Mycolactone: A Broad Spectrum Multitarget Antiviral Active in the Picomolar Range for COVID-19 Prevention and Cure.

We have previously shown computationally that Mycolactone (MLN), a toxin produced by Mycobacterium ulcerans, strongly binds to Munc18b and other proteins, presumably blocking degranulation and exocytosis of blood platelets and mast cells. We investigated the effect of MLN on endocytosis using similar approaches, and it bound strongly to the N-terminal of the clathrin protein and a novel SARS-CoV-2 fusion protein. Experimentally, we found 100% inhibition up to 60 nM and 84% average inhibition at 30 nM in SARS-CoV-2 live viral assays. MLN was also 10× more potent than remdesivir and molnupiravir. MLN's toxicity against human alveolar cell line A549, immortalized human fetal renal cell line HEK293, and human hepatoma cell line Huh7.1 were 17.12%, 40.30%, and 36.25%, respectively. The cytotoxicity IC50 breakpoint ratio versus anti-SARS-CoV-2 activity was more than 65-fold. The IC50 values against the alpha, delta, and Omicron variants were all below 0.020 µM, and 134.6 nM of MLN had 100% inhibition in an entry and spread assays. MLN is eclectic in its actions through its binding to Sec61, AT2R, and the novel fusion protein, making it a good drug candidate for treating and preventing COVID-19 and other similarly transmitted enveloped viruses and pathogens.

Disulfide engineering of human Kunitz-type serine protease inhibitors enhances proteolytic stability and target affinity toward mesotrypsin.

Serine protease inhibitors of the Kunitz-bovine pancreatic trypsin inhibitor (BPTI) family are ubiquitous biological regulators of proteolysis. These small proteins are resistant to proteolysis, but can be slowly cleaved within the protease-binding loop by target proteases, thereby compromising their activity. For the human protease mesotrypsin, this cleavage is especially rapid. Here, we aimed to stabilize the Kunitz domain structure against proteolysis through disulfide engineering. Substitution within the Kunitz inhibitor domain of the amyloid precursor protein (APPI) that incorporated a new disulfide bond between residues 17 and 34 reduced proteolysis by mesotrypsin 74-fold. Similar disulfide engineering of tissue factor pathway inhibitor-1 Kunitz domain 1 (KD1TFPI1) and bikunin Kunitz domain 2 (KD2bikunin) likewise stabilized these inhibitors against mesotrypsin proteolysis 17- and 6.6-fold, respectively. Crystal structures of disulfide-engineered APPI and KD1TFPI1 variants in a complex with mesotrypsin at 1.5 and 2.0 Å resolution, respectively, confirmed the formation of well-ordered disulfide bonds positioned to stabilize the binding loop. Long all-atom molecular dynamics simulations of disulfide-engineered Kunitz domains and their complexes with mesotrypsin revealed conformational stabilization of the primed side of the inhibitor-binding loop by the engineered disulfide, along with global suppression of conformational dynamics in the Kunitz domain. Our findings suggest that the Cys-17-Cys-34 disulfide slows proteolysis by dampening conformational fluctuations in the binding loop and minimizing motion at the enzyme-inhibitor interface. The generalizable approach developed here for the stabilization against proteolysis of Kunitz domains, which can serve as important scaffolds for therapeutics, may thus find applications in drug development.

Design and Evaluation of PEGylated Liposomal Formulation of a Novel Multikinase Inhibitor for Enhanced Chemosensitivity and Inhibition of Metastatic Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has one of the highest mortality rates among cancers. Chemotherapy is the standard first-line treatment, but only modest survival benefits are observed. With the advent of targeted therapies, epidermal growth factor receptor (EGFR) has been acknowledged as a prospective target in PDAC since it is overexpressed in up to 60% of cases. Similarly, the tyrosine-protein kinase Met (cMET) is also overexpressed in PDAC (27-60%) and is a prognostic marker for poor survival. Interestingly, EGFR and cMET share some common signaling pathways including PI3K/Akt and MAPK pathways. Small molecule inhibitors or bispecific antibodies that can target both EGFR and cMET are therefore emerging as novel options for cancer therapy. We previously developed a dual EGFR and cMET inhibitor (N19) that was able to inhibit tumor growth in nonsmall cell lung cancer models resistant to EGFR tyrosine kinase inhibitors (TKI). Here, we report the development of a novel liposomal formulation of N19 (LN19) and showed significant growth inhibition and increased sensitivity toward gemcitabine in the pancreatic adenocarcinoma orthotopic xenograft model. Taken together, our results suggest that LN19 can be valued as an effective combination therapy with conventional chemotherapy such as gemcitabine for PDAC patients.

Genomic Observations of a Rare/Pathogenic SMAD3 Variant in Loeys⁻Dietz Syndrome 3 Confirmed by Protein Informatics and Structural Investigations.

Background and objectives: Loeys-Dietz syndrome 3, also known as aneurysms--osteoarthritis syndrome, is an autosomal dominant genetic connective tissue disease caused by pathogenic variants in SMAD3, a transcription factor involved in TGF-ß signaling. This disorder is characterized by early-onset osteoarthritis and arterial aneurysms. Common features include scoliosis, uvula abnormalities, striae, and velvety skin. Materials and Methods: The pathogenicity of a variant of uncertain significance in the SMAD3 gene was evaluated (variant c.220C > T) through personalized protein informatics and molecular studies. Results: The case of a 44-year-old male, who was originally presumed to have Marfan syndrome, is presented. An expanded gene panel determined the probable cause to be a variant in SMAD3, c.220C > T (p.R74W). His case was complicated by a history of stroke, but his phenotype was otherwise characteristic for Loeys-Dietz syndrome 3. Conclusion: This case emphasizes the importance of comprehensive genetic testing to evaluate patients for connective tissue disorders, as well as the potential benefit of utilizing a protein informatics platform for the assessment of variant pathogenicity.

Heterozygous PINK1 p.G411S increases risk of Parkinson's disease via a dominant-negative mechanism.

SEE GANDHI AND PLUN-FAVREAU DOI101093/AWW320 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: It has been postulated that heterozygous mutations in recessive Parkinson's genes may increase the risk of developing the disease. In particular, the PTEN-induced putative kinase 1 (PINK1) p.G411S (c.1231G>A, rs45478900) mutation has been reported in families with dominant inheritance patterns of Parkinson's disease, suggesting that it might confer a sizeable disease risk when present on only one allele. We examined families with PINK1 p.G411S and conducted a genetic association study with 2560 patients with Parkinson's disease and 2145 control subjects. Heterozygous PINK1 p.G411S mutations markedly increased Parkinson's disease risk (odds ratio = 2.92, P = 0.032); significance remained when supplementing with results from previous studies on 4437 additional subjects (odds ratio = 2.89, P = 0.027). We analysed primary human skin fibroblasts and induced neurons from heterozygous PINK1 p.G411S carriers compared to PINK1 p.Q456X heterozygotes and PINK1 wild-type controls under endogenous conditions. While cells from PINK1 p.Q456X heterozygotes showed reduced levels of PINK1 protein and decreased initial kinase activity upon mitochondrial damage, stress-response was largely unaffected over time, as expected for a recessive loss-of-function mutation. By contrast, PINK1 p.G411S heterozygotes showed no decrease of PINK1 protein levels but a sustained, significant reduction in kinase activity. Molecular modelling and dynamics simulations as well as multiple functional assays revealed that the p.G411S mutation interferes with ubiquitin phosphorylation by wild-type PINK1 in a heterodimeric complex. This impairs the protective functions of the PINK1/parkin-mediated mitochondrial quality control. Based on genetic and clinical evaluation as well as functional and structural characterization, we established p.G411S as a rare genetic risk factor with a relatively large effect size conferred by a partial dominant-negative function phenotype.

TRIO gene segregation in a family with cerebellar ataxia.

AIM OF THE STUDY: To report a family with a novel TRIO gene mutation associated with phenotype of cerebellar ataxia. MATERIALS AND METHODS: Seven family members of Caribbean descent were recruited through our ataxia research protocol; of the family members, the mother and all 3 children were found to be affected with severe young-onset and rapidly progressive truncal and appendicular ataxia leading to early disability. Array comparative genomic hybridization, mitochondrial DNA analysis, and whole-exome sequencing were performed on 3 of the family members (mother and 2 daughters). RESULTS: While the maternal grandmother, great uncle and great aunt were unaffected, the mother and 3 children displayed cognitive dysfunction, severe ataxia, spasticity, and speech disturbances. Age of onset ranged between 3 and 17 years, with average current disease duration of 21 years. Whole-exome sequencing showed a variant p.A1214V in exon 22 of the TRIO gene in 3 of the family members. Array comparative genomic hybridization and mitochondrial DNA analysis were normal. The same variant was later discovered in all but one family member. CONCLUSIONS AND CLINICAL IMPLICATIONS: The TRIO p.A1214V variant is associated with cerebellar ataxia in the studied family; it was present in all affected and unaffected family members. Phenotype is severe and broad. Anticipation seems to be present (based on 2 affected generations). It is warranted to screen additional familial early-onset and rapidly progressive ataxia cases for this genotype. TRIO gene mutations may well represent a novel spinocerebellar ataxia subtype.

An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis.

The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. Although considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here, we examine the importance of substrate dynamics in the cleavage of Kunitz-bovine pancreatic trypsin inhibitor protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4-Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. By using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning 3 orders of magnitude, we identify global and local dynamic features of substrates on the nanosecond-microsecond time scale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substrate-like and product-like states, linking substrate dynamics on the nanosecond-microsecond time scale with large collective substrate motions on the much slower time scale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis.

(Patho-)physiological relevance of PINK1-dependent ubiquitin phosphorylation.

Mutations in PINK1 and PARKIN cause recessive, early-onset Parkinson's disease (PD). Together, these two proteins orchestrate a protective mitophagic response that ensures the safe disposal of damaged mitochondria. The kinase PINK1 phosphorylates ubiquitin (Ub) at the conserved residue S65, in addition to modifying the E3 ubiquitin ligase Parkin. The structural and functional consequences of Ub phosphorylation (pS65-Ub) have already been suggested from in vitro experiments, but its (patho-)physiological significance remains unknown. We have generated novel antibodies and assessed pS65-Ub signals in vitro and in cells, including primary neurons, under endogenous conditions. pS65-Ub is dependent on PINK1 kinase activity as confirmed in patient fibroblasts and postmortem brain samples harboring pathogenic mutations. We show that pS65-Ub is reversible and barely detectable under basal conditions, but rapidly induced upon mitochondrial stress in cells and amplified in the presence of functional Parkin. pS65-Ub accumulates in human brain during aging and disease in the form of cytoplasmic granules that partially overlap with mitochondrial, lysosomal, and total Ub markers. Additional studies are now warranted to further elucidate pS65-Ub functions and fully explore its potential for biomarker or therapeutic development.

Targeted manipulation of the sortilin-progranulin axis rescues progranulin haploinsufficiency.

Progranulin (GRN) mutations causing haploinsufficiency are a major cause of frontotemporal lobar degeneration (FTLD-TDP). Recent discoveries demonstrating sortilin (SORT1) is a neuronal receptor for PGRN endocytosis and a determinant of plasma PGRN levels portend the development of enhancers targeting the SORT1-PGRN axis. We demonstrate the preclinical efficacy of several approaches through which impairing PGRN's interaction with SORT1 restores extracellular PGRN levels. Our report is the first to demonstrate the efficacy of enhancing PGRN levels in iPSC neurons derived from frontotemporal dementia (FTD) patients with PGRN deficiency. We validate a small molecule preferentially increases extracellular PGRN by reducing SORT1 levels in various mammalian cell lines and patient-derived iPSC neurons and lymphocytes. We further demonstrate that SORT1 antagonists and a small-molecule binder of PGRN588₋593, residues critical for PGRN-SORT1 binding, inhibit SORT1-mediated PGRN endocytosis. Collectively, our data demonstrate that the SORT1-PGRN axis is a viable target for PGRN-based therapy, particularly in FTD-GRN patients.

Structural and Functional Impact of Parkinson Disease-Associated Mutations in the E3 Ubiquitin Ligase Parkin.

Mutations in the PARKIN/PARK2 gene that result in loss-of-function of the encoded, neuroprotective E3 ubiquitin ligase Parkin cause recessive, familial early-onset Parkinson disease. As an increasing number of rare Parkin sequence variants with unclear pathogenicity are identified, structure-function analyses will be critical to determine their disease relevance. Depending on the specific amino acids affected, several distinct pathomechanisms can result in loss of Parkin function. These include disruption of overall Parkin folding, decreased solubility, and protein aggregation. However pathogenic effects can also result from misregulation of Parkin autoinhibition and of its enzymatic functions. In addition, interference of binding to coenzymes, substrates, and adaptor proteins can affect its catalytic activity too. Herein, we have performed a comprehensive structural and functional analysis of 21 PARK2 missense mutations distributed across the individual protein domains. Using this combined approach, we were able to pinpoint some of the pathogenic mechanisms of individual sequence variants. Similar analyses will be critical in gaining a complete understanding of the complex regulations and enzymatic functions of Parkin. These studies will not only highlight the important residues, but will also help to develop novel therapeutics aimed at activating and preserving an active, neuroprotective form of Parkin.

The dual functions of the extreme N-terminus of TDP-43 in regulating its biological activity and inclusion formation.

TAR DNA-binding protein-43 (TDP-43) is the principal component of ubiquitinated inclusions in amyotrophic lateral sclerosis (ALS) and the most common pathological subtype of frontotemporal dementia-frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP). To date, the C-terminus of TDP-43, which is aggregation-prone and contains almost all ALS-associated mutations, has garnered much attention while the functions of the N-terminus of TDP-43 remain largely unknown. To bridge this gap in our knowledge, we utilized novel cell culture and computer-assisted models to evaluate which region(s) of TDP-43 regulate its folding, self-interaction, biological activity and aggregation. We determined that the extreme N-terminus of TDP-43, specifically the first 10 residues, regulates folding of TDP-43 monomers necessary for proper homodimerization and TDP-43-regulated splicing. Despite such beneficial functions, we discovered an interesting dichotomy: full-length TDP-43 aggregation, which is believed to be a pathogenic process, also requires the extreme N-terminus of TDP-43. As such, we provide new insight into the structural basis for TDP-43 function and aggregation, and we suggest that stabilization of TDP-43 homodimers, the physiologically active form of TDP-43, may be a promising therapeutic strategy for ALS and FTLD-TDP.

Targeted inhibition of the deubiquitinating enzymes, USP14 and UCHL5, induces proteotoxic stress and apoptosis in Waldenström macroglobulinaemia tumour cells.

Deubiquitinase enzymes (DUBs) of the proteasomal 19S regulatory particle are emerging as important therapeutic targets in several malignancies. Here we demonstrate that inhibition of two proteasome-associated DUBs (USP14 and UCHL5) with the small molecule DUB inhibitor b-AP15, results in apoptosis of human Waldenström macroglobulinaemia (WM) cell lines and primary patient-derived WM tumour cells. Importantly, b-AP15 produced proteotoxic stress and apoptosis in WM cells that have acquired resistance to the proteasome inhibitor bortezomib. In silico modelling identified protein residues that were critical for the binding of b-AP15 with USP14 or UCHL5 and proteasome enzyme activity assays confirmed that b-AP15 does not affect the proteolytic capabilities of the 20S proteasome ß-subunits. In vitro toxicity from b-AP15 appeared to result from a build-up of ubiquitinated proteins and activation of the endoplasmic reticulum stress response in WM cells, an effect that also disrupted the mitochondria. Focused transcriptome profiling of b-AP15-treated WM cells revealed modulation of several genes regulating cell stress and NF-κB signalling, the latter whose protein translocation and downstream target activation was reduced by b-AP15 in vitro. This is the first report to define the effects and underlying mechanisms associated with inhibition of USP14 and UCHL5 DUB activity in WM tumour cells.

Activation of the E3 ubiquitin ligase Parkin.

The PINK1 (phosphatase and tensin homologue-induced putative kinase 1)/Parkin-dependent mitochondrial quality control pathway mediates the clearance of damaged organelles, but appears to be disrupted in Parkinson's disease (PD) [Springer and Kahle (2011) Autophagy 7, 266-278]. Upon mitochondrial stress, PINK1 activates the E3 ubiquitin (Ub) ligase Parkin through phosphorylation of the Ub-like (UBL) domain of Parkin and of the small modifier Ub itself at a conserved residue [Sauvé and Gehring (2014) Cell Res. 24, 1025-1026]. Recently resolved partial crystal structures of Parkin showed a 'closed', auto-inhibited conformation, consistent with its notoriously weak enzymatic activity at steady state [Wauer and Komander (2013) EMBO J. 32, 2099-2112; Riley et al. (2013) Nat. Commun. 4, 1982; Trempe et al. (2013) Science 340, 1451-1455; Spratt et al. (2013) Nat. Commun. 4, 1983]. It has thus become clear that Parkin must undergo major structural rearrangements in order to unleash its catalytic functions. Recent published findings derived from X-ray structures and molecular modelling present a complete structural model of human Parkin at an all-atom resolution [Caulfield et al. (2014) PLoS Comput. Biol. 10, e1003935]. The results of the combined in silico simulations-based and experimental assay-based study indicates that PINK1-dependent Ser65 phosphorylation of Parkin is required for its activation and triggering of 'opening' conformations. Indeed, the obtained structures showed a sequential release of Parkin's intertwined domains and allowed docking of an Ub-charged E2 coenzyme, which could enable its enzymatic activity. In addition, using cell-based screening, select E2 enzymes that redundantly, cooperatively or antagonistically regulate Parkin's activation and/or enzymatic functions at different stages of the mitochondrial autophagy (mitophagy) process were identified [Fiesel et al. (2014) J. Cell Sci. 127, 3488-3504]. Other work that aims to pin-point the particular pathogenic dysfunctions of Parkin mis-sense mutations have been recently disseminated (Fabienne C. Fiesel, Thomas R. Caulfield, Elisabeth L. Moussaud-Lamodiere, Daniel F.A.R. Dourado, Kotaro Ogaki, Owen A. Ross, Samuel C. Flores, and Wolfdieter Springer, submitted). Such a structure-function approach provides the basis for the dissection of Parkin's regulation and a targeted drug design to identify small-molecule activators of this neuroprotective E3 Ub ligase.

Phosphorylation by PINK1 releases the UBL domain and initializes the conformational opening of the E3 ubiquitin ligase Parkin.

Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson's disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin's enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased) molecular dynamics simulation, Monte Carlo algorithms, and minimal-biasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin activators through targeted drug design.

Long-range electrostatic complementarity governs substrate recognition by human chymotrypsin C, a key regulator of digestive enzyme activation.

Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5' subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2' positions of CTRC, although acidic P2' residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels.

Aggregation-prone c9FTD/ALS poly(GA) RAN-translated proteins cause neurotoxicity by inducing ER stress.

The occurrence of repeat-associated non-ATG (RAN) translation, an atypical form of translation of expanded repeats that results in the synthesis of homopolymeric expansion proteins, is becoming more widely appreciated among microsatellite expansion disorders. Such disorders include amyotrophic lateral sclerosis and frontotemporal dementia caused by a hexanucleotide repeat expansion in the C9ORF72 gene (c9FTD/ALS). We and others have recently shown that this bidirectionally transcribed repeat is RAN translated, and the "c9RAN proteins" thusly produced form neuronal inclusions throughout the central nervous system of c9FTD/ALS patients. Nonetheless, the potential contribution of c9RAN proteins to disease pathogenesis remains poorly understood. In the present study, we demonstrate that poly(GA) c9RAN proteins are neurotoxic and may be implicated in the neurodegenerative processes of c9FTD/ALS. Specifically, we show that expression of poly(GA) proteins in cultured cells and primary neurons leads to the formation of soluble and insoluble high molecular weight species, as well as inclusions composed of filaments similar to those observed in c9FTD/ALS brain tissues. The expression of poly(GA) proteins is accompanied by caspase-3 activation, impaired neurite outgrowth, inhibition of proteasome activity, and evidence of endoplasmic reticulum (ER) stress. Of importance, ER stress inhibitors, salubrinal and TUDCA, provide protection against poly(GA)-induced toxicity. Taken together, our data provide compelling evidence towards establishing RAN translation as a pathogenic mechanism of c9FTD/ALS, and suggest that targeting the ER using small molecules may be a promising therapeutic approach for these devastating diseases.

Cryo-EM structures of pathogenic fibrils and their impact on neurodegenerative disease research.

Neurodegenerative diseases are commonly associated with the formation of aberrant protein aggregates within the brain, and ultrastructural analyses have revealed that the proteins within these inclusions often assemble into amyloid filaments. Cryoelectron microscopy (cryo-EM) has emerged as an effective method for determining the near-atomic structure of these disease-associated filamentous proteins, and the resulting structures have revolutionized the way we think about aberrant protein aggregation and propagation during disease progression. These structures have also revealed that individual fibril conformations may dictate different disease conditions, and this newfound knowledge has improved disease modeling in the lab and advanced the ongoing pursuit of clinical tools capable of distinguishing and targeting different pathogenic entities within living patients. In this review, we summarize some of the recently developed cryo-EM structures of ex vivo α-synuclein, tau, ß-amyloid (Aß), TAR DNA-binding protein 43 (TDP-43), and transmembrane protein 106B (TMEM106B) fibrils and discuss how these structures are being leveraged toward mechanistic research and therapeutic development.

A novel histone deacetylase inhibitor W2A-16 improves the barrier integrity in brain vascular endothelial cells.

The maturation of brain microvascular endothelial cells leads to the formation of a tightly sealed monolayer, known as the blood-brain barrier (BBB). The BBB damage is associated with the pathogenesis of age-related neurodegenerative diseases including vascular cognitive impairment and Alzheimer's disease. Growing knowledge in the field of epigenetics can enhance the understanding of molecular profile of the BBB and has great potential for the development of novel therapeutic strategies or targets to repair a disrupted BBB. Histone deacetylases (HDACs) inhibitors are epigenetic regulators that can induce acetylation of histones and induce open chromatin conformation, promoting gene expression by enhancing the binding of DNA with transcription factors. We investigated how HDAC inhibition influences the barrier integrity using immortalized human endothelial cells (HCMEC/D3) and the human induced pluripotent stem cell (iPSC)-derived brain vascular endothelial cells. The endothelial cells were treated with or without a novel compound named W2A-16. W2A-16 not only activates Wnt/ß-catenin signaling but also functions as a class I HDAC inhibitor. We demonstrated that the administration with W2A-16 sustained barrier properties of the monolayer of endothelial cells, as evidenced by increased trans-endothelial electrical resistance (TEER). The BBB-related genes and protein expression were also increased compared with non-treated controls. Analysis of transcript profiles through RNA-sequencing in hCMEC/D3 cells indicated that W2A-16 potentially enhances BBB integrity by influencing genes associated with the regulation of the extracellular microenvironment. These findings collectively propose that the HDAC inhibition by W2A-16 plays a facilitating role in the formation of the BBB. Pharmacological approaches to inhibit HDAC may be a potential therapeutic strategy to boost and/or restore BBB integrity.

In Silico Investigation of Parkin-Activating Mutations Using Simulations and Network Modeling.

Complete loss-of-function mutations in the PRKN gene are a major cause of early-onset Parkinson's disease (PD). PRKN encodes the Parkin protein, an E3 ubiquitin ligase that works in conjunction with the ubiquitin kinase PINK1 in a distinct quality control pathway to tag damaged mitochondria for autophagic clearance, i.e., mitophagy. According to previous structural investigations, Parkin protein is typically kept in an inactive conformation via several intramolecular, auto-inhibitory interactions. Here, we performed molecular dynamics simulations (MDS) to provide insights into conformational changes occurring during the de-repression of Parkin and the gain of catalytic activity. We analyzed four different Parkin-activating mutations that are predicted to disrupt certain aspects of its auto-inhibition. All four variants showed greater conformational motions compared to wild-type protein, as well as differences in distances between domain interfaces and solvent-accessible surface area, which are thought to play critical roles as Parkin gains catalytic activity. Our findings reveal that the studied variants exert a notable influence on Parkin activation as they alter the opening of its closed inactive structure, a finding that is supported by recent structure- and cell-based studies. These findings not only helped further characterize the hyperactive variants but overall improved our understanding of Parkin's catalytic activity and nominated targets within Parkin's structure for potential therapeutic designs.