Cryo-EM Structure of the Full-length hnRNPA1 Amyloid Fibril

Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is a multifunctional RNA-binding protein that is associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis and multisystem proteinopathy. In this study, we have used cryo-electron microscopy to investigate the three-dimensional structure of amyloid fibrils from full-length hnRNPA1 protein. We find that the fibril core is formed by a 45-residue segment of the prion-like low-complexity domain of the protein, whereas the remaining parts of the protein (275 residues) form a fuzzy coat around the fibril core. The fibril consists of two fibril protein stacks that are arranged into a pseudo-2₁ screw symmetry. The ordered core harbors several of the positions that are known to be affected by disease-associated mutations, but does not encompass the most aggregation-prone segments of the protein. These data indicate that the structures of amyloid fibrils from full-length proteins may be more complex than anticipated by current theories on protein misfolding.     

FASN inhibitor TVB-3166 prevents S-acylation of the spike protein of human coronaviruses

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other coronaviruses mediates host cell entry and is S-acylated on multiple phylogenetically conserved cysteine residues. Multiple protein acyltransferase enzymes have been reported to post-translationally modify spike proteins; however, strategies to exploit this modification are lacking. Using resin-assisted capture MS, we demonstrate that the spike protein is S-acylated in SARS-CoV-2-infected human and monkey epithelial cells. We further show that increased abundance of the acyltransferase ZDHHC5 associates with increased S-acylation of the spike protein, whereas ZDHHC5 knockout cells had a 40% reduction in the incorporation of an alkynyl-palmitate using click chemistry detection. We also found that the S-acylation of the spike protein is not limited to palmitate, as clickable versions of myristate and stearate were also labelled the protein. Yet, we observed that ZDHHC5 was only modified when incubated with alkyne-palmitate, suggesting it has specificity for this acyl-CoA, and that other ZDHHC enzymes may use additional fatty acids to modify the spike protein. Since multiple ZDHHC isoforms may modify the spike protein, we also examined the ability of the FASN inhibitor TVB-3166 to prevent S-acylation of the spike proteins of SARS-CoV-2 and human CoV-229E. We show that treating cells with TVB-3166 inhibited S-acylation of expressed spike proteins and attenuated the ability of SARS-CoV-2 and human CoV-229E to spread in vitro. Our findings further substantiate the necessity of CoV spike protein S-acylation and demonstrate that de novo fatty acid synthesis is critical for the proper S-acylation of the spike protein.     

A comparative Proteomics Analysis Identified Differentially Expressed Proteins in Pancreatic Cancer–Associated Stellate Cell Small Extracellular Vesicles

Human pancreatic stellate cells (HPSCs) are an essential stromal component and mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles involved in cell-to-cell communications and are released from stromal cells within PDAC. A detailed comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs) remains a gap in our current knowledge regarding stellate cells and PDAC. We hypothesized there would be differences in sEVs secretion and protein expression that might contribute to PDAC biology. To test this hypothesis, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. We report here our initial observations. First, HPSC cells derived from PDAC tumors secrete a higher volume of sEVs when compared to normal pancreatic stellate cells (HPaStec). Although our data revealed that both normal and tumor-derived sEVs demonstrated no significant biological effect on cancer cells, we observed efficient uptake of sEVs by both normal and cancer epithelial cells. Additionally, intact membrane-associated proteins on sEVs were essential for efficient uptake. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography–tandem mass spectrometry. Most of the 1481 protein groups identified were shared with the exosome database, ExoCarta. Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted p value ≤0.05) between HPSC and HPaStec sEVs. Of note, HPSC sEVs contained dramatically more CSE1L (chromosome segregation 1–like protein), a described marker of poor prognosis in patients with pancreatic cancer. Based on our results, we have demonstrated unique populations of sEVs originating from stromal cells with PDAC and suggest that these are significant to cancer biology. Further studies should be undertaken to gain a deeper understanding that could drive novel therapy.     

RAN proteins in neurodegenerative disease: Repeating themes and unifying therapeutic strategies

Microsatellite-expansion mutations cause >50 neurological diseases but there are no effective treatments. Mechanistic studies have historically focused on protein loss-of-function and protein or RNA gain-of-function effects. It is now clear that many expansion mutations are bidirectionally transcribed producing two toxic expansion RNAs, which can produce up to six mutant proteins by repeat associated non-AUG (RAN) translation. Multiple types of RAN proteins have been shown to be toxic in cell and animal models, to lead to common types of neuropathological changes, and to dysregulate key pathways. How RAN proteins are produced without the canonical AUG or close-cognate AUG-like initiation codons is not yet completely understood but RNA structure, flanking sequences and stress pathways have been shown to be important. Here, we summarize recent progress in understanding the role of RAN proteins, mechanistic insights into their production, and the identification of novel therapeutic strategies that may be applicable across these neurodegenerative disorders.     

New Insights on Effects of Glucocorticoids in Patients With SARS-CoV-2 Infection

ObjectiveSince January 2020, the highly contagious novel coronavirus SARS-CoV-2 has caused a global pandemic. Severe COVID-19 leads to a massive release of proinflammatory mediators, leading to diffuse damage to the lung parenchyma, and the development of acute respiratory distress syndrome. Treatment with the highly potent glucocorticoid (GC) dexamethasone was found to be effective in reducing mortality in severely affected patients.MethodsTo review the effects of glucocorticoids in the context of COVID-19 we performed a literature search in the PubMed database using the terms COVID-19 and glucocorticoid treatment. We identified 1429 article publications related to COVID-19 and glucocorticoid published from 1.1.2020 to the present including 238 review articles and 36 Randomized Controlled Trials. From these studies, we retrieved 13 Randomized Controlled Trials and 86 review articles that were relevant to our review topics. We focused on the recent literature dealing with glucocorticoid metabolism in critically ill patients and investigating the effects of glucocorticoid therapy on the immune system in COVID-19 patients with severe lung injury.ResultsIn our review, we have discussed the regulation of the hypothalamic-pituitary-adrenal axis in patients with critical illness, selection of a specific GC for critical illness-related GC insufficiency, and recent studies that investigated hypothalamic-pituitary-adrenal dysfunction in patients with COVID-19. We have also addressed the specific activation of the immune system with chronic endogenous glucocorticoid excess, as seen in patients with Cushing syndrome, and, finally, we have discussed immune activation due to coronavirus infection and the possible mechanisms leading to improved outcomes in patients with COVID-19 treated with GCs.ConclusionFor clinical endocrinologists prescribing GCs for their patients, a precise understanding of both the molecular- and cellular-level mechanisms of endogenous and exogenous GCs is imperative, including timing of administration, dosage, duration of treatment, and specific formulations of GCs.     

Osilodrostat: A Review of Recent Clinical Studies and Practical Recommendations for its Use in the Treatment of Cushing Disease

ObjectiveCushing disease (CD) is characterized by chronic hypercortisolism caused by an adrenocorticotropic hormone-secreting pituitary adenoma. Surgery remains the first-line treatment option; however, medical therapy is essential if surgery is contraindicated or fails to achieve remission or when recurrence occurs after surgical remission. Osilodrostat (Isturisa), a novel steroidogenic inhibitor, is now approved for the treatment of CD in the United States and Cushing syndrome in Europe. Herein, we review pharmacology and data on the efficacy, safety, and clinical use of osilodrostat and provide guidance on its use in treating patients with CD.MethodsWe reviewed the literature and published clinical trial data of osilodrostat use in patients with Cushing syndrome. Detailed information related to the clinical assessment of osilodrostat use, potential drug-to-drug interactions, drug initiation, dose titration, and the monitoring of drug tolerability were discussed.ResultsClinical trial data demonstrated that osilodrostat, by virtue of inhibiting 11-β hydroxylase, potently and rapidly decreased the 24-hour urinary free cortisol levels and sustained these reductions, with improved glycemia, blood pressure, body weight, and quality of life as well as lessened depression. Osilodrostat may interact with certain drugs, resulting in QT prolongation, which requires careful assessment of concomitant medications and periodic monitoring using electrocardiogram, respectively. The common adverse effects include adrenal insufficiency, hypokalemia, edema, and hyperandrogenic symptoms, which can be minimized using a slower up-titration dosing regimen.ConclusionOsilodrostat is an effective, new treatment option for CD, with positive effects on cardiovascular and quality of life parameters as well as tolerable adverse effects. This article provides a review of the pharmacology of osilodrostat and offers practical recommendations on the use of osilodrostat to treat CD.     

Design and engineering of allosteric communications in proteins

Allostery in proteins plays an important role in regulating protein activities and influencing many biological processes such as gene expression, enzyme catalysis, and cell signaling. The process of allostery takes place when a signal detected at a site on a protein is transmitted via a mechanical pathway to a functional site and, thus, influences its activity. The pathway of allosteric communication consists of amino acids that form a network with covalent and non-covalent bonds. By mutating residues in this allosteric network, protein engineers have successfully established novel allosteric pathways to achieve desired properties in the target protein. In this review, we highlight the most recent and state-of-the-art techniques for allosteric communication engineering. We also discuss the challenges that need to be overcome and future directions for engineering protein allostery.     

Precision materials: Computational design methods of accurate protein materials

Nature has evolved a vast repertoire of structures and functions based on an ordered, orchestrated, protein building-blocks assembly. For decades these sophisticated materials have been studied, mimicked, and repurposed, yet recently, computational protein engineering methods provided an alternative route: creating protein materials de-novo, surpassing evolutionary constraints and optimized for specific tasks. We highlight two areas of research that fundamentally accelerate design of structurally well-defined programmable protein materials. First, implementations of hierarchical assembly and geometric sampling (docking) strategies to create designable backbones under pre-specified symmetry constraints. Second, progress in protein–protein interfaces and sequence design methods, using Rosetta, that drive programmable supramolecular assemblies. These approaches have proven effective in generating diverse protein assemblies in 0-, 1-, 2-, and 3-dimensional architectures (constituting single or multiple components), and as part of a synthetic or a biological system. We expect these methods shall transform the toolbox of protein designers developing next generation synthetic and biological materials.     

Heteroarylamide smoothened inhibitors: Discovery of N-[2,4-dimethyl-5-(1-methylimidazol-4-yl)phenyl]-4-(2-pyridylmethoxy)benzamide (AZD8542) and N-[5-(1H-imidazol-2-yl)-2,4-dimethyl-phenyl]-4-(2- pyridylmethoxy)benzamide (AZD7254)

Aberrant hedgehog (Hh) pathway signaling is implicated in multiple cancer types and targeting the Smoothened (SMO) receptor, a key protein of the Hh pathway, has proven effective in treating metastasized basal cell carcinoma. Our lead optimization effort focused on a series of heteroarylamides. We observed that a methyl substitution ortho to the heteroaryl groups on an aniline core significantly improved the potency of this series of compounds. These findings predated the availability of SMO crystal structure in 2013. Here we retrospectively applied quantum mechanics calculations to demonstrate the o-Me substitution favors the bioactive conformation by inducing a dihedral twist between the heteroaryl rings and the core aniline. The o-Me also makes favorable hydrophobic interactions with key residue side chains in the binding pocket. From this effort, two compounds (AZD8542 and AZD7254) showed excellent pharmacokinetics across multiple preclinical species and demonstrated in vivo activity in abrogating the Hh paracrine pathway as well as anti- tumor effects.     

Validation of low-dose lung cancer PET-CT protocol and PET image improvement using machine learning

PurposeTo conduct a simplified lesion-detection task of a low-dose (LD) PET-CT protocol for frequent lung screening using 30% of the effective PETCT dose and to investigate the feasibility of increasing clinical value of low-statistics scans using machine learning.MethodsWe acquired 33 SD PET images, of which 13 had actual LD (ALD) PET, and simulated LD (SLD) PET images at seven different count levels from the SD PET scans. We employed image quality transfer (IQT), a machine learning algorithm that performs patch-regression to map parameters from low-quality to high-quality images. At each count level, patches extracted from 23 pairs of SD/SLD PET images were used to train three IQT models – global linear, single tree, and random forest regressions with cubic patch sizes of 3 and 5 voxels. The models were then used to estimate SD images from LD images at each count level for 10 unseen subjects. Lesion-detection task was carried out on matched lesion-present and lesion-absent images.ResultsLD PET-CT protocol yielded lesion detectability with sensitivity of 0.98 and specificity of 1. Random forest algorithm with cubic patch size of 5 allowed further 11.7% reduction in the effective PETCT dose without compromising lesion detectability, but underestimated SUV by 30%.ConclusionLD PET-CT protocol was validated for lesion detection using ALD PET scans. Substantial image quality improvement or additional dose reduction while preserving clinical values can be achieved using machine learning methods though SUV quantification may be biased and adjustment of our research protocol is required for clinical use.     

Smart de novo Macromolecular Structure Modeling from Cryo-EM Maps

The study of macromolecular structures has expanded our understanding of the amazing cell machinery and such knowledge has changed how the pharmaceutical industry develops new vaccines in recent years. Traditionally, X-ray crystallography has been the main method for structure determination, however, cryogenic electron microscopy (cryo-EM) has increasingly become more popular due to recent advancements in hardware and software. The number of cryo-EM maps deposited in the EMDataResource (formerly EMDatabase) since 2002 has been dramatically increasing and it continues to do so. De novo macromolecular complex modeling is a labor-intensive process, therefore, it is highly desirable to develop software that can automate this process. Here we discuss our automated, data-driven, and artificial intelligence approaches including map processing, feature extraction, modeling building, and target identification. Recently, we have enabled DNA/RNA modeling in our deep learning-based prediction tool, DeepTracer. We have also developed DeepTracer-ID, a tool that can identify proteins solely based on the cryo-EM map. In this paper, we will present our accumulated experiences in developing deep learning-based methods surrounding macromolecule modeling applications.     

The Lysine Acetylation Modification in the Porin Aha1 of Aeromonas hydrophila Regulates the Uptake of Multidrug Antibiotics

Protein lysine acetylation (Kac) modification plays important roles in diverse physiological functions. However, there is little evidence on the role of Kac modification in bacterial antibiotic resistance. Here, we compared the differential expressions of whole-cell proteins and Kac peptides in oxytetracycline sensitive and oxytetracycline resistance (OXY^R) strains of Aeromonas hydrophila using quantitative proteomics technologies. We observed a porin family protein Aha1 downregulated in the OXY^R strain, which may have an important role in the OXY resistance. Interestingly, seven of eight Kac peptides of Aha1 decreased abundance in OXY^R as well. Microbiologic assays showed that the K57R, K187R, and K197R Aha1 mutants significantly increased antibiotic resistance to OXY and reduced the intracellular OXY accumulation in OXY stress. Moreover, these Aha1 mutants displayed multidrug resistance features to tetracyclines and β-lactam antibiotics. The 3D model prediction showed that the Kac states of K57, K187, and K197 sites located at the extracellular pore vestibule of Aha1 may be involved in the uptake of specific types of antibiotics. Overall, our results indicate a novel antibiotic resistance mechanism mediated by Kac modification, which may provide a clue for the development of antibiotic therapy strategies.     

Integrating structure-based approaches in generative molecular design

Generative molecular design for drug discovery and development has seen a recent resurgence promising to improve the efficiency of the design-make-test-analyse cycle; by computationally exploring much larger chemical spaces than traditional virtual screening techniques. However, most generative models thus far have only utilized small-molecule information to train and condition de novo molecule generators. Here, we instead focus on recent approaches that incorporate protein structure into de novo molecule optimization in an attempt to maximize the predicted on-target binding affinity of generated molecules. We summarize these structure integration principles into either distribution learning or goal-directed optimization and for each case whether the approach is protein structure-explicit or implicit with respect to the generative model. We discuss recent approaches in the context of this categorization and provide our perspective on the future direction of the field.     

Recent advances in the structural biology of modular polyketide synthases and nonribosomal peptide synthetases

Polyketides and nonribosomal peptides are an important class of natural products with useful bioactivities. These compounds are similarly biosynthesized using enzymes with modular structures despite having different physicochemical properties. These enzymes are attractive targets for bioengineering to produce “unnatural” natural products owing to their modular structures. Therefore, their structures have been studied for a long time; however, the main focus was on truncated-single domains. Surprisingly, there is an increasing number of the structures of whole modules reported, most of which have been enabled through the recent advances in cryogenic electron microscopy technology. In this review, we have summarized the recent advances in the structural elucidation of whole modules.     

Better Cryo-EM Specimen Preparation: How to Deal with the Air–Water Interface?

Cryogenic electron microscopy (cryo-EM) is now one of the most powerful and widely used methods to determine high-resolution structures of macromolecules. A major bottleneck of cryo-EM is to prepare high-quality vitrified specimen, which still faces many practical challenges. During the conventional vitrification process, macromolecules tend to adsorb at the air–water interface (AWI), which is known unfriendly to biological samples. In this review, we outline the nature of AWI and the problems caused by it, such as unpredictable or uneven particle distribution, protein denaturation, dissociation of complex and preferential orientation. We review and discuss the approaches and underlying mechanisms to deal with AWI: 1) Additives, exemplified by detergents, forming a protective layer at AWI and thus preserving the native folds of target macromolecules. 2) Fast vitrification devices based on the idea to freeze in-solution macromolecules before their touching of AWI. 3) Thin layer of continuous supporting films to adsorb macromolecules, and when functionalized with affinity ligands, to specifically anchor the target particles away from the AWI. Among these supporting films, graphene, together with its derivatives, with negligible background noise and mechanical robustness, has emerged as a new generation of support. These strategies have been proven successful in various cases and enable us a better handling of the problems caused by the AWI in cryo-EM specimen preparation.     

Influence of halloysite nanotubes on the efficiency of Asparaginase against mice Ehrlich solid carcinoma

Herein, the impact of the halloysite nanotubes to suppress the side effects of Asparaginase (ANase) cellular proliferation was investigated. Methods: A total of 100 adult male mice was employed. These mice were divided into four equal groups; Group 1 (control), Group 2 (ESC group) of a single dose of 0.15 ml Ehrlich cells (2 × 10⁶) intraperitoneal infusion(IP), Group 3 (ESC + ANase group) received six doses equal treatments of Intratumoral (IT) 0.07 ml Aspragnase (7 mg/kg) over two weeks. For two weeks, Group 4 (ESC + ASNase + HNTs) received an IT administration of 0.07 ml Asparaginase stocked on Halloysite nanotubes (HNTs) (30 mg/kg) three times per week. A blood specimen was collected, and the liver was removed to be investigated histologically. Results: TEM measurements for the Halloysite nanoclay showed their tubular cylindrical shape with a mean diameter of 50 nm and an average length of 1 μm, whereas The X-ray diffraction pattern of the Halloysite nanoclay showed their characteristic peaks. ESC increases the serum levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and bilirubin than control and other groups, even as albumin and total protein were decreasing. After using Halloysite Nanotube, the rates of these variables were enhanced up to 75%. The hepatocytes histological studies showed protection against Ehrlich Solid carcinoma-induced degenerative, necrotic, and inflammatory changes up to 70%. In conclusion, halloysite nanotubes have demonstrated effective removal of Ehrlich solid carcinoma in mice using an ASNase delivery system. It promoted the ASNase to inhibit the adverse effect of ANase's on the liver and remove the tumour cells.     

Novel oxindole compounds inhibit the aggregation of amyloidogenic proteins associated with neurodegenerative diseases

Amyloidogenic proteins form aggregates in cells, thereby leading to neurodegenerative disorders, including Alzheimer's and prion's disease, amyotrophic lateral sclerosis (ALS) in humans, and degenerative myelopathy (DM) and cognitive dysfunction in dogs. Hence, many small-molecule compounds have been screened to examine their inhibitory effects on amyloidogenic protein aggregation. However, no effective drug suitable for transition to clinical use has been found. Here we examined several novel oxindole compounds (GIF compounds) for their inhibitory effects on aggregate formation of the canine mutant superoxide dismutase 1 (cSOD1 E40K), a causative mutation resulting in DM, using Thioflavin-T fluorescence. Most GIF compounds inhibited the aggregation of cSOD1 E40K. Among the compounds, GIF-0854-r and GIF-0890-r were most effective. Their inhibitory effects were also observed in cSOD1 E40K-transfected cells. Additionally, GIF-0890-r effectively inhibited the aggregate formation of human SOD1 G93A, a causative mutation of ALS. GIF-0827-r and GIF-0856-r also effectively inhibited aggregate formation of human prion protein (hPrP). Subsequently, the correlation between their inhibitory effects on cSOD1 and hPrP aggregation was shown, indicating GIF compounds inhibited the aggregate formation of multiple amyloidogenic proteins. Conclusively, the novel oxindole compounds (GIF-0827-r, GIF-0854-r, GIF-0856-r, and GIF-0890-r) are proposed as useful therapeutic candidates for amyloidogenic neurodegenerative disorders.