically important for protein function. Therefore, we sought to define whether ASC specks comprise active inflammasome complexes or are simply the end stage of exhausted ASC polymers. Using a THP-1 cell–lysing model of caspase-1 activation that is ASC dependent, we compared caspase-1 activation induced by preassembled insoluble ASC specks and soluble monomeric forms of ASC. Unexpectedly, after controlling for the concentration dependence of ASC oligomerization, we found that only insoluble forms of ASC promoted caspase-1 autocatalysis. This link to insolubility was recapitulated with recombinant ASC. We show that purified recombinant ASC spontaneously precipitated and was functional, whereas the maltose-binding protein–ASC fusion to ASC (promoting enhanced solubility) was inactive until induced to insolubility by binding to amylose beads. This functional link to insolubility also held true for the Y146A mutation of the CARD of ASC, which avoids insolubility and caspase-1 activation. Thus, we conclude that the role of ASC insolubility in inflammasome function is inextricably linked to its pyrin domain–mediated and CARD-mediated polymerizations. These findings will support future studies into the molecular mechanisms controlling ASC solubility.

New Aβ(1–42) ligands from anti-amyloid antibodies: Design, synthesis, and structural interaction

Alzheimer's disease (AD), is the most common neurodegenerative disorder of the aging population resulting in progressive cognitive and functional decline. Accumulation of amyloid plaques around neuronal cells is considered a critical pathogenetic event and, in most cases, a hallmark of the pathology. In the attempt to identify anti-AD drug candidates, hundreds of molecules targeting Aβ peptides have been screened. Peptide molecules have been widely explored, appreciating chemical stability, biocompatibility, and low production cost. More recently, many anti-Aβ(1–42) monoclonal antibodies have been developed, given the excellent potential of immunotherapy for treating or preventing AD.Antibodies are versatile ligands that bind a large variety of molecules with high affinity and specificity; however, their extensive therapeutic application is complex and requires huge economic investments. Novel approaches to identify alternative antibody formats are considered with great interest.In this context, taking advantage of the favorable peptide properties and the availability of Aβ-antibodies structural data, we followed an innovative research approach to identify short peptide sequences on the model of the binding sites of Aβ(1–42)/antibodies. WAibH and SYSTPGK were designed as mimics of solanezumab and aducanumab, respectively. Circular dichroism and nuclear magnetic resonance analysis reveal that the antibody-derived peptides interact with Aβ(1–42) in the soluble monomeric form. Moreover, AFM microscopy imaging shows that WAibH and SYSTPGK are capable of controlling the Aβ(1–42) aggregation. The strategy to identify WAibH and SYSTPGK is innovative and can be widely applied for new anti-Aβ antibody mimicking peptides.

Neoadjuvant Chemoradiotherapy Changes the Landscape of Soluble Immune Checkpoint Molecules in Patients With Locally Advanced Rectal Cancer.

BackgroundWe aimed to investigate clinical implications of specific soluble immune checkpoint molecules (sICMs) in locally advanced rectal cancer (LARC) patients treated with neoadjuvant chemoradiotherapy (nCRT).MethodsWe prospectively enrolled 30 LARC patients treated with nCRT and collected blood samples from them before, during, and after nCRT for prospective studies. Immune checkpoints often refer to T cell surface molecules influencing the immune response. Immune checkpoints, in the form of a soluble monomeric form, is widely present in blood. In the study, eight immune checkpoint-related plasma proteins, including programmed death-ligand 1 (PD-L1), CD80, CD86, CD28, CD27, glucocorticoid-induced tumor necrosis factor receptor (GITR), GITR ligand (GITRL), and inducible T-cell costimulator (ICOS), were measured using the Luminex platform. Two independent pathologists categorized patients as the good responders and the poor responders according to Dworak tumor regression grade (TRG).ResultsOf the 30 patients, the levels of sPD-L1, sCD80, sCD86, sCD28, sGITR, sGITRL, sCD27, and sICOS decreased during nCRT (Pre-nCRT vs. During-nCRT, all p<0.05) but were restored after nCRT treatment (Pre-nCRT vs. Post-nCRT, all p>0.05). In the 14 good responders, the levels of sICMs, other than sGITR (p=0.081) and sGITRL (p=0.071), decreased significantly during nCRT (Pre-nCRT vs. During-nCRT, p<0.05), but they were all significantly increased after nCRT (During-nCRT vs. Post-nCRT, all p<0.05). In the 16 poor responders, only sCD80 was significantly reduced during nCRT (Pre-nCRT vs. During-nCRT, p<0.05), and none was significantly increased after nCRT (During-nCRT vs. Post-nCRT, all p<0.05). High levels of sICMs before nCRT were associated with poor response (all OR≥1). The Pre-model that incorporated the 8 sICMs before nCRT yielded a good predictive value (AUC, 0.848) and was identified as an independent predictor of treatment response (OR, 2.62; 95% CI, 1.11-6.18; p=0.027).ConclusionOur results suggest chemoradiotherapy could influence the change of sPD-L1, sCD80, sCD86, sCD28, sGITR, sGITRL, sCD27, and sICOS in patients with LARC. The levels of the majority of soluble immune checkpoint molecules were reduced during nCRT and then restored at the end of nCRT, particularly in patients who responded well to nCRT. Combined baseline sICMs can be developed to predict treatment response.

Structural basis of Alzheimer β-amyloid peptide recognition by engineered lipocalin proteins with aggregation-blocking activity.

We describe the structural analysis of two Anticalin® proteins that tightly bind Aβ40, a peptide involved in the pathophysiology of Alzheimer's disease. These anticalins, US7 and H1GA, were engineered on the basis of the human lipocalin 2, thus yielding compact single-domain binding proteins as an alternative to antibodies. Albeit selected under different conditions and mutually deviating in 13 amino acid positions within the binding pocket (of 17 mutated residues in total), both crystallised anticalins recognize the same epitope in the middle of the β-amyloid peptide. In the two complexes with the Aβ40 peptide, its central part comprising residues LysP16 to LysP28 shows well defined electron density whereas the flanking regions appear structurally disordered. The compact zigzag-bend conformation which is seen in both structures may indicate a role during conversion of the soluble monomeric form into pathogenic Aβ state(s) and, thus, explain the aggregation-inhibiting effect of the anticalins. In contrast to solanezumab, which targets the same Aβ region in a different conformation, the anticalin H1GA does not show cross-reactivity with sequence-related human plasma proteins. Consequently, anticalins offer promising reagents to prevent oligomerization of Aβ peptides to neurotoxic species in vivo and their small size may enable new routes for brain delivery.

Identification of alpha‐Synuclein Disaggregator from Camellia sp. Insight of Molecular Docking and Molecular Dynamics Simulations

AbstractMisfolded protein formation and aggregation are the central hallmarks for various neurodegenerative disorders. When it comes to Parkinson's disease (PD), alpha‐synuclein (α‐syn) is the culprit protein. The presence of α‐syn protein in lewy bodies and lewy neurites confirmed its presence in the occurrence of PD. The protein is natively present in the soluble monomeric forms, but certain factors such as oxidative stress convert the structure into insoluble oligomeric formats. This study focuses on the inhibitory effects of various antioxidant compounds on α‐syn oligomerization. We had collected the list of compounds present in the Camellia sp. plant. Using a computational approach, we found the potential interaction sites between the antioxidant compounds and α‐syn using a computational approach. Molecular docking and simulation studies suggest that the compound Theaflavin‐3‐3‐digallate shows best interactions with −7.1 kcal/mol and can reduce the alpha‐sheet structure of α‐syn structure to loop region.

Molecular and kinetic characterization of two acetylcholinesterases with particular focus on the roles of two amino acid substitutions (Y390N and F392W) in Bemisia tabaci

Two acetylcholinesterases (AChEs) are present in Bemisia tabaci (BtAChE1 and BtAChE2). A conserved AChE mutation conferring organophosphate (OP) resistance (F392W in BtAChE1) is saturated in field populations despite its potential fitness cost, and a highly conserved amino acid residue forming the backdoor of AChE is substituted with a unique amino acid (Y390N in BtAChE1) in B. tabaci. Thus, the roles and relationships of the two amino acid substitutions in the evolutionary adaptation of B. tabaci remain to be elucidated, and little information is available on the catalytic and molecular properties of BtAChE1 and BtAChE2. To determine which AChE is a more relevant target of OPs and carbamates, the molecular and kinetic properties of BtAChE1 and BtAChE2 were investigated. Both BtAChE1 and BtAChE2 were exclusively expressed in head and thorax but not in abdomen, bound to the membrane via GPI anchoring, and present as dimeric forms. Soluble monomeric form was detected only in BtAChE2. The catalytic activity of baculovirus-expressed BtAChE1 was 19.5-fold higher than that of BtAChE2. The inhibition assay revealed that the F392W mutation in BtAChE1 enhanced resistance to OPs. The artificial substitution of N390 (wild form) to Y (putative ancient form) led to reduced catalytic efficiency and increased inhibition by glycoalkaloids, suggesting that the Y390N substitution in BtAChE1 may have been required for Solanaceae host adaptation. BtAChE1 was proven to function as a main catalytic enzyme for ACh hydrolysis, thus being the main target of OPs and carbamates.

High-speed atomic force microscopy to study pore-forming proteins.

Pore-forming proteins (PFPs) include virulence factors that are produced by many pathogenic bacteria. However, PFPs also comprise non-virulence factors, such as apoptotic Bcl2-like proteins, and also occur in non-pathogenic bacteria and indeed in all kingdoms of life. Pore-forming proteins are an ancient class of proteins, which are tremendously powerful in damaging cell membranes. In general, upon binding to lipid membranes, they convert from the soluble monomeric form into an oligomeric state, and then undergo a dramatic conformational change to form transmembrane pores. Thus, PFPs render the plasma membrane of their target cells permeable to solutes, potentially leading to cell death, or to more subtle manipulations of cellular functions. Recent cryo-EM and X-ray crystallography studies revealed high-resolution structures of several PFPs in their pre-pore and pore states, however many aspects regarding the cues that induce pore formation, the pre-pore to pore conformational transition, the mechanism of membrane permeation and associated dynamics are still less well understood, and direct visualization of the dynamics of these transitions are missing. Using high-speed atomic force microscopy (HS-AFM), the kinetics of oligomerization and the pre-pore to pore transition dynamics of various PFPs, such as Listeriolysin O (LLO), lysenin, and Perforin-2 (PFN2), could be studied. These studies revealed that LLO does not form pores of regular shape or size, but rather forms membrane inserted arcs that propagate and damage lipid membranes as lineactants. In contrast, lysenin forms stable pre-pore and pore nonameric rings and HS-AFM allowed to study their diffusion on and the pH-dependent insertion into the membrane. Similarly, PFN2 underwent pre-pore to pore transition upon acidification. The openness of the HS-AFM system allowed the transition to be imaged in real time and revealed that all observed molecules transitioned into the pore state within 3s. In this chapter, we detail protocols to prepare lipids, form supported lipid bilayers, and provide guidelines for real-time, real-space HS-AFM observations of PFPs in action.

Going full circle: Determining the structures of complement component 9.

Pore forming proteins (PFPs) undergo dramatic conformational changes to punch holes in the target membrane. These PFPs have the ability to self-assemble, by way of oligomerization, and have the capacity to transform from a water soluble state (commonly referred to as fluid phase) to a membrane adhered form. Accordingly, PFPs are metastable, that is they are inert until the right conditions cause the release of potential energy stored in the conformational fold leading to a vast structural rearrangement into a membrane-inserted oligomeric form. However, the metastable state of PFPs poses a problem of leading to aggregation and precipitation in conditions typically required for structural biology techniques. Here, we discuss the protein chemistry of the MACPF protein complement component 9 (C9). C9 is part of a larger complex assembly known as the membrane attack complex (MAC) that has been studied extensively for its ability to form pores in bacteria. An unusual artifact of human C9 is the ability to form a soluble oligomeric state of the channel portion of the MAC, called polyC9. PolyC9 formation does not require the presence of membranes or other complement factors. It is only in recent years that structural studies of the MAC have become successful owing to improved recombinant DNA expression systems and the improvement of high-resolution techniques (both X-ray crystallography and single particle cryo-EM). We discuss the expression and purification of recombinant C9, crystallization of the soluble monomeric form of C9 and the preparation of the oligomeric polyC9.

13C and 15N chemical shift assignments of A117V and M129V human Y145Stop prion protein amyloid fibrils.

The C-terminally truncated Y145Stop variant of prion protein (PrP23-144) has been linked to a heritable prionopathy in humans and is also capable of triggering a transmissible prion disease in mice. PrP23-144 can be converted from soluble monomeric form to amyloid under physiological conditions, providing an in vitro model for investigating the molecular basis of amyloid strains and cross-seeding barriers. Here, we use magic-angle spinning solid-state NMR to establish the sequential backbone and sidechain 13C and 15N chemical shift assignments for amyloid fibrils formed by the A117V and M129V mutants of human PrP23-144, which in the context of full length PrP in vivo are among the specific residues associated with development of Gerstmann-Straüssler-Scheinker disease. The chemical shift data are utilized to identify amino acids comprising the rigid amyloid core regions and to predict the protein secondary structures for human PrP23-144 A117V and M129V fibrils.

Purification and interactions of the MucA\u2019 and MucB proteins constituting the DNA polymerase RI

BackgroundThe MucA’ and MucB proteins comprise the core of DNA polymerase RI which is a strong mutator utilized in mutagenicity assays such as the standard Ames test. A close relative DNA polymerase V, composed of the homologous UmuD’ and UmuC proteins, is considered to be an ortholog of the mammalian DNA polymerase η. The catalytic subunits of these polymerases belong to the Y-family which specializes in the translesion DNA synthesis across various DNA adducts to rescue stalled chromosomal replication at the expense of mutations. Based on genetic evidence, DNA polymerase RI possesses the greatest ability to induce various types of mutations among all so far characterized members of the Y-superfamily. The exceptionally high mutagenic potential of MucA’B has been taken advantage of in numerous bacterial mutagenicity assays incorporating the conjugative plasmid pKM101 carrying the mucAB operon such as the Ames Test.ResultsWe established new procedures for the purification of MucB protein as well as its accessory protein MucA’ using the refolding techniques. The purified MucA’ protein behaved as a molecular dimer which was fully stable in solution. The soluble monomeric form of MucB protein was obtained after refolding on a gel-filtration column and remained stable in a nondenaturing buffer containing protein aggregation inhibitors. Using the surface plasmon resonance technique, we demonstrated that the purified MucA’ and MucB proteins interacted and that MucB protein preferentially bound to single-stranded DNA. In addition, we revealed that MucB protein interacted with the β-subunit of DNA polymerase III holoenzyme of E. coli.ConclusionThe MucA’ and MucB proteins can be isolated from inclusion bodies and solubilized in vitro. The refolded MucB protein interacts with its MucA’ partner as well as with DNA what suggests it retains biological activity. The interaction of MucB with the processivity subunit of DNA polymerase III may imply the role of the subunit as an accessory protein to MucB during the translesion DNA synthesis.

Non-Specific Porins of Yersinia pseudotuberculosis as Inductors of Experimental Hyperthyroidism in Mice.

In vivo experiments showed that antibodies to OmpC and OmpF porins of Yersinia pseudotuberculosis increased thyroxine (T4) level in the blood of experimental animals. The mice were immunized with different antigens: recombinant OmpF porin in a soluble monomeric form, trimers of OmpC and OmpF porins isolated from the outer membrane, or antibodies to them. The level of thyroxine in the blood of mice immunized with OmpF and OmpC porins increased by 5.47 and 22.3 times, respectively; after immunization with antibodies to these proteins, blood thyroxine increased by 9.28 and 14.29 times. Immunization with recombinant OmpF porin induced no reliable increase in thyroxine level. Hence, the serum to recombinant OmpF porin contains no antibodies specific to conformational antigenic determinants that are present in the protein trimer and, according to our previous findings from molecular docking studies, determine cross-reactions between OmpF porin of Y. pseudotuberculosis and thyroidstimulating hormone receptor.

Molecular and biochemical characterization of the bed bug salivary gland cholinesterase as an acetylcholine-sequestering enzyme

The common bed bug, Cimex lectularius, possesses a cholinesterase expressed exclusively in the salivary gland (ClSChE). In this study, we investigated the molecular forms, tissue distribution patterns and biochemical properties of ClSChE and showed that ClSChE exists as a soluble monomeric form or a soluble dimeric form connected by a disulfide bridge. Immunohistochemical analysis confirmed that ClSChE was expressed in the epithelial cells of both the salivary gland and the duct. In addition, the secretion of monomeric ClSChE through the proboscis during feeding was confirmed by western blotting using a ClSChE-specific antibody. To predict the role of ClSChE injected into the tissue of an animal host, we analyzed the extent of hydrolysis of acetylcholine (ACh) by ClSChE by ultra-performance liquid chromatography-tandem mass spectrometry. ClSChE binding to ACh was not clearly resolved in the binding assay format used in this study, probably due to the weak but detectable ACh-hydrolytic activity of ClSChE. Nevertheless, kinetic analysis revealed that ClSChE possesses extremely low Km (high affinity to ACh) and Vmax values. These findings suggest that ClSChE functions virtually as an ACh-sequestering protein by having a very strong affinity to ACh but an extremely long turnover time. Given that ACh regulates a wide variety of host physiologies, we discuss the tentative roles of ClSChE in blood vessel constriction and itch/pain regulation in the host.

ANALYSIS OF DOMAIN SPECIFICITY OF THE PROTECTIVE CHIMERIC ANTIBODY ch14D5a AGAINST GLYCOPROTEIN E OF TICK-BORNE ENCEPHALITIS VIRUS

A drug for the prevention and therapy of tick-borne encephalitis virus is being developed on the basis of the protective chimeric antibody ch14D5a. At the same time, the epitope recognized by this antibody on the surface of glycoprotein E has not been localized yet. The aim of this work was to identify the domain of glycoprotein E, to which the protective antibody ch14D5a binds. As a result, four recombinant variants of glycoprotein E were generated using the bacterial expression system: (1) the rE protein containing the domains D1, D2, and D3 of glycoprotein E; (2) the rED1+2 protein containing domains D1 and D2; (3) the rED3_301 protein, which is domain D3 of glycoprotein E, and (4) the rED3_294 protein comprising domain D3 and a hinge region connecting domains D1 and D3. The rED3_294 and rED3_301 proteins were obtained in soluble monomeric form. The rE and rED1+2 proteins were extracted from the inclusion bodies of Escherichia coli. Using Western blot analysis and surface plasmon resonance analysis, it was demonstrated that the protective chimeric antibody ch14D5a and its Fab fragment bound specifically to domain D3 of glycoprotein E. Since the antibodies recognizing epitopes on the surface of domain D3 do not tend to cause antibody-dependent enhancement of the infection as compared to antibodies directed to domains D1 and D2, the data obtained confirm the promise of using the antibody ch14D5a in the development of a therapeutic preparation against the tick-borne encephalitis virus.