Competition between inside-out unfolding and pathogenic aggregation in an amyloid-forming ß-propeller.

Studies of folded-to-misfolded transitions using model protein systems reveal a range of unfolding needed for exposure of amyloid-prone regions for subsequent fibrillization. Here, we probe the relationship between unfolding and aggregation for glaucoma-associated myocilin. Mutations within the olfactomedin domain of myocilin (OLF) cause a gain-of-function, namely cytotoxic intracellular aggregation, which hastens disease progression. Aggregation by wild-type OLF (OLFWT) competes with its chemical unfolding, but only below the threshold where OLF loses tertiary structure. Representative moderate (OLFD380A) and severe (OLFI499F) disease variants aggregate differently, with rates comparable to OLFWT in initial stages of unfolding, and variants adopt distinct partially folded structures seen along the OLFWT urea-unfolding pathway. Whether initiated with mutation or chemical perturbation, unfolding propagates outward to the propeller surface. In sum, for this large protein prone to amyloid formation, the requirement for a conformational change to promote amyloid fibrillization leads to direct competition between unfolding and aggregation.

DnaJs are enriched in tau regulators.

The aberrant accumulation of tau protein is implicated as a pathogenic factor in many neurodegenerative diseases. Tau seeding may underlie its predictable spread in these diseases. Molecular chaperones can modulate tau pathology, but their effects have mainly been studied in isolation. This study employed a semi-high throughput assay to identify molecular chaperones influencing tau seeding using Tau RD P301S FRET Biosensor cells, which express a portion of tau containing the frontotemporal dementia-related P301S tau mutation fused to a FRET biosensor. Approximately fifty chaperones from five major families were screened using live cell imaging to monitor FRET-positive tau seeding. Among the tested chaperones, five exhibited significant effects on tau in the primary screen. Notably, three of these were from the DnaJ family. In subsequent studies, overexpression of DnaJA2, DnaJB1, and DnaJB6b resulted in significant reductions in tau levels. Knockdown experiments by shRNA revealed an inverse correlation between DnaJB1 and DnaJB6b with tau levels. DnaJB6b overexpression, specifically, reduced total tau levels in a cellular model with a pre-existing pool of tau, partially through enhanced proteasomal degradation. Further, DnaJB6b interacted with tau complexes. These findings highlight the potent chaperone activity within the DnaJ family, particularly DnaJB6b, towards tau.

Benzothiazole Substitution Analogs of Rhodacyanine Hsp70 Inhibitors Modulate Tau Accumulation.

The accumulation and aggregation of the microtubule-associated protein tau (tau) into intracellular neuronal tangles are a hallmark of a range of progressive neurodegenerative tauopathies, including Alzheimer's disease (AD), frontotemporal dementia, Pick's disease, and progressive supranuclear palsy. The aberrant phosphorylation of tau is associated with tau aggregates in AD. Members of the heat shock protein 70 kDa (Hsp70) family of chaperones bind directly to tau and modulate tau clearance and aggregation. Small molecules that inhibit the Hsp70 family of chaperones have been shown to reduce the accumulation of tau, including phosphorylated tau. Here, eight analogs of the rhodacyanine inhibitor, JG-98, were synthesized and evaluated. Like JG-98, many of the compounds inhibited ATPase activity of the cytosolic heat shock cognate 70 protein (Hsc70) and reduced total, aggregated, and phosphorylated tau accumulation in cultured cells. Three compounds, representing divergent clogP values, were evaluated for in vivo blood-brain barrier penetration and tau reduction in an ex vivo brain slice model. AL69, the compound with the lowest clogP and the lowest membrane retention in a parallel artificial membrane permeability assay (PAMPA), reduced phosphorylated tau accumulation. Our results suggest that benzothiazole substitutions of JG-98 that increase hydrophilicity may increase the efficacy of these Hsp70 inhibitors to reduce phosphorylated tau.

Genetically engineered mouse models of FK506-binding protein 5.

FK506 binding protein 51 (FKBP51) is a molecular chaperone that influences stress response. In addition to having an integral role in the regulation of steroid hormone receptors, including glucocorticoid receptor, FKBP51 has been linked with several biological processes including metabolism and neuronal health. Genetic and epigenetic alterations in the gene that encodes FKBP51, FKBP5, are associated with increased susceptibility to multiple neuropsychiatric disorders, which has fueled much of the research on this protein. Because of the complexity of these processes, animal models have been important in understanding the role of FKBP51. This review examines each of the current mouse models of FKBP5, which include whole animal knockout, conditional knockout, overexpression, and humanized mouse models. The generation of each model and observational details are discussed, including behavioral phenotypes, molecular changes, and electrophysiological alterations basally and following various challenges. While much has been learned through these models, there are still many aspects of FKBP51 biology that remain opaque and future studies are needed to help illuminate these current gaps in knowledge. Overall, FKBP5 continues to be an exciting potential target for stress-related disorders.

Chaperoning of specific tau structure by immunophilin FKBP12 regulates the neuronal resilience to extracellular stress.

Alzheimer's disease and related tauopathies are characterized by the pathogenic misfolding and aggregation of the microtubule-associated protein tau. Understanding how endogenous chaperones modulate tau misfolding could guide future therapies. Here, we show that the immunophilin FKBP12, the 12-kDa FK506-binding protein (also known as FKBP prolyl isomerase 1A), regulates the neuronal resilience by chaperoning a specific structure in monomeric tau. Using a combination of mouse and cell experiments, in vitro aggregation experiments, nuclear magnetic resonance-based structural analysis of monomeric tau, site-specific phosphorylation and mutation, as well as structure-based analysis using the neural network-based structure prediction program AlphaFold, we define the molecular factors that govern the binding of FKBP12 to tau and its influence on tau-induced neurotoxicity. We further demonstrate that tyrosine phosphorylation of tau blocks the binding of FKBP12 to two highly specific structural motifs in tau. Our data together with previous results demonstrating FKBP12/tau colocalization in neurons and neurofibrillary tangles support a critical role of FKBP12 in regulating tau pathology.

Small molecule targeting long noncoding RNA GAS5 administered intranasally improves neuronal insulin signaling and decreases neuroinflammation in an aged mouse model.

Shifts in normal aging set stage for neurodegeneration and dementia affecting 1 in 10 adults. The study demonstrates that lncRNA GAS5 is decreased in aged and Alzheimer's disease brain. The role and targets of lncRNA GAS5 in the aging brain were elucidated using a GAS5-targeting small molecule NPC86, a frontier in lncRNA-targeting therapeutic. Robust techniques such as molecular dynamics simulation of NPC86 binding to GAS5, in vitro functional assays demonstrating that GAS5 regulates insulin signaling, neuronal survival, phosphorylation of tau, and neuroinflammation via toll-like receptors support the role of GAS5 in maintaining healthy neurons. The study demonstrates the safety and efficacy of intranasal NPC86 treatment in aged mice to improve cellular functions with transcriptomic analysis in response to NPC86. In summary, the study demonstrates that GAS5 contributes to pathways associated with neurodegeneration and NPC86 has tremendous therapeutic potential to prevent the advent of neurodegenerative diseases and dementias.

Chaperoning activity of the cyclophilin family prevents tau aggregation.

Tauopathies, such as Alzheimer's disease, are characterized by the misfolding and progressive accumulation of the microtubule associated protein tau. Chaperones, tasked with maintaining protein homeostasis, can become imbalanced with age and contribute to the progression of neurodegenerative disease. Cyclophilins are a promising pool of underinvestigated chaperones with peptidyl-prolyl isomerase activity that may play protective roles in regulating tau aggregation. Using a Thioflavin T fluorescence-based assay to monitor in vitro tau aggregation, all eight cyclophilins, which include PPIA to PPIH prevent tau aggregation, with PPIB, PPIC, PPID, and PPIH showing the greatest inhibition. The low thermal stability of PPID and the strong heparin binding of PPIB undermines the simplistic interpretation of reduced tau aggregation. In a cellular model of tau accumulation, all cyclophilins, except PPID and PPIH, reduce insoluble tau. PPIB, PPIC, PPIE, and PPIF also reduce soluble tau levels with PPIC exclusively protecting cells from tau seeding. Overall, this study demonstrates cyclophilins prevent tau fibril formation and many reduce cellular insoluble tau accumulation with PPIC having the greatest potential as a molecular tool to mitigate tau seeding and accumulation.

Recombinant antibodies recognize conformation-dependent epitopes of the leucine zipper of misfolding-prone myocilin.

Recombinant antibodies with well-characterized epitopes and known conformational specificities are critical reagents to support robust interpretation and reproducibility of immunoassays across biomedical research. For myocilin, a protein prone to misfolding that is associated with glaucoma and an emerging player in other human diseases, currently available antibodies are unable to differentiate among the numerous disease-associated protein states. This fundamentally constrains efforts to understand the connection between myocilin structure, function, and disease. To address this concern, we used protein engineering methods to develop new recombinant antibodies that detect the N-terminal leucine zipper structural domain of myocilin and that are cross-reactive for human and mouse myocilin. After harvesting spleens from immunized mice and in vitro library panning, we identified two antibodies, 2A4 and 1G12. 2A4 specifically recognizes a folded epitope while 1G12 recognizes a range of conformations. We matured antibody 2A4 for improved biophysical properties, resulting in variant 2H2. In a human IgG1 format, 2A4, 1G12, and 2H2 immunoprecipitate full-length folded myocilin present in the spent media of human trabecular meshwork (TM) cells, and 2H2 can visualize myocilin in fixed human TM cells using fluorescence microscopy. These new antibodies should find broad application in glaucoma and other research across multiple species platforms.

A New Contact Killing Toxin Permeabilizes Cells and Belongs to a Broadly Distributed Protein Family.

Vibrio cholerae is an aquatic Gram-negative bacterium that causes severe diarrheal cholera disease when ingested by humans. To eliminate competitor cells in both the external environment and inside hosts, V. cholerae uses the type VI secretion system (T6SS). The T6SS is a macromolecular contact-dependent weapon employed by many Gram-negative bacteria to deliver cytotoxic proteins into adjacent cells. In addition to canonical T6SS gene clusters encoded by all sequenced V. cholerae isolates, strain BGT49 encodes another locus, which we named auxiliary (Aux) cluster 4. The Aux 4 cluster is located on a mobile genetic element and can be used by killer cells to eliminate both V. cholerae and Escherichia coli cells in a T6SS-dependent manner. A putative toxin encoded in the cluster, which we name TpeV (type VI permeabilizing effector Vibrio), shares no homology to known proteins and does not contain motifs or domains indicative of function. Ectopic expression of TpeV in the periplasm of E. coli permeabilizes cells and disrupts the membrane potential. Using confocal microscopy, we confirm that susceptible target cells become permeabilized when competed with killer cells harboring the Aux 4 cluster. We also determine that tpiV, the gene located immediately downstream of tpeV, encodes an immunity protein that neutralizes the toxicity of TpeV. Finally, we show that TpeV homologs are broadly distributed across important human, animal, and plant pathogens and are localized in proximity to other T6SS genes. Our results suggest that TpeV is a toxin that belongs to a large family of T6SS proteins. IMPORTANCE Bacteria live in polymicrobial communities where competition for resources and space is essential for survival. Proteobacteria use the T6SS to eliminate neighboring cells and cause disease. However, the mechanisms by which many T6SS toxins kill or inhibit susceptible target cells are poorly understood. The sequence of the TpeV toxin that we describe here is unlike any previously described protein. We demonstrate that it has antimicrobial activity by permeabilizing cells, eliminating membrane potentials, and causing severe cytotoxicity. TpeV homologs are found near known T6SS genes in human, animal, and plant bacterial pathogens, indicating that the toxin is a representative member of a broadly distributed protein family. We propose that TpeV-like toxins contribute to the fitness of many bacteria. Finally, since antibiotic resistance is a critical global health threat, the discovery of new antimicrobial mechanisms could lead to the development of new treatments against resistant strains.

Optically Modulated and Optically Activated Delayed Fluorescent Proteins through Dark State Engineering.

Modulating fluorescent protein emission holds great potential for increasing readout sensitivity for applications in biological imaging and detection. Here, we identify and engineer optically modulated yellow fluorescent proteins (EYFP, originally 10C, but renamed EYFP later, and mVenus) to yield new emitters with distinct modulation profiles and unique, optically gated, delayed fluorescence. The parent YFPs are individually modulatable through secondary illumination, depopulating a long-lived dark state to dynamically increase fluorescence. A single point mutation introduced near the chromophore in each of these YFPs provides access to a second, even longer-lived modulatable dark state, while a different double mutant renders EYFP unmodulatable. The naturally occurring dark state in the parent YFPs yields strong fluorescence modulation upon long-wavelength-induced dark state depopulation, allowing selective detection at the frequency at which the long wavelength secondary laser is intensity modulated. Distinct from photoswitches, however, this near IR secondary coexcitation repumps the emissive S1 level from the long-lived triplet state, resulting in optically activated delayed fluorescence (OADF). This OADF results from secondary laser-induced, reverse intersystem crossing (RISC), producing additional nanosecond-lived, visible fluorescence that is delayed by many microseconds after the primary excitation has turned off. Mutation of the parent chromophore environment opens an additional modulation pathway that avoids the OADF-producing triplet state, resulting in a second, much longer-lived, modulatable dark state. These Optically Modulated and Optically Activated Delayed Fluorescent Proteins (OMFPs and OADFPs) are thus excellent for background- and reference-free, high sensitivity cellular imaging, but time-gated OADF offers a second modality for true background-free detection. Our combined structural and spectroscopic data not only gives additional mechanistic details for designing optically modulated fluorescent proteins but also provides the opportunity to distinguish similarly emitting OMFPs through OADF and through their unique modulation spectra.

Structural Arrangement within a Peptide Fibril Derived from the Glaucoma-Associated Myocilin Olfactomedin Domain.

Myocilin-associated glaucoma is a new addition to the list of diseases linked to protein misfolding and amyloid formation. Single point variants of the ∼257-residue myocilin olfactomedin domain (mOLF) lead to mutant myocilin aggregation. Here, we analyze the 12-residue peptide P1 (GAVVYSGSLYFQ), corresponding to residues 326-337 of mOLF, previously shown to form amyloid fibrils in vitro and in silico. We applied solid-state NMR structural measurements to test the hypothesis that P1 fibrils adopt one of three predicted structures. Our data are consistent with a U-shaped fibril arrangement for P1, one that is related to the U-shape predicted previously in silico. Our data are also consistent with an antiparallel fibril arrangement, likely driven by terminal electrostatics. Our proposed structural model is reminiscent of fibrils formed by the Aß(1-40) Iowa mutant peptide, but with a different arrangement of molecular turn regions. Taken together, our results strengthen the connection between mOLF fibrils and the broader amylome and contribute to our understanding of the fundamental molecular interactions governing fibril architecture and stability.

Hsp22 with an N-Terminal Domain Truncation Mediates a Reduction in Tau Protein Levels.

Misfolding, aggregation and accumulation of proteins are toxic elements in the progression of a broad range of neurodegenerative diseases. Molecular chaperones enable a cellular defense by reducing or compartmentalizing these insults. Small heat shock proteins (sHsps) engage proteins early in the process of misfolding and can facilitate their proper folding or refolding, sequestration, or clearance. Here, we evaluate the effects of the sHsp Hsp22, as well as a pseudophosphorylated mutant and an N-terminal domain deletion (NTDΔ) variant on tau aggregation in vitro and tau accumulation and aggregation in cultured cells. Hsp22 wild-type (WT) protein had a significant inhibitory effect on heparin-induced aggregation in vitro and the pseudophosphorylated mutant Hsp22 demonstrated a similar effect. When co-expressed in a cell culture model with tau, these Hsp22 constructs significantly reduced soluble tau protein levels when transfected at a high ratio relative to tau. However, the Hsp22 NTDΔ protein drastically reduced the soluble protein expression levels of both tau WT and tau P301L/S320F even at lower transfection ratios, which resulted in a correlative reduction of the triton-insoluble tau P301L/S320F aggregates.

Calcium-ligand variants of the myocilin olfactomedin propeller selected from invertebrate phyla reveal cross-talk with N-terminal blade and surface helices.

Olfactomedins are a family of modular proteins found in multicellular organisms that all contain five-bladed ß-propeller olfactomedin (OLF) domains. In support of differential functions for the OLF propeller, the available crystal structures reveal that only some OLF domains harbor an internal calcium-binding site with ligands derived from a triad of residues. For the myocilin OLF domain (myoc-OLF), ablation of the ion-binding site (triad Asp, Asn, Asp) by altering the coordinating residues affects the stability and overall structure, in one case leading to misfolding and glaucoma. Bioinformatics analysis reveals a variety of triads with possible ion-binding characteristics lurking in OLF domains in invertebrate chordates such as Arthropoda (Asp-Glu-Ser), Nematoda (Asp-Asp-His) and Echinodermata (Asp-Glu-Lys). To test ion binding and to extend the observed connection between ion binding and distal structural rearrangements, consensus triads from these phyla were installed in the myoc-OLF. All three protein variants exhibit wild-type-like or better stability, but their calcium-binding properties differ, concomitant with new structural deviations from wild-type myoc-OLF. Taken together, the results indicate that calcium binding is not intrinsically destabilizing to myoc-OLF or required to observe a well ordered side helix, and that ion binding is a differential feature that may underlie the largely elusive biological function of OLF propellers.

Stable calcium-free myocilin olfactomedin domain variants reveal challenges in differentiating between benign and glaucoma-causing mutations.

Nonsynonymous gene mutations can be beneficial, neutral, or detrimental to the stability, structure, and biological function of the encoded protein, but the effects of these mutations are often not readily predictable. For example, the ß-propeller olfactomedin domain of myocilin (mOLF) exhibits a complex interrelationship among structure(s), stability, and aggregation. Numerous mutations within mOLF are linked to glaucoma; the resulting variants are less stable, aggregation-prone, and sequestered intracellularly, causing cytotoxicity. Here, we report the first stable mOLF variants carrying substitutions in the calcium-binding site that exhibit solution characteristics indistinguishable from those of glaucoma variants. Crystal structures of these stable variants at 1.8-2.0-Å resolution revealed features that we could not predict by molecular dynamics simulations, including loss of loop structure, helix unwinding, and a blade shift. Double mutants that combined a stabilizing substitution and a selected glaucoma-causing single-point mutant rescued in vitro folding and stability defects. In the context of full-length myocilin, secretion of stable single variants was indistinguishable from that of the WT protein, and the double mutants were secreted to varying extents. In summary, our finding that mOLF can tolerate particular substitutions that render the protein stable despite a conformational switch emphasizes the complexities in differentiating between benign and glaucoma-causing variants and provides new insight into the possible biological function of myocilin.

Differential Misfolding Properties of Glaucoma-Associated Olfactomedin Domains from Humans and Mice.

Mutations in myocilin, predominantly within its olfactomedin (OLF) domain, are causative for the heritable form of open angle glaucoma in humans. Surprisingly, mice expressing Tyr423His mutant myocilin, corresponding to a severe glaucoma-causing mutation (Tyr437His) in human subjects, exhibit a weak, if any, glaucoma phenotype. To address possible protein-level discrepancies between mouse and human OLFs, which might lead to this outcome, biophysical properties of mouse OLF were characterized for comparison with those of human OLF. The 1.55 Å resolution crystal structure of mouse OLF reveals an asymmetric 5-bladed ß-propeller that is nearly indistinguishable from previous structures of human OLF. Wild-type and selected mutant mouse OLFs mirror thermal stabilities of their human OLF counterparts, including characteristic stabilization in the presence of calcium. Mouse OLF forms thioflavin T-positive aggregates with a similar end-point morphology as human OLF, but amyloid aggregation kinetic rates of mouse OLF are faster than human OLF. Simulations and experiments support the interpretation that kinetics of mouse OLF are faster because of a decreased charge repulsion arising from more neutral surface electrostatics. Taken together, phenotypic differences observed in mouse and human studies of mutant myocilin could be a function of aggregation kinetics rates, which would alter the lifetime of putatively toxic protofibrillar intermediates.

Simulations and Experiments Delineate Amyloid Fibrilization by Peptides Derived from Glaucoma-Associated Myocilin.

Mutant myocilin aggregation is associated with inherited open angle glaucoma, a prevalent optic neuropathy leading to blindness. Comprehension of mutant myocilin aggregation is of fundamental importance to glaucoma pathogenesis and ties glaucoma to amyloid diseases such as Alzheimer's. Here, we probe the aggregation properties of peptides derived from the myocilin olfactomedin domain. Peptides P1 (residues 326-337) and P3 (residues 426-442) were identified previously to form amyloids. Coarse-grained discontinuous molecular dynamics simulations using the PRIME20 force field (DMD/PRIME20) predict that P1 and P3 are aggregation-prone; P1 consistently forms fibrillar aggregates with parallel in-register ß-sheets, whereas P3 forms ß-sheet-containing aggregates without distinct order. Natural abundance 13C solid-state NMR spectra validate that aggregated P1 exhibits amyloid signatures and is more homogeneous than aggregated P3. DMD/PRIME20 simulations provide a viable method to predict peptide aggregation propensities and aggregate structure/order which cannot be accessed by bioinformatics or readily attained experimentally.

Epitope mapping of commercial antibodies that detect myocilin.

The presence of myocilin is often used in the process of validating trabecular meshwork (TM) cells and eye tissues, but the antibody reagents used for detection are poorly characterized. Indeed, for over a century, researchers have been using antibodies to track proteins of interest in a variety of biological contexts, but many antibodies remain ill-defined at the molecular level and in their target epitope. Such issues have prompted efforts from major funding agencies to validate reagents and combat reproducibility issues across biomedical sciences. Here we characterize the epitopes recognized by four commercial myocilin antibodies, aided by structurally and biochemically characterized myocilin fragments. All four antibodies recognize enriched myocilin secreted from human TM cell media. The detection of myocilin fragments by ELISA and Western blot reveal a variety of epitopes across the myocilin polypeptide chain. A more precise understanding of myocilin antibody targets, including conformational specificity, should aid the community in standardizing protocols across laboratories and in turn, lead to a better understanding of eye physiology and disease.

Structure and Misfolding of the Flexible Tripartite Coiled-Coil Domain of Glaucoma-Associated Myocilin.

Glaucoma-associated myocilin is a member of the olfactomedins, a protein family involved in neuronal development and human diseases. Molecular studies of the myocilin N-terminal coiled coil demonstrate a unique tripartite architecture: a Y-shaped parallel dimer-of-dimers with distinct tetramer and dimer regions. The structure of the dimeric C-terminal 7-heptad repeats elucidates an unexpected repeat pattern involving inter-strand stabilization by oppositely charged residues. Molecular dynamics simulations reveal an alternate accessible conformation in which the terminal inter-strand disulfide limits the extent of unfolding and results in a kinked configuration. By inference, full-length myocilin is also branched, with two pairs of C-terminal olfactomedin domains. Selected variants within the N-terminal region alter the apparent quaternary structure of myocilin but do so without compromising stability or causing aggregation. In addition to increasing our structural knowledge of naturally occurring extracellular coiled coils and biomedically important olfactomedins, this work broadens the scope of protein misfolding in the pathogenesis of myocilin-associated glaucoma.

Metal ion coordination in the E. coli Nudix hydrolase dihydroneopterin triphosphate pyrophosphatase: New clues into catalytic mechanism.

Dihydroneopterin triphosphate pyrophosphatase (DHNTPase), a member of the Mg2+ dependent Nudix hydrolase superfamily, is the recently-discovered enzyme that functions in the second step of the pterin branch of the folate biosynthetic pathway in E. coli. DHNTPase is of interest because inhibition of enzymes in bacterial folate biosynthetic pathways is a strategy for antibiotic development. We determined crystal structures of DHNTPase with and without activating, Mg2+-mimicking metals Co2+ and Ni2+. Four metal ions, identified by anomalous scattering, and stoichiometrically confirmed in solution by isothermal titration calorimetry, are held in place by Glu56 and Glu60 within the Nudix sequence motif, Glu117, waters, and a sulfate ion, of which the latter is further stabilized by a salt bridge with Lys7. In silico docking of the DHNTP substrate reveals a binding mode in which the pterin ring moiety is nestled in a largely hydrophobic pocket, the ß-phosphate activated for nucleophilic attack overlays with the crystallographic sulfate and is in line with an activated water molecule, and remaining phosphate groups are stabilized by all four identified metal ions. The structures and binding data provide new details regarding DHNTPase metal requirements, mechanism, and suggest a strategy for efficient inhibition.