Multimeric Protein Research Articles

LDL and HDL Have Opposing Effects on VWF Self-Association

Von Willebrand Factor (VWF) is a large, multimeric plasma protein with roles in primary hemostasis and thrombosis. VWF unfolds in response to shear stress and self-associates into adhesive fibers that can capture and hold platelets at sites of vascular injury, initiating platelet plug formation. VWF has also been implicated in pathologies associated with microvascular dysfunction and occlusion, and dysregulation of VWF can produce the catastrophic microvascular thrombotic disorder thrombotic thrombocytopenic purpura. VWF self-association is regulated by several factors: it is enhanced by shear stress, it is decreased by regulatory cleavage by ADAMTS13 metalloprotease[Dong et al. Blood, 2002, 100:4033], and our group recently showed that high density lipoprotein (HDL) attenuates VWF self-association [Chung et al. Blood, 2016, 127:637]. High levels of HDL are associated with decreased risk of thrombosis, whereas high levels of low density lipoprotein (LDL) is a strong risk factor. This led us to investigate whether LDL also influences VWF self-association and fiber formation.We studied the effects of lipoproteins on VWF self-association by applying shear stress to VWF solutions by vortexing, which caused VWF loss from solution through a process of adsorption and self-association [Chung et al. Blood, 2016, 127:637]. We inhibited the ADAMTS13 in citrated-plasma with EDTA, sheared the treated plasma in the presence of exogenous HDL or LDL, and measured the VWF remaining in solution by sandwich ELISA. HDL attenuated VWF self-association in plasma, whereas LDL had the opposite effect, enhancing self-association. Using purified components, we sheared recombinant VWF in the presence of a fixed concentration of HDL that stabilized the VWF, and added increasing amounts of LDL to attain LDL/HDL ratios found in physiological and pathophysiological conditions. As the molar ratio of LDL/HDL particles increased from 0.17 to 0.67, VWF self-association also increased. When we repeated these assays with double the concentrations of both lipoproteins, the effect on VWF self-association was comparable. We tested EDTA-treated platelet-poor-plasma with known LDL and HDL concentrations and found that the LDL/HDL ratio regulated VWF self-association in a similar manner, but the effect was less pronounced than with the purified components.To further assess the effect of LDL and HDL on VWF self-association under shear, we perfused purified plasma VWF in a microcolumn impingement device [Herbig & Diamond JTH, 2015, 13:1699] at a wall shear rate of 10,000/s, in the presence of PBS buffer, LDL, HDL, and LDL/HDL molar ratios of 0.17 and 0.67. VWF fiber formation was monitored by differential interference contrast and immunofluorescence microscopy. The width of the bundles of VWF fibers were measured and total fluorescence was quantified using image analysis software. The presence of LDL dramatically enhanced the size of VWF fibers and the rate of formation and deposition on the microcolumn, indicating that LDL interacts directly with VWF to enhance self-association. Perfusing HDL with VWF had the opposite effect and attenuated self-association. When VWF was perfused with LDL and HDL together, the LDL/HDL ratio regulated the thickness and length of the VWF fibers formed. Our findings reveal a novel role by which LDL, by enhancing VWF self-association, may contribute to VWF-mediated thrombosis. DisclosuresNo relevant conflicts of interest to declare.

Prognostic Impacts and Dynamic Changes of Cohesin Complex Gene Mutations in De Novo Acute Myeloid Leukemia

BackgroundPatients with acute myeloid leukemia (AML) have remarkable heterogeneity in the pathogenesis, clinical features, and treatment outcomes. Risk-stratification is one of the most critical factors in the treatment of these patients. Cohesin is a multimeric protein complex, composed of four core subunits, including SMC1A, SMC3, RAD21 and either STAG1 or STAG2 proteins. Studies regarding the clinical and prognostic relevance and dynamic changes of cohesin gene mutations in de novo AML patients are limited and the findings are controversial. In this study, we aimed to investigate the clinical, biological and prognostic implications of cohesin gene mutations in a large cohort of de novo AML patients.MethodWe analyzed cohesin gene mutations and their clinico-biological associations in 391 adult patients with newly diagnosed de novo non-M3 AML. To evaluate the dynamic changes of cohesin and co-occurring gene mutations, sequential analyses of gene mutations by targeted next generation sequencing using HiSeq platform (Illumina) were performed in 386 samples from 116 patients during follow-ups. Furthermore, to investigate the pathophysiological pathways associated with cohesin gene mutations, gene expression microarray analysis was performed in 173 patients, including 10 cohesin-mutated and 163 wild-type patients, who had enough mRNA for analysis.ResultsWe found 37 patients (9.5%) had cohesin gene mutations, most prevalent in RAD21 (3.8%) and STAG2 (3.1%). Except for one patient with concurrent mutations in STAG2 and RAD21, the mutations in these component genes were mutually exclusive, suggesting a convergence of biological effects of these mutations (Figure 1). Mutations in STAG2 and RAD21 were mainly truncations or frameshift mutations (10/12, 83.3% and 12/15, 80%, respectively) while those in SMC1A and SMC3 were mostly missense mutations (7/8, 87.5% and 2/2, 100%, respectively). Cohesin mutations were inversely associated with complex karyotype (P=0.023) and unfavorable-risk cytogenetics (P=0.003). Interestingly, six (16.7%) of the 38 patients with t(8;21) had cohesin gene mutations, all in RAD21, while none of the patients with inv(16) had any cohesin mutation. Compared with other cohesin genes mutations, STAG2 mutations more frequently co-occurred with RUNX1 mutations (27.3% vs. 0%, P=0.023) and tended to co-occurred with ASXL1 mutations (25.0% vs. 4.0%, P=0.084), but less frequently with NPM1 mutations (0% vs. 32.0%, P=0.036) (Figure 1) . In multivariate Cox proportional hazards regression analyses for overall survival (OS) and disease-free survival (DFS), the presence of cohesin gene mutations was an independent favorable factor (P=0.047 and 0.028, respectively). A scoring system incorporating cohesin mutations and other prognostic factors could well stratify the patients into different risk groups, which could be validated in another independent cohort. Serial analyses by next generation sequencing showed that two patients lost the original cohesin mutations during disease evolution, while none of the patients without the mutation acquired a novel one at relapse. We applied Bradley-Terry model to evaluate the temporal order of gene mutations in cohesin-mutated patients. The STAG2 mutations occurred as an early event, while RAD21 and SMC1A mutations occurred relatively late. Ingenuity Pathway Analysis revealed ERK1/2 as a hub gene in cohesin mutation-associated biological pathways. Furthermore, Gene Set Enrichment Analysis demonstrated that cohesin gene mutations were closely associated with several biological pathways involved in hematopoietic/myeloid/blood cell-related functions, including differentiation of blood cells (P=7.96×10-11), proliferation of blood cells (P=3.22×10-10), apoptosis (P=1.39×10-8), and cell death of blood cells (P=9.12×10-8).ConclusionCohesin gene mutations were independent prognostic factors in de novo AML. Further, cohesin gene mutations were strongly associated with the biological function related to proliferation and differentiation of blood cells. Sequential analyses showed cohesin gene mutations might be lost during disease evolution, but none of the patients without the mutation acquired a novel one during the clinical course indicating they may not be critical in disease progression. Further prospective studies with larger cohorts are warranted to confirm our findings. [Display omitted] DisclosuresTsai:Celgene International Sàrl: Research Funding. Hou:Celgene International Sàrl: Research Funding. Tang:Celgene International Sàrl: Research Funding. Kuo:Celgene International Sàrl: Research Funding. Lin:Celgene International Sàrl: Research Funding. Yao:Celgene International Sàrl: Research Funding. Li:Celgene International Sàrl: Research Funding. Huang:Celgene International Sàrl: Research Funding. Ko:Celgene International Sàrl: Research Funding. Lin:Celgene International Sàrl: Research Funding. Wu:Celgene International Sàrl: Research Funding. Chen:Celgene International Sàrl: Research Funding. Tsai:Celgene International Sàrl: Research Funding. Tien:Celgene International Sàrl: Research Funding.

Nelfinavir Blocks Export of Newly Synthesized Protein from the ER and Interacts with ER-Resident and Mitochondrial Proteins in an Activity-Dependent Fashion

The majority of multiple myeloma (MM) patients initially respond to proteasome inhibitor (PI)-based therapy, but later develop PI resistance and ultimately dies from PI-refractory MM. PI sensitivity of MM cells correlates with the activation status of the unfolded protein response (UPR) and its major activating axis IRE1/XBP1. PI-refractory MM cells downregulate IRE1/XBP1, and high IRE1/XBP1 expression correlates with clinical sensitivity to PI treatment. The anti-HIV drug nelfinavir induces IRE1/XBP1 and overcomes PI-resistance of MM in vitro . Nelfinavir showed unprecedented clinical activity in PI-refractory, heavily pretreated MM with ORR 65% in combination with bortezomib and dexamethasone. The molecular target of nelfinavir and its mechanism of action in MM cells are unknown. We synthesized structurally modified nelfinavir analogues equipped with a covalently protein-binding, photo-reactive moiety and functional groups that allow affinity purification of the nelfinavir analogue bound to its interacting cellular protein (SC-441), together with appropriate controls where the active center of nelfinavir was sterically occupied (SC-451). Isolation and identification of nelfinavir-interacting proteins in AMO-1 MM cells and cells adapted to carfilzomib (AMO-CFZ) was achieved with these tools by photoactivation in living cells, affinity purification and tandem mass spectrometry. We identified a small set of proteins that specifically bound to active nelfinavir. The majority of these were endoplasmic reticulum (ER)-resident proteins involved in protein quality control and transport from ER to Golgi apparatus. In addition, individual proteins from mitochondria or the nucleus were identified. We thus hypothesized that nelfinavir interferes with intracellular protein transport, in line with its UPR-activating properties. Using a construct of ST-SBP-TNFa-EGFP engineered with an ER-retention signal (KDEL) that can be released upon biotin treatment of cells (RUSH-system), in conjunction with confocal microscopy and flow cytometry in U2OS cells, we show that nelfinavir, but not its analogue SC-451, which lacks proteasome inhibitor-sensitizing activity, retains TNFa-EGFP in the ER and prevents its transport to the Golgi and into the extracellular space. We confirmed in MM cells that active nelfinavir, but not SC-451, likewise retains newly synthesized IgA by preventing its secretion, and similarly decreases MHC class I export to the cell surface.Functionally depletion of the putative ER-targets of nelfinavir by siRNA in U2OS cells sensitized cells to nelfinavir and proteasome inhibitors. The mitochondrial targets of nelfinavir were identified as constituents of the mitochondria permeability transition pore (mPTP), a multimeric protein complex that regulates the permeability of mitochondrial membranes. The formation of reactive oxygen species (ROS) and inhibition of ABCB1 efflux, known for nelfinavir, can be functionally linked to mPTP modulation.In conclusion, we demonstrate that nelfinavir blocks export of newly synthetized protein from the ER, consistent with its activity-dependent binding to ER proteins. The block in protein export from the ER by nelfinavir is consistent with its ER stress-inducing, IRE1/XBP1-activating, and PI-sensitizing effect in the clinic. We further identify mPTP as an additional protein target of nelfinavir, consistent with its ROS-inducing and ABCB1-modulating activity. The block in ER protein export represents a novel mechanism of action to sensitize MM cells for proteasome inhibitor treatment. DisclosuresNo relevant conflicts of interest to declare.

TERT and its binding protein: overexpression of GABPA/B in high grade gliomas.

Enhanced expression of TERT in gliomas is a result of two hotspot mutations, C228T and C250T, at the promoter region. GA-binding proteins selectively bind at these positions, respectively, causing an activation of the promoter and overexpression of TERT. GABP is a multimeric protein consisting of GABPA and GABPB with its isoforms GABPB1, GABPB1-L, GABPB1-S, GABPB2.In this study, we investigated the mRNA expression and association between TERT and GABPA/B isoforms in tumor samples of different glioma grades. The expression was determined by quantitative real-time PCR and the results were statistically analyzed.We present that TERT is mainly expressed in primary glioblastomas. All GA-binding proteins progress through the glioma grades and have the highest expression levels in secondary glioblastomas. In secondary glioblastomas after chemotherapy, GABPB1 and GABPB1-L are expressed on a lower level than without treatment. In high grades, TERT and GABPA, GAPB1, GABPB1-L, GABPB1-S are upregulated compared to low grades. Between primary and secondary glioblastomas with and without chemotherapy, TERT is elevated in the former while GABPB1 is increased in the secondary glioblastomas. GABPA and GABPB1, GABPB1-L and GABPB1-S positive correlate in primary glioblastomas.The present study confirms the upregulation of TERT in primary glioblastomas while all GABP proteins rise with the malignancy of the gliomas. Further investigations must be made to elucidate the relation between TERT and all GABP proteins as it may play a key role in the gliomagenesis.

Leishmania mexicana recombinant filamentous acid phosphatase as carrier for Toxoplasma gondii surface antigen 1 expression in Leishmania tarentolae.

Leishmania tarentolae has been used to produce recombinant intracellular and secreted proteins for their easy handling and posttranslational modifications. Filamentous acid phosphatase is a multimeric protein complex composed of many subunits assembled in a linear highly glycosylated filament, which is secreted in vast amounts into the culture supernatant via the flagellar pocket of Leishmania mexicana promastigotes. This suggested that the protein could be used as a carrier for the Surface Antigen1 protein of a Toxoplasma gondii (SAG1) for easy purification to generate a protein with multiple SAG1 subunits suitable for immunisation. SAG1 has an immunodominant structure that is involved in binding to host cells. Previous studies used this surface protein for vaccination for its immunological importance for triggering a type 1 immune response in the host. This study aims to determine the production of recombinant filamentous protein carried subunits of the surface protein of Toxoplasma gondii for vaccination purposes. Leishmania codon-optimised SAG1 was cloned as a fusion construct into pLEXSY-ble2.1 plasmid and introduced into Leishmania tarentolae to generate recombinant cell lines expressing a filamentous fusion protein called SAP2SAG1. PCR confirmed the correct integration into the small ribosomal subunit RNA gene locus of Leishmania tarentolae. Immunofluorescences and Immunoblot analyses were used to detect the fusion protein in the sediment of culture supernatants of recombinant L. tarentolae promastigotes after purification by ultracentrifugation. The yield of purified protein was low that suggested further investigations of other methods for scaling large production of secreted protein.

Development of a Modular Vaccine Platform for Multimeric Antigen Display Using an Orthobunyavirus Model.

Emerging infectious diseases represent an increasing threat to human and animal health. Therefore, safe and effective vaccines that could be available within a short time frame after an outbreak are required for adequate prevention and control. Here, we developed a robust and versatile self-assembling multimeric protein scaffold particle (MPSP) vaccine platform using lumazine synthase (LS) from Aquifex aeolicus. This scaffold allowed the presentation of peptide epitopes by genetic fusion as well as the presentation of large antigens by bacterial superglue-based conjugation to the pre-assembled particle. Using the orthobunyavirus model Schmallenberg virus (SBV) we designed MPSPs presenting major immunogens of SBV and assessed their efficacy in a mouse model as well as in cattle, a target species of SBV. All prototype vaccines conferred protection from viral challenge infection and the multivalent presentation of the selected antigens on the MPSP markedly improved their immunogenicity compared to the monomeric subunits. Even a single shot vaccination protected about 80% of mice from an otherwise lethal dose of SBV. Most importantly, the MPSPs induced a virtually sterile immunity in cattle. Altogether, LS represents a promising platform for modular and rapid vaccine design.

Channeling plant immunity

Plant intracellular NLR proteins detect pathogen effectors and then form multimeric protein complexes ("resistosomes") that activate immune responses and cell death through unknown mechanisms. In this issue of Cell, Bi etal. show that the ZAR1 resistosome exhibits cation channel activity, enabling calcium influx that activates defense mechanisms and culminates in cell death.

Heterodimer Formation of the Homodimeric ABC Transporter OpuA.

Many proteins have a multimeric structure and are composed of two or more identical subunits. While this can be advantageous for the host organism, it can be a challenge when targeting specific residues in biochemical analyses. In vitro splitting and re-dimerization to circumvent this problem is a tedious process that requires stable proteins. We present an in vivo approach to transform homodimeric proteins into apparent heterodimers, which then can be purified using two-step affinity-tag purification. This opens the door to both practical applications such as smFRET to probe the conformational dynamics of homooligomeric proteins and fundamental research into the mechanism of protein multimerization, which is largely unexplored for membrane proteins. We show that expression conditions are key for the formation of heterodimers and that the order of the differential purification and reconstitution of the protein into nanodiscs is important for a functional ABC-transporter complex.

Chaperonin point mutation enhances cadmium endurance in Saccharomyces cerevisiae.

To study the effect of the mutation in conserved G412E in Cct7p subunit of CCT complex on its cellular fate. TriC/CCT is a dynamic multimeric protein that assists in protein folding in an energy-dependent manner. A point mutation in the ATP binding pocket in the equatorial domain of the Cct7p subunit delays the doubling time. The cell size was twice the wild type, and the formation of protein aggregates suggests disturbed folding of the proteins. Upon growing in stressful conditions of arsenous acid and cadmium chloride, the mutant was lethal in As3+ but grew well in Cd2+ with 10.5µg cadmium uptake mg-1 compared to the wild type. The increased expression of vacuole transporters YCF1 and BPT1 by ten-fold and two-fold in mutant indicates the metal transportation to the vacuole. CCT complex was vulnerable to the mutation in G412E in the Cct7p subunit of protein folding molecular machinery. Interestingly, already stressed cells provided robustness against oxidative stress and cadmium sequestration in the vacuole.

Von Willebrand Factor Facilitates Intravascular Dissemination of Microsporidia Encephalitozoon hellem.

Microsporidia are a group of spore-forming, fungus-related pathogens that can infect both invertebrates and vertebrates including humans. The primary infection site is usually digestive tract, but systemic infections occur as well and cause damages to organs such as lung, brain, and liver. The systemic spread of microsporidia may be intravascular, requiring attachment and colonization in the presence of shear stress. Von Willebrand Factor (VWF) is a large multimeric intravascular protein and the key attachment sites for platelets and coagulation factors. Here in this study, we investigated the interactions between VWF and microsporidia Encephalitozoon hellem (E. hellem), and the modulating effects on E. hellem after VWF binding. Microfluidic assays showed that E. hellem binds to ultra-large VWF strings under shear stress. In vitro germination assay and infection assay proved that E. hellem significantly increased the rates of germination and infection, and these effects would be reversed by VWF blocking antibody. Mass spectrometry analysis further revealed that VWF-incubation altered various aspects of E. hellem including metabolic activity, levels of structural molecules, and protein maturation. Our findings demonstrated that VWF can bind microsporidia in circulation, and modulate its pathogenicity, including promoting germination and infection rate. VWF facilitates microsporidia intravascular spreading and systemic infection.

Self-assembly of protein superstructures by physical interactions under cytoplasm-like conditions

The structure-driven assembly of multimeric protein complexes and the formation of intracellular phase-like protein condensates have been the subject of intense research. However, the assembly of larger superstructures comprising cellular components, such as protein nanoparticles driven by general physical rather than specific biochemical interactions, remains relatively uncharacterized. Here, we use gas vesicles (GVs)—genetically encoded protein nanoparticles that form ordered intracellular clusters—as a model system to study the forces driving multiparticle assembly under cytoplasm-like conditions. Our calculations and experimental results show that the ordered assembly of GVs can be achieved by screening their mutual electrostatic repulsion with electrolytes and creating a crowding force with dissolved macromolecules. The precise balance of these forces results in different packing configurations. Biomacromolecules such as polylysine and DNA are capable of driving GV clustering. These results provide basic insights into how physically driven interactions affect the formation of protein superstructures, offer guidance for manipulating nanoparticle assembly in cellular environments through synthetic biology methods, and inform research on the biotechnology applications of GVs.

A super-potent tetramerized ACE2 protein displays enhanced neutralization of SARS-CoV-2 virus infection

Approaches are needed for therapy of the severe acute respiratory syndrome from SARS-CoV-2 coronavirus (COVID-19). Interfering with the interaction of viral antigens with the angiotensin converting enzyme 2 (ACE-2) receptor is a promising strategy by blocking the infection of the coronaviruses into human cells. We have implemented a novel protein engineering technology to produce a super-potent tetravalent form of ACE2, coupled to the human immunoglobulin γ1 Fc region, using a self-assembling, tetramerization domain from p53 protein. This high molecular weight Quad protein (ACE2-Fc-TD) retains binding to the SARS-CoV-2 receptor binding spike protein and can form a complex with the spike protein plus anti-viral antibodies. The ACE2-Fc-TD acts as a powerful decoy protein that out-performs soluble monomeric and dimeric ACE2 proteins and blocks both SARS-CoV-2 pseudovirus and SARS-CoV-2 virus infection with greatly enhanced efficacy. The ACE2 tetrameric protein complex promise to be important for development as decoy therapeutic proteins against COVID-19. In contrast to monoclonal antibodies, ACE2 decoy is unlikely to be affected by mutations in SARS-CoV-2 that are beginning to appear in variant forms. In addition, ACE2 multimeric proteins will be available as therapeutic proteins should new coronaviruses appear in the future because these are likely to interact with ACE2 receptor.

Integrating on-grid immunogold labeling and cryo-electron tomography to reveal photosystem II structure and spatial distribution in thylakoid membranes

A long-standing challenge in cell biology is elucidating the structure and spatial distribution of individual membrane-bound proteins, protein complexes and their interactions in their native environment. Here, we describe a workflow that combines on-grid immunogold labeling, followed by cryo-electron tomography (cryoET) imaging and structural analyses to identify and characterize the structure of photosystem II (PSII) complexes. Using an antibody specific to a core subunit of PSII, the D1 protein (uniquely found in the water splitting complex in all oxygenic photoautotrophs), we identified PSII complexes in biophysically active thylakoid membranes isolated from a model marine diatom Phaeodactylum tricornutum. Subsequent cryoET analyses of these protein complexes resolved two PSII structures: supercomplexes and dimeric cores. Our integrative approach establishes the structural signature of multimeric membrane protein complexes in their native environment and provides a pathway to elucidate their high-resolution structures.

Neutralizing antibody vaccine for pandemic and pre-emergent coronaviruses.

Betacoronaviruses caused the outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome, as well as the current pandemic of SARS coronavirus2 (SARS-CoV-2)1-4. Vaccines that elicit protective immunity against SARS-CoV-2 and betacoronaviruses that circulate in animals have the potential to prevent future pandemics. Here we show that the immunization of macaques with nanoparticles conjugated with the receptor-binding domain of SARS-CoV-2, and adjuvanted with 3M-052 and alum, elicits cross-neutralizing antibody responses against bat coronaviruses, SARS-CoV and SARS-CoV-2 (including the B.1.1.7, P.1 and B.1.351 variants). Vaccination of macaques with these nanoparticles resulted in a 50% inhibitory reciprocal serum dilution (ID50) neutralization titre of 47,216 (geometric mean) for SARS-CoV-2, as well as in protection against SARS-CoV-2 in the upper and lower respiratory tracts. Nucleoside-modified mRNAs that encode a stabilized transmembrane spike or monomeric receptor-binding domain also induced cross-neutralizing antibody responses against SARS-CoV and bat coronaviruses, albeit at lower titres than achieved with the nanoparticles. These results demonstrate that current mRNA-based vaccines may provide some protection from future outbreaks of zoonotic betacoronaviruses, and provide a multimeric proteinplatform for the further development of vaccines against multiple (or all) betacoronaviruses.

Exploring Directionality and Kinetics of a Bacterial Type IV Secretion System

Type IV Secretion Systems (T4SSs) are large multimeric protein complexes that secrete diverse substrates across a donor cell membrane. While some pathogenic bacteria use T4SSs to secrete virulence factors into host cells, many bacteria utilize T4SSs to transfer DNA from donor to recipient cell during the process of conjugation. T4SSs can not only transfer their cognate conjugative DNA element, they can mobilize resident plasmids that do not encode their own dedicated conjugation machinery. Here, we have studied the kinetics and directionality of DNA transfer by the T4SS encoded by the conjugative element ICEBs1 of the bacterium Bacillus subtilis. Prior research has shown that ICEBs1 supports very high conjugation and plasmid mobilization frequencies. We compared the timing and frequency of ICEBs1-dependent conjugation and plasmid mobilization. We mixed donor and recipients cells, each containing a mobilizable plasmid with a different antibiotic resistance marker, and placed them on solid filters for various periods of time to allow for DNA transfer. The cell mixtures were then transferred to selective media to measure the frequency of DNA transfer. Mating and plasmid mobilization were found to occur within 2 minutes and peaked after 1-2 hours. Reverse mobilization, in which the donor receives DNA from the recipient, was found to occur after 1-2 hours and was at least 2 logs less frequent than mating. As expected, reverse mobilization involved a two-step process, in which the conjugative DNA element is first transferred from the donor to the recipient, allowing the recipient to then produce its own T4SS and transfer DNA back to the donor.

Increased VWF and Decreased ADAMTS-13 in COVID-19: Creating a Milieu for (Micro)Thrombosis.

von Willebrand factor (VWF) is a large adhesive multimeric protein involved in hemostasis. The larger the size (or number of VWF multimers), the greater the functionality of the protein. A deficiency or defect of VWF can lead to von Willebrand disease (VWD) and cause bleeding. Conversely, an increase in VWF may create an environment that promotes thrombosis. ADAMS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), sometimes called VWF-cleaving protease, is primarily responsible for controlling the size of VWF. The most severe deficiency (<10% of normal levels) of ADAMTS-13 arises in thrombotic thrombocytopenic purpura, a condition characterized by the presence of ultralarge VWF and clinically resulting in enhanced risk of thrombosis. However, ADAMTS-13 deficiency may result from other pathological processes. Of relevance is the recent finding that COVID-19 (coronavirus disease 2019) is associated with both increased levels and activity of VWF as well as generally decreased (or occasionally normal) activity levels of ADAMTS-13. Thus, in COVID-19 there is an alteration in the VWF/ADAMTS-13 axis, most often described by increased VWF/ADAMTS-13 ratio (or reduced ADAMTS-13/VWF ratio). COVID-19 is also associated with high prothrombotic risk. Thus, the imbalance of VWF and ADAMTS-13 in COVID-19 may be providing a milieu that promotes (micro)thrombosis, in a clinical picture resembling a secondary thrombotic microangiopathy in some patients. This review therefore assesses the literature on VWF, ADAMTS-13, and COVID-19. Whenever reported in COVID-19, VWF has always been identified as raised (compared with normal reference ranges or control populations). Reports have included VWF level (i.e., VWF antigen) and in some cases one or more VWF "activity" (e.g., collagen binding; platelet glycoprotein Ib [GPIb] binding, using ristocetin cofactor or more modern versions including VWF:GPIbR [recombinant] and VWF:GPIbM [mutant]). Whenever reported, ADAMTS-13 has been reported as "normal" or reduced; however, it should be recognized that "normal" levels may still identify a relative reduction in individual cases. Some reports also discuss the raised VWF/ADAMTS-13 (or reduced ADAMTS-13/VWF) ratio, but very few provide actual numerical data.

Dynamic Protease Activation on a Multimeric Synthetic Protein Scaffold via Adaptable DNA-Based Recruitment Domains.

Hexameric hemoprotein (HTHP) is employed as a scaffold protein for the supramolecular assembly and activation of the apoptotic signalling enzyme caspase‐9, using short DNA elements as modular recruitment domains. Caspase‐9 assembly and activation on the HTHP platform due to enhanced proximity is followed by combinatorial inhibition at high scaffold concentrations. The DNA recruitment domains allow for reversible switching of the caspase‐9 assembly and activity state using short modulatory DNA strands. Tuning of the recruitment domain affinity allows for generating kinetically trapped active enzyme complexes, as well as for dynamic repositioning of caspases over scaffold populations and inhibition using monovalent sink platforms. The conceptual combination of a highly structured multivalent protein platform with modular DNA recruitment domains provides emergent biomimicry properties with advanced levels of control over protein assembly.

Dynamic Protease Activation on a Multimeric Synthetic Protein Scaffold via Adaptable DNA‐Based Recruitment Domains

AbstractHexameric hemoprotein (HTHP) is employed as a scaffold protein for the supramolecular assembly and activation of the apoptotic signalling enzyme caspase‐9, using short DNA elements as modular recruitment domains. Caspase‐9 assembly and activation on the HTHP platform due to enhanced proximity is followed by combinatorial inhibition at high scaffold concentrations. The DNA recruitment domains allow for reversible switching of the caspase‐9 assembly and activity state using short modulatory DNA strands. Tuning of the recruitment domain affinity allows for generating kinetically trapped active enzyme complexes, as well as for dynamic repositioning of caspases over scaffold populations and inhibition using monovalent sink platforms. The conceptual combination of a highly structured multivalent protein platform with modular DNA recruitment domains provides emergent biomimicry properties with advanced levels of control over protein assembly.

Dimerization Drives Proper Folding of Human Alanine:Glyoxylate Aminotransferase But Is Dispensable for Peroxisomal Targeting.

Peroxisomal matrix proteins are transported into peroxisomes in a fully-folded state, but whether multimeric proteins are imported as monomers or oligomers is still disputed. Here, we used alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal 5′-phosphate (PLP)-dependent enzyme, whose deficit causes primary hyperoxaluria type I (PH1), as a model protein and compared the intracellular behavior and peroxisomal import of native dimeric and artificial monomeric forms. Monomerization strongly reduces AGT intracellular stability and increases its aggregation/degradation propensity. In addition, monomers are partly retained in the cytosol. To assess possible differences in import kinetics, we engineered AGT to allow binding of a membrane-permeable dye and followed its intracellular trafficking without interfering with its biochemical properties. By fluorescence recovery after photobleaching, we measured the import rate in live cells. Dimeric and monomeric AGT displayed a similar import rate, suggesting that the oligomeric state per se does not influence import kinetics. However, when dimerization is compromised, monomers are prone to misfolding events that can prevent peroxisomal import, a finding crucial to predicting the consequences of PH1-causing mutations that destabilize the dimer. Treatment with pyridoxine of cells expressing monomeric AGT promotes dimerization and folding, thus, demonstrating the chaperone role of PLP. Our data support a model in which dimerization represents a potential key checkpoint in the cytosol at the crossroad between misfolding and correct targeting, a possible general mechanism for other oligomeric peroxisomal proteins.

Designing of a chimeric protein contains StxB, intimin and EscC against toxicity and adherence of enterohemorrhagic Escherichia coli O157:H7 and evaluation of serum antibody titers against it

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain is known as one of the major human foodborne pathogens. Lack of effective clinical treatment for human diarrheal diseases confirms the need for vaccine production against enteric bacteria such as E.coli O157:H7. Shiga-like toxin (Stx), EscC, and Intimin are the main important virulent factors of this enteric pathogen. In the present study, a comparative Omics analysis was conducted to identify most invasion EHEC antigenic factors as a potential immunogen. SEI (Stx-EscC-Intimin) trivalent chimeric protein was designed from the exposed and epitope rich part of these virulence factors. Sequence optimization, physicochemical properties, mRNA folding, three-dimensional structure and immunoinformatics data were investigated. The chimeric gene was synthesized with codon bias of E. coli. Recombinant protein was expressed and confirmed by western blot analysis. To evaluate the immunogenicity of the designed protein, the protein was administered to BALB/c mice and the serum IgG was determined by ELISA. Based on the Ramachandran plot, the validation data showed that 90.1 % of residues lie in the favored region. The high antigenicity of the multimeric protein was predicted by the immunoinformatic analysis. Epitope prediction had shown the proper distribution of linear and conformational B-cell epitopes and the competition of T-cell epitopes to bind MHC molecules too. Recombinant ESI Protein with 74.5 kDa was expressed in E. coli. Western blot analysis by anti-Stx antibody, confirmed a single band of chimeric protein. Consequently, the chimeric gene was designed and constructed after assessments. From in silico approach, the protein deduced from this cassette can be an immunogen candidate, and act against toxicity and adherence of EHEC.