Full-length Protein Research Articles

The homodimerization domain of the Stl repressor is crucial for efficient inhibition of mycobacterial dUTPase

The dUTPase is a key DNA repair enzyme in Mycobacterium tuberculosis, and it may serve as a novel promising anti-tuberculosis target. Stl repressor from Staphylococcus aureus was shown to bind to and inhibit dUTPases from various sources, and its expression in mycobacterial cells interfered with cell growth. To fine-tune and optimize Stl-induced inhibition of mycobacterial dUTPase, we aimed to decipher the molecular details of this interaction. Structural background of the complex between dUTPase and a truncated Stl lacking the repressor C-terminal homodimerization domain has been described, however, the effects of this truncation of Stl on enzyme binding and inhibition are still not known. Using several independent biophysical, structural and enzyme kinetic methods, here we show that lack of the repressor homodimerization domain strongly perturbs both enzyme binding and inhibition. We also investigated the role of a mycobacteria-specific loop in the Stl-interaction. Our results show that removal of this loop leads to a ten-fold increase in the apparent inhibition constant of Stl. We present a high-resolution three-dimensional structure of mycobacterial dUTPase lacking the genus-specific loop for structural insight. Our present data suggest that potent inhibition of mycobacterial dUTPase by Stl requires the wild-type full-length protein context.

SOP-MULTI: A Self-Organized Polymer-Based Coarse-Grained Model for Multidomain and Intrinsically Disordered Proteins with Conformation Ensemble Consistent with Experimental Scattering Data.

Multidomain proteins with long flexible linkers and full-length intrinsically disordered proteins (IDPs) are best defined as an ensemble of conformations rather than a single structure. Determining high-resolution ensemble structures of such proteins poses various challenges by using tools from experimental structural biophysics. Integrative approaches combining available low-resolution ensemble-averaged experimental data and in silico biomolecular reconstructions are now often used for the purpose. However, extensive Boltzmann weighted conformation sampling for large proteins, especially for ones where both the folded and disordered domains exist in the same polypeptide chain, remains a challenge. In this work, we present a 2-site per amino-acid resolution SOP-MULTI force field for simulating coarse-grained models of multidomain proteins. SOP-MULTI combines two well-established self-organized polymer models─: (i) SOP-SC models for folded systems and (ii) SOP-IDP for IDPs. For the SOP-MULTI, we introduce cross-interaction terms between the beads belonging to the folded and disordered regions to generate conformation ensembles for full-length multidomain proteins such as hnRNP A1, TDP-43, G3BP1, hGHR-ECD, TIA1, HIV-1 Gag, polyubiquitin, and FUS. When back-mapped to all-atom resolution, SOP-MULTI trajectories faithfully recapitulate the scattering data over the range of the reciprocal space. We also show that individual folded domains preserve native contacts with respect to solved folded structures, and root-mean-square fluctuations of residues in folded domains match those obtained from all-atom molecular dynamics simulation trajectories of the same folded systems. SOP-MULTI force field is made available as a LAMMPS-compatible user package along with setup codes for generating the required files for any full-length protein with folded and disordered regions.

A single dose recombinant AAV based CHIKV vaccine elicits robust and durable protective antibody responses in mice.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that is responsible for Chikungunya fever, which is characterized by fever, rash, and debilitating polyarthralgia. Since its re-emergence in 2004, CHIKV has continued to spread to new regions and become a severe health threat to global public. Development of safe and single dose vaccines that provide durable protection is desirable to control the spread of virus. The recombinant adeno-associated virus (rAAV) vectors represent promising vaccine platform to provide prolonged protection with a single-dose immunization. In this study, we developed a rAAV capsid serotype 1 vector based CHIKV vaccine and evaluated its protection effect against CHIKV challenge. The recombinant AAV1 encoding the full-length structural proteins of CHIKV (named as rAAV1-CHIKV-SP) was generated in vitro by transfecting the plasmids of AAV helper-free system into HEK-293T cells. The safety and immunogenicity of rAAV1-CHIKV-SP were tested in 4-week-old C57BL/6 mice. The antibody responses of the mice receiving prime-boost or single-dose immunization of the vaccine were determined by ELISA and plaque reduction neutralizing test. The immunized mice were challenged with CHIKV to evaluate the protection effect of the vaccine. The rAAV1-CHIKV-SP showed remarkable safety and immunogenicity in C57BL/6 mice. A single dose intramuscular injection of rAAV1-CHIKV-SP elicited high level and long-lasting antibody responses, and conferred complete protection against a heterologous CHIKV strain challenge. These results suggest rAAV1-CHIKV-SP represents a promising vaccine candidate against different CHIKV clades with a simplified immunization strategy.

The structures of protein kinase A in complex with CFTR: Mechanisms of phosphorylation and noncatalytic activation

Protein kinase A (PKA) is a key regulator of cellular functions by selectively phosphorylating numerous substrates, including ion channels, enzymes, and transcription factors. It has long served as a model system for understanding the eukaryotic kinases. Using cryoelectron microscopy, we present complex structures of the PKA catalytic subunit (PKA-C) bound to a full-length protein substrate, the cystic fibrosis transmembrane conductance regulator (CFTR)-an ion channel vital to human health. CFTR gating requires phosphorylation of its regulatory (R) domain. Unphosphorylated CFTR engages PKA-C at two locations, establishing two "catalytic stations" near to, but not directly involving, the R domain. This configuration, coupled with the conformational flexibility of the R domain, permits transient interactions of the eleven spatially separated phosphorylation sites. Furthermore, we determined two structures of the open-pore CFTR stabilized by PKA-C, providing a molecular basis to understand how PKA-C stimulates CFTR currents even in the absence of phosphorylation.

In silico analysis of R2R3-MYB transcription factors in the basal eudicot model, Aquilegia coerulea

R2R3-MYBs are an important group of transcription factors that regulate crucial developmental processes across the plant kingdom; yet no comprehensive analysis of the R2R3-MYBs in the early-diverging eudicot clade of Ranunculaceae has been conducted so far. In the present study, Aquilegia coerulea is chosen to understand the extent of conservation and divergence of R2R3-MYBs as a representative of the family by analysing the genomic distribution, organization, gene structure, physiochemical properties, protein architecture, evolution and possible mode of expansion. Genome-wide analysis showed the presence of 82 putative homologues classified into 21 subgroups, based on phylogenetic analysis of full-length protein sequences. The domain has remained largely conserved across all homologues with few differences from the characterized Arabidopsis thaliana R2R3-MYBs. The topology of the phylogenetic tree remains the same when full-length protein sequences are used, indicating that the evolution of R2R3-MYBs is driven by the domain region only. This is supported by the presence of similar structures of exon–intron and conserved motifs within the same subgroup. Furthermore, comparisons of the AqcoeR2R3-MYB members with monocots and core-eudicots revealed the evolutionary expansion of a few functional clades, such as A. thaliana R2R3-MYB subgroup 6 (SG6), the upstream regulatory factors of floral pigment biosynthesis and floral color. The reconstructed evolutionary history of SG6-like genes across angiosperms highlights the occurrence of independent duplication events in the genus Aquilegia. AqcoeR2R3-MYB genes are present in all seven chromosomes of A. coerulea, most of which result from local and segmental duplications. Selection analysis of these duplicated gene pairs indicates purifying selection except one, and the physiochemical analyses of R2R3-MYBs reveal differences among the MYBs signifying their functional diversification. This study paves the way for further investigation of paralogous copies and their probable role in the evolution of different floral traits in A. coerulea. It lays the foundation for functional genomic studies of R2R3-MYBs in the basal eudicots and facilitates comparative studies among angiosperms. The work also provides a framework for deciphering novel genetic regulatory pathways that govern the diversity of floral morphology.

Dysfunctional atrial fibroblast processing of filamin A in atrial fibrillation

Abstract Background/Introduction Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, is associated with atrial structural remodelling, hallmarked by fibrosis. Atrial fibrosis is instigated by atrial fibroblasts (AFBs). It was previously uncovered the hormone calcitonin (CT) is produced in the heart and, via binding to the CT-receptors (CTR), inhibits atrial fibrogenesis and fibroblast profibrotic activity. [1] In AF, abnormally increased CTR internalisation causes loss of physiological surface CTR and prevents fibroblast activation by CT. The molecular mechanisms of this phenomenon in ACFs are unknown. Aim To explore the role of filamin A (FLNa), an actin cross-linking protein that was previously shown to interact with CTR, [2] in the regulation of CTR trafficking. Methods AFBs were enzymatically isolated from left atrial appendage biopsies collected from 30 patients in persistent AF or normal sinus rhythm (SR, controls) undergoing elective heart surgery. Immunostaining assessed the localisation of CTR and its overlap with the nucleus (stained with DAPI) and FLNa in AFBs. Co-localisation was quantified by the JACoP BIOP plugin for ImageJ. Target mRNA expression was assessed by RT-qPCR and protein levels by immunoblot. Immunoprecipitation (IP) then immunoblot (IB) validated protein-protein interactions. Calpain activity was assessed with a fluorescence-based assay. The effects of calpains on FLNa proteolytic processing was examined with calpain inhibitor calpeptin (10nM–100nM vs DMSO loading control). Results Immunostaining revealed aberrant, intracellular localisation of CTR in AF-AFBs (A) with increased nuclear localisation (p=0.03) and reduced co-localisation with FLNa (p=0.03). FLNa gene expression showed a trend towards reduction in AF (by 36%, p=0.06; B). Meanwhile, although full-length FLNa protein was unaltered in AF, there was a striking 67% reduction (p=0.02) in the expression of FLNa’s c-terminal fragment (FLNaCT), which contains the CTR binding domain (C). Co-immunoprecipitation confirmed a physical interaction between CTR and FLNaCT (D) in both SR and AF AFBs. FLNaCT is typically generated when FLNa is cleaved by the protease calpain 1, whose protein (F) but not mRNA (E), was significantly downregulated (71%, p<0.001) in AF. Puzzlingly, a ~70% loss of calpain 1 protein was associated with unchanged calpain activity (G) and FLNaCT levels in AFBs treated with calpain inhibitor calpeptin (H). Conclusions Persistent AF is associated with an aberrant CTR localisation and FLNa proteolytic processing, not attributable to altered calpain activity. Further characterisation of the molecular determinants of the altered CTR localisation may uncover novel and potentially druggable facets of structural remodelling in AF.

Modulating TTN gene expression in human iPSC-CMs to investigate mechanisms and therapies in TTN-associated dilated cardiomyopathy

Abstract Background Familial dilated cardiomyopathy (DCM) is a common and harmful condition, and no available therapies target the underlying genetic mechanisms. Truncating variants in TTN are the most commonly identified genetic cause of DCM. However, it remains unclear how TTN variants drive disease. Contemporary thinking identifies haploinsufficiency, dominant negative effects, or a combination of both as likely driving mechanisms. There is competing evidence as to whether I-band and A-band mutations differ in severity or in the relative roles of haploinsufficiency and dominant-negative effects. Clarifying disease mechanisms will help pave the way to targeted genetic therapies. Purpose To modulate TTN gene expression in human-induced stem cell-derived cardiomyocyte (hiPSC-CM) models of TTN-associated DCM carrying I-band or A-band variants to provide insight into the comparative roles of haploinsufficiency and dominant negative mechanisms in DCM, and to identify novel therapeutic strategies. Methods We used CRISPR-Cas9 gene-editing to generate hiPSC lines with known disease-causing heterozygous truncating mutations in the I-band or A-band of TTN. These were screened for off-target mutations and pluripotency. hiPSCs were differentiated into hiPSC-CMs for phenotyping. We assayed TTN mRNA and titin protein levels. We trialled approaches for modulating TTN expression including CRISPR activation. Results Two hiPSC lines with heterozygous mutations in the I-band and A-band of TTN were generated using CRISPR/Cas-9 gene-editing. These hiPSC lines were successfully differentiated into hiPSC-CMs. Both cell lines demonstrated unchanged TTN mRNA expression compared to wildtype controls, but had reduced expression of full-length titin protein and identifiable truncated protein products. TTN mRNA expression was subsequently increased in these models using modulators of gene expression. Conclusions hiPSC-CM models of DCM with truncating mutations in the I-band or A-band of TTN were generated and phenotyped. These models were then used to trial gene expression modulation methods. This provides a human cellular system that can be used to better understand disease mechanisms of TTN-associated DCM, with the ultimate aim of establishing gene therapies for TTN-associated DCM.

Self-association and multimer formation in AtLEA4-5, a desiccation-induced intrinsically disordered protein from plants.

During seed maturation, plants may experience severe desiccation, leading to the accumulation of late embryogenesis abundant (LEA) proteins. These intrinsically disordered proteins also accumulate in plant tissues under water deficit. Functional roles of LEA proteins have been proposed based on in vitro studies, where monomers are considered as the functional units. However, the potential formation of homo-oligomers has been little explored. In this work, we investigated the potential self-association of Arabidopsis thaliana group 4 LEA proteins (AtLEA4) using in vitro and in vivo approaches. LEA4 proteins represent a compelling case of study due to their high conservation throughout the plant kingdom. This protein family is characterized by a conserved N-terminal region, with a high alpha-helix propensity and invitro protective activity, as compared to the highly disordered and low-conserved C-terminal region. Our findings revealed that full-length AtLEA4 proteins oligomerize and that both terminal regions are sufficient for self-association in vitro. However, the ability of both amino and carboxy regions of AtLEA4-5 to self-associate invivo is significantly lower than that of the entire protein. Using high-resolution and quantitative fluorescence microscopy, we were able to disclose the unreported ability of LEA proteins to form high-order oligomers in planta. Additionally, we found that high-order complexes require the simultaneous engagement of both terminal regions, indicating that the entire protein is needed to attain such structural organization. This research provides valuable insights into the self-association of LEA proteins in plants and emphasizes the role of protein oligomer formation.

AlphaFold-Multimer accurately captures interactions and dynamics of intrinsically disordered protein regions

Interactions mediated by intrinsically disordered protein regions (IDRs) pose formidable challenges in structural characterization. IDRs are highly versatile, capable of adopting diverse structures and engagement modes. Motivated by recent strides in protein structure prediction, we embarked on exploring the extent to which AlphaFold-Multimer can faithfully reproduce the intricacies of interactions involving IDRs. To this end, we gathered multiple datasets covering the versatile spectrum of IDR binding modes and used them to probe AlphaFold-Multimer's prediction of IDR interactions and their dynamics. Our analyses revealed that AlphaFold-Multimer is not only capable of predicting various types of bound IDR structures with high success rate, but that distinguishing true interactions from decoys, and unreliable predictions from accurate ones is achievable by appropriate use of AlphaFold-Multimer's intrinsic scores. We found that the quality of predictions drops for more heterogeneous, fuzzy interaction types, most likely due to lower interface hydrophobicity and higher coil content. Notably though, certain AlphaFold-Multimer scores, such as the Predicted Aligned Error and residue-ipTM, are highly correlated with structural heterogeneity of the bound IDR, enabling clear distinctions between predictions of fuzzy and more homogeneous binding modes. Finally, our benchmarking revealed that predictions of IDR interactions can also be successful when using full-length proteins, but not as accurate as with cognate IDRs. To facilitate identification of the cognate IDR of a given partner, we established "minD," which pinpoints potential interaction sites in a full-length protein. Our study demonstrates that AlphaFold-Multimer can correctly identify interacting IDRs and predict their mode of engagement with a given partner.

Functional characterization of chlorophyll b reductase NON-YELLOW COLORING 1 in Medicago truncatula

Chlorophyll degradation is a characteristic process of leaf senescence. Two mutant lines, which showed green leaves and seeds during senescence, were identified by screening a Tnt-1 retrotransposon-tagged population of Medicago truncatula. Genetic and molecular analyses indicated that the mutated gene is NON-YELLOW COLORING 1 (MtNYC1) in M. truncatula. MtNYC1 encoded a chlorophyll b reductase, characterized by three transmembrane domains and a catalytic site (Y***K). Our investigation further identified three splicing variants of MtNYC1, encoding a full-length protein (MtNYC1A) and two truncated proteins (MtNYC1B, MtNYC1C). Genetic evidence indicated that the catalytic site and the third transmembrane domain were critical domains for chlorophyll b reductase. The coordinated action of three splicing variants plays a pivotal role in the degradation of chlorophyll during the senescence of leaves. This discovery provides precise target sites for the development of stay-green legume cultivars.

Headless hemagglutinin-containing influenza viral particles direct immune responses toward more conserved epitopes.

Seasonal influenza vaccines provide mostly strain-specific protection due to the elicitation of antibody responses focused on evolutionarily plastic antigenic sites in the hemagglutinin head domain. To direct the humoral response toward more conserved epitopes, we generated an influenza virus particle where the full-length hemagglutinin protein was replaced with a membrane-anchored, "headless" variant while retaining the normal complement of other viral structural proteins such as the neuraminidase as well as viral RNAs. We found that a single administration of a headless virus particle-based vaccine elicited high titers of antibodies that recognized more conserved epitopes on the major viral glycoproteins. Furthermore, the vaccine could elicit these responses even in the presence of pre-existing, hemagglutinin (HA) head-focused influenza immunity. Importantly, these antibody responses mediated protective, but non-neutralizing functions such as neuraminidase inhibition and antibody-dependent cellular cytotoxicity. Additionally, we show the vaccine can provide protection from homologous and heterologous challenges in mouse models of severe influenza without any measurable HA head-directed antibody responses. Thus, headless hemagglutinin containing viral particles may represent a tool to drive the types of antibody responses predicted to increase influenza vaccine breadth and durability.IMPORTANCECurrent seasonal influenza vaccines provide incomplete protection from disease. This is partially the result of the antibody response being directed toward parts of the virus that are tolerant of mutations. Redirecting the immune response to more conserved regions of the virus has been a central strategy of next-generation vaccine designs and approaches. Here, we develop and test a vaccine based on a modified influenza virus particle that expresses a partially deleted hemagglutinin protein along with the other viral structural proteins. We demonstrate this vaccine elicits antibodies that recognize the more conserved viral epitopes of the hemagglutinin stalk and neuraminidase protein to facilitate protection against influenza viruses despite a lack of classical viral neutralization activity.

Tackling APOE's structural challenges via in silico modeling in the era of neural networks: Can AlphaFold II help circumvent the problem of lacking full-length protein structure?

The APOE gene, encoding apolipoprotein E, is a predictor of longevity and age-related diseases. Despite numerous genetic studies, the data on molecular mechanisms by which apolipoprotein E affects the human phenotype remain incomplete due to the structural properties of the protein. Recently, a number of studies have used in silico drug discovery techniques based on protein-ligand docking, further highlighting the issue of lacking 3D structure of apolipoprotein E. Using molecular dynamics simulation, we found that AlphaFold II models of apolipoprotein E conformationally significantly differ both from the only available NMR structure, 2L7B, and structures obtained through circular dichroism spectroscopy: the ε4 isoform lacks the salt bridge between R61 and E255, while the ε2 and ε3 isoforms have extensive networks of interdomain interactions. Our findings challenge the benefits of using AlphaFold II for obtaining starting conformations for molecular docking.

Binding mode of cyclic chemerin-9 peptide and chemerinS157 protein at CMKLR.

The chemokine-like receptor 1 (CMKLR1) is activated by the adipokine and chemoattractant protein chemerin. Cryo-EM structures of chemerin-9-CMKLR1-Gi have been published, where chemerin-9 is the nonapeptide of the C terminus of chemerinS157. Chemerin-9 is as active as the full-length protein in Ca2+-release but shows differences in equilibrium read-outs. An equally potent cyclic chemerin-9 variant (cC9) was reported previously. Now, we have built a computational model of CMKLR1 to investigate the binding mode of cC9 and chemerinS157 in comparison to chemerin-9. Differences were investigated using CMKLR1 variants. Double-mutant cycle analysis identified CMKLR1-F2.53 as the relevant position for Phe8-binding of cC9. Energy contribution revealed slight differences in Phe8-binding to CMKLR1-F2.53 and space for larger residues. This was confirmed as the chemerin-9 variant with 1-naphthyl-L-alanine at position 8 showed a 4-fold increased potency of 2 nM (pEC50=8.6±0.15). While chemerin-9 and cC9 share their interactions at the CMKLR1, chemerinS157 tolerates most mutations of CMKLR1 in the deep binding site. The computational model of chemerinS157 suggests a β-sheet interaction between the N-terminal CMKLR1-segment I25VVL28 and the β-sheet D108KVLGRLVH116 of ChemS157, which was confirmed experimentally. Our data expand the knowledge by identifying the binding mode of chemerinS157 and cC9 at CMKLR1 facilitating future structure-based drug design.

Hepatitis Delta Antigen Retains the Assembly Domain as the Only Rigid Entity.

The hepatitis delta virus (HDV) S-HDAg and L-HDAg antigens are the two isoforms of the single protein encoded by the viral genome. Together with the double-stranded RNA genome they form the HDV ribonucleoprotein (RNP) complex. In the context of a divide-and-conquer approach, we used a combination of cell-free protein synthesis and proton (1H)-detected fast magic angle spinning solid-state NMR at highest magnetic field to characterize S-HDAg. We sequentially assigned denovo its isolated N-terminal assembly domain using less than 1 mg of fully protonated protein. Our results show that the assembly domain is the sole rigid component in S-HDAg, with its structure remaining fully conserved within the full-length protein. In contrast, the rest of the protein remains dynamic. This work provides the necessary foundation for future studies of the viral RNP.

Design of a Cereblon construct for crystallographic and biophysical studies of protein degraders

The ubiquitin E3 ligase cereblon (CRBN) is the target of therapeutic drugs thalidomide and lenalidomide and is recruited by most targeted protein degraders (PROTACs and molecular glues) in clinical development. Biophysical and structural investigation of CRBN has been limited by current constructs that either require co-expression with the adaptor DDB1 or inadequately represent full-length protein, with high-resolution structures of degrader ternary complexes remaining rare. We present the design of CRBNmidi, a construct that readily expresses from E. coli with high yields as soluble, stable protein without DDB1. We benchmark CRBNmidi for wild-type functionality through a suite of biophysical techniques and solve high-resolution co-crystal structures of its binary and ternary complexes with degraders. We qualify CRBNmidi as an enabling tool to accelerate structure-based discovery of the next generation of CRBN based therapeutics.

Structure and Antigenicity of the Porcine Astrovirus 4 Capsid Spike

Porcine astrovirus 4 (PoAstV4) has been recently associated with respiratory disease in pigs. In order to understand the scope of PoAstV4 infections and to support the development of a vaccine to combat PoAstV4 disease in pigs, we designed and produced a recombinant PoAstV4 capsid spike protein for use as an antigen in serological assays and for potential future use as a vaccine antigen. Structural prediction of the full-length PoAstV4 capsid protein guided the design of the recombinant PoAstV4 capsid spike domain expression plasmid. The recombinant PoAstV4 capsid spike was expressed in Escherichia coli, purified by affinity and size-exclusion chromatography, and its crystal structure was determined at 1.85 Å resolution, enabling structural comparisons to other animal and human astrovirus capsid spike structures. The recombinant PoAstV4 capsid spike protein was also used as an antigen for the successful development of a serological assay to detect PoAstV4 antibodies, demonstrating that the recombinant PoAstV4 capsid spike retains antigenic epitopes found on the native PoAstV4 capsid. These studies lay a foundation for seroprevalence studies and the development of a PoAstV4 vaccine for swine.

Delivering large genes using adeno-associated virus and the CRE-lox DNA recombination system.

Adeno-associated virus (AAV) is a safe and efficient gene delivery vehicle for gene therapies. However, its relatively small packaging capacity limits its use as a gene transfer vector. Here, we describe a strategy to deliver large genes that exceed the AAV's packaging capacity using up to four AAV vectors and the CRE-lox DNA recombination system. We devised novel lox sites by combining non-compatible and reaction equilibrium-modifying lox site variants. These lox sites facilitate sequence-specific and near-unidirectional recombination of AAV vector genomes, enabling efficient reconstitution of up to 16kb of therapeutic genes in a pre-determined configuration. Using this strategy, we have developed AAV gene therapy vectors to deliver IFT140, PCDH15, CEP290, and CDH23 and demonstrate efficient production of full-length proteins in cultured mammalian cells and mouse retinas. Notably, AAV-IFT140 gene therapy vectors ameliorated retinal degeneration and preserved visual functions in an IFT140-associated retinitis pigmentosa mouse model. The CRE-lox approach described here provides a simple, flexible, and effective platform for generating AAV gene therapy vectors beyond AAV's packaging capacity.

Molecular Interactions between Tau Protein and TIA1: Distinguishing Physiological Condensates from Pathological Fibrils.

The interaction of tau protein with other key proteins essential for stress granule formation determines their functional and pathological impact. In a biological framework, the synergy between Alzheimer's associated tau protein and the stress granule core protein TIA1 is widely recognized. However, the molecular details of this association remain unclear. In this study, we throw light on the importance of the state in which the TIA1 exists in mediating its association with the tau protein. Investigations were carried out on the three repeat constructs of tau (K19) and different structures formed by TIA1. Specifically, the condensate formed by TIA1 full-length (TIA1-FL) protein as well as fibril formed by low complexity domain of TIA1 (TIA1-LCD). The dynamics of K19 inside TIA1-FL condensates and the aggregation kinetics of K19 in the presence of TIA1-LCD fibrils were examined using various biophysical techniques. Relaxation-based solution NMR spectroscopic investigations suggest a weak interaction with TIA1 condensates and indicated a reduction in the dynamics of K19 within these TIA1 condensates. In contrast, a significant interaction was observed between K19, and TIA1-LCD fibrils primarily mediated through 321KCGS324 and 306VQIVYKPVDLSKV317. Our findings emphasize that the interaction between Tau and TIA1 varies depending on whether TIA1 is in its physiological condensate form or its pathological fibril state.

Production of active human iduronate-2-sulfatase (IDS) enzyme in Nicotiana benthamiana

Many strategies have been developed to produce high levels of biologically active recombinant proteins in plants for biopharmaceutical purposes. However, the production of an active form of human iduronate-2-sulfatase (hIDS) for the treatment of Hunter syndrome by enzyme replacement therapy (ERT) is challenging due to the requirement for cotranslational modification by a formylglycine-producing enzyme encoded by sulfatase modifying factor 1 (hSUMF1) at the Cys84 residue, which converts it to C(alpha)-formylglycine. In this study, we have shown that hIDS can be highly expressed in N. benthamiana by using different constructs. Among them, BiP-GB1-L-dCBD1-2L-8xHis-L-6xHis-3L-EK-hIDS-HDEL (GB1-CBD1-hIDS) showed a high expression level when transiently co-expressed with the turnip crinkle virus gene silencing suppressor P38 and GB1-fused human calreticulin (GB1-CRT1) as a folding enhancer. The hSUMF1 was co-expressed with hIDS for cotranslational modification. The full-length recombinant proteins were purified using Ni2+-NTA affinity resin followed by enterokinase treatment to obtain tag-free hIDS. The N-terminal fragment was removed using microcrystalline cellulose (MCC) beads. The purified active form of hIDS can successfully cleave the sulfate group from an artificial substrate, 4-nitrocatechol sulfate, at a similar level to commercial hIDS expressed in animal cells. These results suggest that plants could be a promising platform for the production of recombinant hIDS.

Refinement and optimisation of Neisseria gonorrhoeae NHBA and MetQ vaccine candidates

Neisseria gonorrhoeae has a significant impact on reproductive health with an estimated 82 million new cases of infection per year worldwide. Due to the ongoing emergence of multidrug-resistant N. gonorrhoeae strains, the high number of asymptomatic cases, and the risk of disease sequelae, the development of a gonococcal vaccine is urgently needed. We have previously described two potential gonococcal vaccine antigens, cNHBA (C-terminal fragment of the Neisseria Heparin Binding Antigen) and MetQ (methionine-binding protein). This study aimed to optimise these antigens for improved immune responses and to facilitate vaccine production, by investigating cNHBA fusions with the full-length MetQ protein or N-terminal and C-terminal MetQ fragments (Met1 and Met2, respectively) adjuvanted with aluminium hydroxide. The cNHBA and MetQ fragments and fusion antigens were all immunogenic in mice, generating a predominantly IgG1 response. Antibodies mediated bacterial killing via both serum bactericidal activity (SBA) and opsonophagocytic activity (OPA), and reduced adherence to cervical and urethral epithelial cells. Among the antigen fusions tested, MetQ-cNHBA and cNHBA-Met2 generated the highest SBA, OPA and adherence blocking titres and are proposed as promising optimised antigens for N. gonorrhoeae vaccine development.