Plasma Leptin Reflects Progression of Neurofibrillary Pathology in Animal Model of Tauopathy.

The close relationship between Alzheimer's disease (AD) and obesity was recognized many years ago. However, complete understanding of the pathological mechanisms underlying the interactions between degeneration of CNS and fat metabolism is still missing. The leptin a key adipokine of white adipose tissue has been suggested as one of the major mediators linking the obesity and AD. Here we investigated the association between peripheral levels of leptin, general metabolic status and stage of the pathogenesis in rat transgenic model of AD. We demonstrate significantly decreased levels of plasma leptin in animals with experimentally induced progressive neurofibrillary pathology, which represents only 62.3% (P = 0.0015) of those observed in normal wild type control animals. More detailed analysis showed a strong and statistically significant inverse correlation between the load of neurofibrillary pathology and peripheral levels of leptin (r = - 0.7248, P = 0.0177). We also observed a loss of body weight during development of neurodegeneration (about 14% less than control animals, P = 0.0004) and decrease in several metabolic parameters such as glucose, insulin, triglycerides and VLDL in plasma of the transgenic animals. Our data suggest that plasma leptin could serve as a convenient peripheral biomarker for tauopathies and Alzheimer's disease. Decrease in gene expression of leptin in fat tissue and its plasma level was found as one of the consequences of experimentally induced neurodegeneration. Our data may help to design rational diagnostic and therapeutic strategies for patients suffering from Alzheimer's disease or other forms of tauopathy.

The structure of the unstructured: mosaic of tau protein linear motifs obtained by high-resolution techniques and molecular simulation.

Intrinsically disordered proteins are flexible molecules with important physiological functions. Their mode of action often involves short segments, called linear motifs, which may exhibit distinct structural propensities. Tau is intrinsically disordered, microtubule-associated protein involved in the pathogenesis of various tauopathies. In this review we analyze the collection of 3D structures of tau local linear motifs gained from the deposited structures of tau complexes with various binding partners as well as of tau-tau complexes; determined by X-ray and electron crystallography, single-particle electron microscopy, NMR spectroscopy and molecular dynamics simulations. Insights into the partially stabilized conformations of tau linear motifs are valuable for understanding the physiological and pathological processes involving tau protein.

Hypertension does not alter disturbances in leptin signalling observed in experimental model of tauopathy.

Neurodegeneration is associated with hypertension and disturbance in fat metabolism. The complex interaction of neurodegenerative processes with both metabolic changes and blood pressure is still not fully elucidated. Here we demonstrate that the experimentally induced tauopathy in hypertensive transgenic animals causes significant downregulation of plasma leptin (53% of control), reduction of body weight by 11%, a 1.2-fold drop of adiposity index, and decrease in HDL cholesterol level, while the fasting glucose and insulin concentration remain unchanged. Despite of these alterations we found the leptin projection circuit including the arcuate nucleus, paraventricular nucleus in hypothalamus, and nucleus tractus solitarius in the brainstem not affected by neurofibrillary pathology. Furthermore, hypertension does not alter disturbances in leptin signalling. The presented data provide further insight into neurodegeneration-induced metabolic alterations relevant for human tauopathies.

Lactoferrin is a natural inhibitor of plasminogen activation.

The plasminogen system is essential for dissolution of fibrin clots, and in addition, it is involved in a wide variety of other physiological processes, including proteolytic activation of growth factors, cell migration, and removal of protein aggregates. On the other hand, uncontrolled plasminogen activation contributes to many pathological processes (e.g. tumor cells' invasion in cancer progression). Moreover, some virulent bacterial species (e.g. Streptococci or Borrelia) bind human plasminogen and hijack the host's plasminogen system to penetrate tissue barriers. Thus, the conversion of plasminogen to the active serine protease plasmin must be tightly regulated. Here, we show that human lactoferrin, an iron-binding milk glycoprotein, blocks plasminogen activation on the cell surface by direct binding to human plasminogen. We mapped the mutual binding sites to the N-terminal region of lactoferrin, encompassed also in the bioactive peptide lactoferricin, and kringle 5 of plasminogen. Finally, lactoferrin blocked tumor cell invasion in vitro and also plasminogen activation driven by Borrelia Our results explain many diverse biological properties of lactoferrin and also suggest that lactoferrin may be useful as a potential tool for therapeutic interventions to prevent both invasive malignant cells and virulent bacteria from penetrating host tissues.

Cost-Effective Protein Production in CHO Cells Following Polyethylenimine-Mediated Gene Delivery Showcased by the Production and Crystallization of Antibody Fabs.

Laboratory production of recombinant mammalian proteins, particularly antibodies, requires an expression pipeline assuring sufficient yield and correct folding with appropriate posttranslational modifications. Transient gene expression (TGE) in the suspension-adapted Chinese Hamster Ovary (CHO) cell lines has become the method of choice for this task. The antibodies can be secreted into the media, which facilitates subsequent purification, and can be glycosylated. However, in general, protein production in CHO cells is expensive and may provide variable outcomes, namely in laboratories without previous experience. While achievable yields may be influenced by the nucleotide sequence, there are other aspects of the process which offer space for optimization, like gene delivery method, cultivation process or expression plasmid design. Polyethylenimine (PEI)-mediated gene delivery is frequently employed as a low-cost alternative to liposome-based methods. In this work, we are proposing a TGE platform for universal medium-scale production of antibodies and other proteins in CHO cells, with a novel expression vector allowing fast and flexible cloning of new genes and secretion of translated proteins. The production cost has been further reduced using recyclable labware. Nine days after transfection, we routinely obtain milligrams of antibody Fabs or human lactoferrin in a 25 mL culture volume. Potential of the platform is established based on the production and crystallization of antibody Fabs and their complexes.

Time to Kill and Time to Heal: The Multifaceted Role of Lactoferrin and Lactoferricin in Host Defense.

Lactoferrin is an iron-binding glycoprotein present in most human exocrine fluids, particularly breast milk. Lactoferrin is also released from neutrophil granules, and its concentration increases rapidly at the site of inflammation. Immune cells of both the innate and the adaptive immune system express receptors for lactoferrin to modulate their functions in response to it. On the basis of these interactions, lactoferrin plays many roles in host defense, ranging from augmenting or calming inflammatory pathways to direct killing of pathogens. Complex biological activities of lactoferrin are determined by its ability to sequester iron and by its highly basic N-terminus, via which lactoferrin binds to a plethora of negatively charged surfaces of microorganisms and viruses, as well as to mammalian cells, both normal and cancerous. Proteolytic cleavage of lactoferrin in the digestive tract generates smaller peptides, such as N-terminally derived lactoferricin. Lactoferricin shares some of the properties of lactoferrin, but also exhibits unique characteristics and functions. In this review, we discuss the structure, functions, and potential therapeutic uses of lactoferrin, lactoferricin, and other lactoferrin-derived bioactive peptides in treating various infections and inflammatory conditions. Furthermore, we summarize clinical trials examining the effect of lactoferrin supplementation in disease treatment, with a special focus on its potential use in treating COVID-19.

The structure and interactions of SpoIISA and SpoIISB, a toxin-antitoxin system in Bacillus subtilis.

Spore formation in Bacillus subtilis begins with an asymmetric cell division, following which differential gene expression is established by alternative compartment-specific RNA polymerase σ factors. The spoIISAB operon of B. subtilis was identified as a locus whose mutation leads to increased activity of the first sporulation-specific sigma factor, σ(F). Inappropriate spoIISA expression causes lysis of vegetatively growing B. subtilis cells and Escherichia coli cells when expressed heterologously, effects that are countered by co-expression of spoIISB, identifying SpoIISA-SpoIISB as a toxin-antitoxin system. SpoIISA has three putative membrane-spanning segments and a cytoplasmic domain. Here, the crystal structure of a cytoplasmic fragment of SpoIISA (CSpoIISA) in complex with SpoIISB has been determined by selenomethionine-multiwavelength anomalous dispersion phasing to 2.5 Å spacing, revealing a CSpoIISA(2)·SpoIISB(2) heterotetramer. CSpoIISA has a single domain α/ß structure resembling a GAF domain with an extended α-helix at its N terminus. The two CSpoIISA protomers form extensive interactions through an intermolecular four-helix bundle. Each SpoIISB chain is highly extended and lacking tertiary structure. The SpoIISB chains wrap around the CSpoIISA dimer, forming extensive interactions with both CSpoIISA protomers. CD spectroscopy experiments indicate that SpoIISB is a natively disordered protein that adopts structure only in the presence of CSpoIISA, whereas surface plasmon resonance experiments revealed that the CSpoIISA·SpoIISB complex is stable with a dissociation constant in the nanomolar range. The results are interpreted in relation to sequence conservation and mutational data, and possible mechanisms of cell killing by SpoIISA are discussed.

Crystallization and preliminary X-ray diffraction analysis of tau protein microtubule-binding motifs in complex with Tau5 and DC25 antibody Fab fragments.

The Alzheimer's disease-associated protein tau is an intrinsically disordered protein with no preferred structure in solution. Under physiological conditions, tau binds to microtubules and regulates their dynamics, whereas during the development of neurodegeneration tau dissociates from microtubules, misfolds and creates highly insoluble deposits. To elucidate the determinants of tau-protein misfolding, tau peptides from microtubule-binding motifs were crystallized in complexes with Fab fragments of specific monoclonal antibodies. The crystals diffracted to 1.69 Šresolution and gave complete data sets using a synchrotron X-ray source. Molecular replacement was used to solve the phase problem.

Crystallization and preliminary X-ray diffraction analysis of two peptides from Alzheimer PHF in complex with the MN423 antibody Fab fragment.

The major constituent of the Alzheimer's disease paired helical filaments (PHF) core is the intrinsically disordered protein (IDP) tau. Globular binding partners, e.g. monoclonal antibodies, can stabilize the fold of disordered tau in complexes. A previously published structure of a proteolytically generated tau fragment in a complex with the PHF-specific monoclonal antibody MN423 revealed a turn-like structure of the PHF core C-terminus [Sevcik et al. (2007). FEBS Lett. 581, 5872-5878]. To examine the structures of longer better-defined PHF segments, crystals of the MN423 Fab fragment were grown in the presence of two synthetic peptides derived from the PHF core C-terminus. For each, X-ray diffraction data were collected at 100 K at a synchrotron source and initial phases were obtained by molecular replacement.

Interaction kinetics reveal distinct properties of conformational ensembles of three-repeat and four-repeat tau proteins.

Tau protein is an intrinsically disordered protein. Its physiological state is best described as a conformational ensemble (CE) of metastable structures interconverting on the local and molecular scale. The monoclonal antibody DC39C recognizes a linear C-terminal tau epitope, and as the tau interaction partner, its binding parameters report about tau CE. Association kinetics of DC39C binding, together with crosslinking mass spectrometry, show differences in the accessibility of the C terminus in CEs of tau isoforms. Furthermore, removal of the C terminus accelerated the aggregation kinetics of three-repeat tau proteins. Our results suggest a novel mechanism of splicing-driven regulation of the tau C-terminal domain with consequences on the specific roles of tau isoforms in microtubule assembly and pathological aggregation.

Blockade of TMPRSS2-mediated priming of SARS-CoV-2 by lactoferricin.

In addition to vaccines, there is an urgent need for supplemental antiviral therapeutics to dampen the persistent COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The transmembrane protease serine 2 (TMPRSS2), that is responsible for proteolytic priming of the SARS-CoV-2 spike protein, appears as a rational therapeutic target. Accordingly, selective inhibitors of TMPRSS2 represent potential tools for prevention and treatment of COVID-19. Previously, we identified the human milk glycoprotein lactoferrin as a natural inhibitor of plasminogen conversion to plasmin, a serine protease homologous to TMPRSS2. Here, we tested whether lactoferrin and lactoferricin, a biologically active natural peptide produced by pepsin-mediated digestion of lactoferrin, together with synthetic peptides derived from lactoferrin, were able to block TMPRSS2 and SARS-CoV-2 infection. Particularly, we revealed that both lactoferricin and the N-terminal synthetic peptide pLF1 significantly inhibited: i) proteolytic activity of TMPRSS2 and plasmin, ii) proteolytic processing of the SARS-CoV-2 spike protein, and iii) SARS-CoV-2 infection of SARS-CoV-2-permissive cells. Thus, natural and synthetic peptides derived from lactoferrin represent feasible candidates for supporting prevention and treatment of COVID-19.

Monoclonal antibodies targeting two immunodominant epitopes on the Spike protein neutralize emerging SARS-CoV-2 variants of concern.

BACKGROUND: The emergence of new SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta) that harbor mutations in the viral S protein raised concern about activity of current vaccines and therapeutic antibodies. Independent studies have shown that mutant variants are partially or completely resistant against some of the therapeutic antibodies authorized for emergency use. METHODS: We employed hybridoma technology, ELISA-based and cell-based S-ACE2 interaction assays combined with authentic virus neutralization assays to develop second-generation antibodies, which were specifically selected for their ability to neutralize the new variants of SARS-CoV-2. FINDINGS: AX290 and AX677, two monoclonal antibodies with non-overlapping epitopes, exhibit subnanomolar or nanomolar affinities to the receptor binding domain of the viral Spike protein carrying amino acid substitutions N501Y, N439K, E484K, K417N, and a combination N501Y/E484K/K417N found in the circulating virus variants. The antibodies showed excellent neutralization of an authentic SARS-CoV-2 virus representing strains circulating in Europe in spring 2020 and also the variants of concern B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). In addition, AX677 is able to bind Omicron Spike protein just like the wild type Spike. The combination of the two antibodies prevented the appearance of escape mutations of the authentic SARS-CoV-2 virus. Prophylactic administration of AX290 and AX677, either individually or in combination, effectively reduced viral burden and inflammation in the lungs, and prevented disease in a mouse model of SARS-CoV-2 infection. INTERPRETATION: The virus-neutralizing properties were fully reproduced in chimeric mouse-human versions of the antibodies, which may represent a promising tool for COVID-19 therapy. FUNDING: The study was funded by AXON Neuroscience SE and AXON COVIDAX a.s.

Purification of MBP fusion proteins using engineered DARPin affinity matrix.

Maltose binding protein (MBP) has a long history as an expression tag with the ability to increase the solubility of fused proteins. A critical step for obtaining a sufficient amount of the MBP fusion protein is purification. Commercially available amylose matrix for the affinity purification of MBP fusion proteins has two main issues: (i) low (micromolar) affinity and (ii) the limited number of uses due to the cleavage of polysaccharide matrix by the amylases, present in the crude cell extract. Here, we present a new affinity purification approach based on the protein-protein interaction. We developed the affinity matrix which contains immobilized Designed Ankyrin Repeat Protein off7 (DARPin off7) - previously identified MBP binder with nanomolar affinity. The functionality of the DARPin affinity matrix was tested on the purification of MBP-tagged green fluorescent protein and flavodoxin. The affinity purification of the MBP fusion proteins, based on the MBP-DARPin off7 interaction, enables the purification of the fusion proteins in a simple two-steps procedure. The DARPin affinity matrix - easy to construct, resistant to amylase, insensitive to maltose contamination, and reusable for multiple purification cycles - provides an alternative approach to commercially available affinity matrices for purification of proteins containing the MBP tag.

NMR Studies of Tau Protein in Tauopathies.

Tauopathies, including Alzheimer's disease (AD), are the most troublesome of all age-related chronic conditions, as there are no well-established disease-modifying therapies for their prevention and treatment. Spatio-temporal distribution of tau protein pathology correlates with cognitive decline and severity of the disease, therefore, tau protein has become an appealing target for therapy. Current knowledge of the pathological effects and significance of specific species in the tau aggregation pathway is incomplete although more and more structural and mechanistic insights are being gained using biophysical techniques. Here, we review the application of NMR to structural studies of various tau forms that appear in its aggregation process, focusing on results obtained from solid-state NMR. Furthermore, we discuss implications from these studies and their prospective contribution to the development of new tauopathy therapies.

Monoclonal antibody MN423 as a stable mold facilitates structure determination of disordered tau protein.

Flexibility of intrinsically disordered tau protein is important for performing its functions. It is believed that alteration of the flexibility is instrumental to the assembly of tau protein into paired helical filaments (PHF) in tauopathies. Tau flexibility represents the main obstacle for structure determination of its conformation in physiology and/or pathology. We have alleviated this inherited difficulty by using specific monoclonal antibodies as tau protein surrogate binding partners. In this work we compare two "antibody mold structures": (1) X-ray structure of the free form of the Alzheimer's disease PHF core-specific antibody MN423 and (2) previously solved structure of the complex of MN423 with the PHF core C-terminal tau peptide. We found that MN423 combining site is in both structures identical. As a consequence, recombinant tau assumes in the complex a fold determined by the antibody combining site. Obtained results show that MN423 functions as a molecular mold for the PHF core segment, and opens the way for structure determination of other PHF core segments providing that other conformation-specific antibodies are available. Data from in silico docking of tau peptide into antibody mold, obtained in this study, show that biochemical data and computational approaches provide results comparable to X-ray crystallography.

Transition of tau protein from disordered to misordered in Alzheimer's disease.

The neuronal protein tau, a member of the class of intrinsically disordered proteins, is characterized by the absence of any firm 3-D structure and high solubility when free in solution. The tau protein forms insoluble fibrils in the brain of people suffering from Alzheimer's disease (AD) and other tauopathies and plays a key role in the neurodegenerative process. Posttranslational events leading to the transition of tau from a disordered highly soluble protein to the insoluble aggregate seem to be associated with hyperphosphorylation, truncation or a combination of both. These modifications are assumed to change the native disorder of tau into a preaggregation state, which then directly initiates the fibrillization process. Conformation-specific anti-tau antibody DC11 detects pathological truncated tau forms present in AD brains but not in the normal human brain. A truncated tau protein selected from this pool of DC11-positive molecules was sufficient to initiate and drive the complete tau cascade of neurofibrillary pathology in a rat model of tauopathy. Thus, DC11 antibody recognizes the AD-specific conformation of tau not found in the normal human brain. We propose the term 'misdisordered' for this distinct conformational state of tau, which is the first step in the transition of tau from a soluble disordered protein to its insoluble, misordered aggregated form.

Humanized tau antibodies promote tau uptake by human microglia without any increase of inflammation.

Immunotherapies targeting pathological tau have recently emerged as a promising approach for treatment of neurodegenerative disorders. We have previously showed that the mouse antibody DC8E8 discriminates between healthy and pathological tau, reduces tau pathology in murine tauopathy models and inhibits neuronal internalization of AD tau species in vitro.Here we show, that DC8E8 and antibodies elicited against the first-in-man tau vaccine, AADvac1, which is based on the DC8E8 epitope peptide, both promote uptake of pathological tau by mouse primary microglia. IgG1 and IgG4 isotypes of AX004, the humanized versions of DC8E8, accelerate tau uptake by human primary microglia isolated from post-mortem aged and diseased brains. This promoting activity requires the presence of the Fc-domain of the antibodies.The IgG1 isotype of AX004 showed greater ability to promote tau uptake compared to the IgG4 isotype, while none of the antibody-tau complexes provoked increased pro-inflammatory activity of microglia. Our data suggest that IgG1 has better suitability for therapeutic development.

MIRRAGGE - Minimum Information Required for Reproducible AGGregation Experiments.

Reports on phase separation and amyloid formation for multiple proteins and aggregation-prone peptides are recurrently used to explore the molecular mechanisms associated with several human diseases. The information conveyed by these reports can be used directly in translational investigation, e.g., for the design of better drug screening strategies, or be compiled in databases for benchmarking novel aggregation-predicting algorithms. Given that minute protocol variations determine different outcomes of protein aggregation assays, there is a strong urge for standardized descriptions of the different types of aggregates and the detailed methods used in their production. In an attempt to address this need, we assembled the Minimum Information Required for Reproducible Aggregation Experiments (MIRRAGGE) guidelines, considering first-principles and the established literature on protein self-assembly and aggregation. This consensus information aims to cover the major and subtle determinants of experimental reproducibility while avoiding excessive technical details that are of limited practical interest for non-specialized users. The MIRRAGGE table (template available in Supplementary Information) is useful as a guide for the design of new studies and as a checklist during submission of experimental reports for publication. Full disclosure of relevant information also enables other researchers to reproduce results correctly and facilitates systematic data deposition into curated databases.

Novel mutations in TLR genes cause hyporesponsiveness to Mycobacterium avium subsp. paratuberculosis infection.

BACKGROUND: Toll like receptors (TLR) play the central role in the recognition of pathogen associated molecular patterns (PAMPs). Mutations in the TLR1, TLR2 and TLR4 genes may change the ability to recognize PAMPs and cause altered responsiveness to the bacterial pathogens. RESULTS: The study presents association between TLR gene mutations and increased susceptibility to Mycobacterium avium subsp. paratuberculosis (MAP) infection. Novel mutations in TLR genes (TLR1- Ser150Gly and Val220Met; TLR2 - Phe670Leu) were statistically correlated with the hindrance in recognition of MAP legends. This correlation was confirmed subsequently by measuring the expression levels of cytokines (IL-4, IL-8, IL-10, IL-12 and IFN-gamma) in the mutant and wild type moDCs (mocyte derived dendritic cells) after challenge with MAP cell lysate or LPS. Further in silico analysis of the TLR1 and TLR4 ectodomains (ECD) revealed the polymorphic nature of the central ECD and irregularities in the central LRR (leucine rich repeat) motifs. CONCLUSION: The most critical positions that may alter the pathogen recognition ability of TLR were: the 9th amino acid position in LRR motif (TLR1-LRR10) and 4th residue downstream to LRR domain (exta-LRR region of TLR4). The study describes novel mutations in the TLRs and presents their association with the MAP infection.