In situ structural insights into the excitation-contraction coupling mechanism of skeletal muscle.

Excitation-contraction coupling (ECC) is a fundamental mechanism in control of skeletal muscle contraction and occurs at triad junctions, where dihydropyridine receptors (DHPRs) on transverse tubules sense excitation signals and then cause calcium release from the sarcoplasmic reticulum via coupling to type 1 ryanodine receptors (RyR1s), inducing the subsequent contraction of muscle filaments. However, the molecular mechanism remains unclear due to the lack of structural details. Here, we explored the architecture of triad junction by cryo-electron tomography, solved the in situ structure of RyR1 in complex with FKBP12 and calmodulin with the resolution of 16.7 Angstrom, and found the intact RyR1-DHPR supercomplex. RyR1s arrange into two rows on the terminal cisternae membrane by forming right-hand corner-to-corner contacts, and tetrads of DHPRs bind to RyR1s in an alternating manner, forming another two rows on the transverse tubule membrane. This unique arrangement is important for synergistic calcium release and provides direct evidence of physical coupling in ECC.

The effect and mechanism of variable particle size microplastics and levofloxacin on the neurotoxicity of Rana nigromaculata based on the microorganism-intestine-brain axis.

Microplastics (MPs) usually appear in the aquatic environment as complex pollutants in combination with other environmental pollutants, such as levofloxacin (LVFX). After a 45-day exposure to LVFX and MPs with different particle sizes at environmental levels, LVFX was neurotoxic to Rana nigromaculata tadpoles. The order of the effects of the exposure treatment on tadpole behavior was: LVFX-MP3>LVFX-MP1>LVFX-MP2 ≥ LVFX. Results of transcriptome analysis of tadpole brain tissue showed that LVFX in combination with 0.10 and 10.00 µm MP interferes with the nervous system through the cell adhesion molecules pathway. Interestingly, the order of effects of the co-exposure on oxidative stress in the intestine was inconsistent with that of tadpole behavior. We found that Paraacteroides might be a microplastic indicator species for the gut microbiota of aquatic organisms. The results of the targeted metabolism of neurotransmitters in the intestine suggest that in the LVFX-MP2 treatment, LVFX alleviated the intestinal microbiota disorder caused by 1.00 µm MP, by regulating intestinal microbiota participating in the TCA cycle VI and gluconeogenesis and tetrapyrrole biosynthesis I, while downregulating Met and Orn, and upregulating 5HIAA, thereby easing the neurotoxicity to tadpoles exposed to LVFX-MP2. This work is of great significance for the comprehensive assessment of the aquatic ecological risks of microplastics-antibiotic compound pollutants.

An Exploration of Multiple Component Peptide Assemblies by Enzyme-Instructed Self-Assembly.

Based on the motifs (RNISY (M) and DEEVELILGDT (D)) in the protein crystal structures of Merlin and CRL4DCAF-1, we phosphorylated the tyrosine residue in M and conjugated M to a self-assembling motif to produce a phosphopeptide (1P) and examined enzyme-instructed self-assembly (EISA) of 1P with and without the presence of D (4). Our results show that EISA of 1P forms a hydrogel at exceedingly low volume fraction (~ 0.03%) even with the presence of the hydrophilic peptide, 4. Unlike 1P, 2P (a diastereomer of 1P) or 3P (the enantiomer of 1P) forms a hydrogel via EISA when their concentration is four or three times that of 1P, respectively. Circular dichroism (CD) spectra show that increasing the concentration of the phosphopeptides lowers the CD signals of the mixtures, and the magnitudes of the CD signals depends on the interaction between M and D. This work contributes insight for understanding multi-component hydrogels formed by self-assembly that involves both specific intermolecular interaction and enzymatic reactions.

A Fluorescence Polarization Assay for Macrodomains Facilitates the Identification of Potent Inhibitors of the SARS-CoV-2 Macrodomain.

Viral macrodomains, which can bind to and/or hydrolyze adenine diphosphate ribose (ADP-ribose or ADPr) from proteins, have been suggested to counteract host immune response and be viable targets for the development of antiviral drugs. Therefore, developing high-throughput screening (HTS) techniques for macrodomain inhibitors is of great interest. Herein, using a novel tracer TAMRA-ADPr, an ADP-ribose compound conjugated with tetramethylrhodamine, we developed a robust fluorescence polarization assay for various viral and human macrodomains including SARS-CoV-2 Macro1, VEEV Macro, CHIKV Macro, human MacroD1, MacroD2, and PARP9 Macro2. Using this assay, we validated Z8539 (IC50 6.4 µM) and GS441524 (IC50 15.2 µM), two literature-reported small-molecule inhibitors of SARS-CoV-2 Macro1. Our data suggest that GS441524 is highly selective for SARS-CoV-2 Macro1 over other human and viral macrodomains. Furthermore, using this assay, we identified pNP-ADPr (ADP-ribosylated p-nitrophenol, IC50 370 nM) and TFMU-ADPr (ADP-ribosylated trifluoromethyl umbelliferone, IC50 590 nM) as the most potent SARS-CoV-2 Macro1 binders reported to date. An X-ray crystal structure of SARS-CoV-2 Macro1 in complex with TFMU-ADPr revealed how the TFMU moiety contributes to the binding affinity. Our data demonstrate that this fluorescence polarization assay is a useful addition to the HTS methods for the identification of macrodomain inhibitors.

Structural elucidation of how ARF small GTPases induce membrane tubulation for vesicle fission.

The ADP-Ribosylation Factor (ARF) small GTPases have been found to act in vesicle fission through a direct ability to tubulate membrane. Here, we have used cryo-electron microscopy (EM) to solve the structure of an ARF6 protein lattice assembled on tubulated membrane to 3.9 Å resolution. ARF6 forms tetramers that polymerize into helical arrays to form this lattice. We identify, and confirm functionally, protein contacts critical for this lattice formation. The solved structure also suggests how the ARF amphipathic helix is positioned in the lattice for membrane insertion, and how a GTPase-activating protein (GAP) docks onto the lattice to catalyze ARF-GTP hydrolysis in completing membrane fission. As ARF1 and ARF6 are structurally conserved, we have also modeled ARF1 onto the ARF6 lattice, which has allowed us to pursue the reconstitution of Coat Protein I (COPI) vesicles to confirm more definitively that the ARF lattice acts in vesicle fission. Our findings are notable for having achieved the first detailed glimpse of how a small GTPase bends membrane and having provided a molecular understanding of how an ARF protein acts in vesicle fission.

Surgical gestures as a method to quantify surgical performance and predict patient outcomes.

How well a surgery is performed impacts a patient's outcomes; however, objective quantification of performance remains an unsolved challenge. Deconstructing a procedure into discrete instrument-tissue "gestures" is a emerging way to understand surgery. To establish this paradigm in a procedure where performance is the most important factor for patient outcomes, we identify 34,323 individual gestures performed in 80 nerve-sparing robot-assisted radical prostatectomies from two international medical centers. Gestures are classified into nine distinct dissection gestures (e.g., hot cut) and four supporting gestures (e.g., retraction). Our primary outcome is to identify factors impacting a patient's 1-year erectile function (EF) recovery after radical prostatectomy. We find that less use of hot cut and more use of peel/push are statistically associated with better chance of 1-year EF recovery. Our results also show interactions between surgeon experience and gesture types-similar gesture selection resulted in different EF recovery rates dependent on surgeon experience. To further validate this framework, two teams independently constructe distinct machine learning models using gesture sequences vs. traditional clinical features to predict 1-year EF. In both models, gesture sequences are able to better predict 1-year EF (Team 1: AUC 0.77, 95% CI 0.73-0.81; Team 2: AUC 0.68, 95% CI 0.66-0.70) than traditional clinical features (Team 1: AUC 0.69, 95% CI 0.65-0.73; Team 2: AUC 0.65, 95% CI 0.62-0.68). Our results suggest that gestures provide a granular method to objectively indicate surgical performance and outcomes. Application of this methodology to other surgeries may lead to discoveries on methods to improve surgery.

Cryo-EM structure and electrophysiological characterization of ALMT from Glycine max reveal a previously uncharacterized class of anion channels.

Aluminum-activated malate transporters (ALMTs) form an anion channel family that plays essential roles in diverse functions in plants. Arabidopsis ALMT12, also named QUAC1 (quick anion channel 1), regulates stomatal closure in response to environmental stimuli. However, the molecular basis of ALMT12/QUAC1 activity remains elusive. Here, we describe the cryo-EM structure of ALMT12/QUAC1 from Glycine max at 3.5-Å resolution. GmALMT12/QUAC1 is a symmetrical dimer, forming a single electropositive T-shaped pore across the membrane. The transmembrane and cytoplasmic domains are assembled into a twisted two-layer architecture, with their associated dimeric interfaces nearly perpendicular. GmALMT12/QUAC1-mediated currents display rapid kinetics of activation/deactivation and a bell-shaped voltage dependency, reminiscent of the rapid (R)-type anion currents. Our structural and functional analyses reveal a domain-twisting mechanism for malate-mediated activation. Together, our study uncovers the molecular basis for a previously uncharacterized class of anion channels and provides insights into the gating and modulation of the ALMT12/QUAC1 anion channel.

VHUT-cryo-FIB, a method to fabricate frozen hydrated lamellae from tissue specimens for in situ cryo-electron tomography.

Cryo-electron tomography (cryo-ET) provides a promising approach to study intact structures of macromolecules in situ, but the efficient preparation of high-quality cryosections represents a bottleneck. Although cryo-focused ion beam (cryo-FIB) milling has emerged for large and flat cryo-lamella preparation, its application to tissue specimens remains challenging. Here, we report an integrated workflow, VHUT-cryo-FIB, for efficiently preparing frozen hydrated tissue lamella that can be readily used in subsequent cryo-ET studies. The workflow includes vibratome slicing, high-pressure freezing, ultramicrotome cryo-trimming and cryo-FIB milling. Two strategies were developed for loading cryo-lamella via a side-entry cryo-holder or an FEI AutoGrid. The workflow was validated by using various tissue specimens, including rat skeletal muscle, rat liver and spinach leaf specimens, and in situ structures of ribosomes were obtained at nanometer resolution from the spinach and liver samples.

Dynamic Continuum of Nanoscale Peptide Assemblies Facilitates Endocytosis and Endosomal Escape.

Alkaline phosphatase (ALP) enables intracellular targeting by peptide assemblies, but how the ALP substrates enter cells remains elusive. Here we show that nanoscale phosphopeptide assemblies cluster ALP to enable caveolae-mediated endocytosis (CME) and endosomal escape. Specifically, fluorescent phosphopeptides undergo enzyme-catalyzed self-assembly to form nanofibers. Live cell imaging unveils that phosphopeptides nanoparticles, coincubated with HEK293 cells overexpressing red fluorescent protein-tagged tissue-nonspecific ALP (TNAP-RFP), cluster TNAP-RFP in lipid rafts to enable CME. Further dephosphorylation of the phosphopeptides produces peptidic nanofibers for endosomal escape. Inhibiting TNAP, cleaving the membrane anchored TNAP, or disrupting lipid rafts abolishes the endocytosis. Decreasing the transformation to nanofibers prevents the endosomal escape. As the first study establishing a dynamic continuum of nanoscale assemblies for cellular uptake, this work illustrates an effective design for enzyme-responsive supramolecular therapeutics and provides mechanism insights for understanding the dynamics of cellular uptake of proteins or exogenous peptide aggregates.

Structural insights into membrane remodeling by SNX1.

The sorting nexin (SNX) family of proteins deform the membrane to generate transport carriers in endosomal pathways. Here, we elucidate how a prototypic member, SNX1, acts in this process. Performing cryoelectron microscopy, we find that SNX1 assembles into a protein lattice that consists of helical rows of SNX1 dimers wrapped around tubular membranes in a crosslinked fashion. We also visualize the details of this structure, which provides a molecular understanding of how various parts of SNX1 contribute to its ability to deform the membrane. Moreover, we have compared the SNX1 structure with a previously elucidated structure of an endosomal coat complex formed by retromer coupled to a SNX, which reveals how the molecular organization of the SNX in this coat complex is affected by retromer. The comparison also suggests insight into intermediary stages of assembly that results in the formation of the retromer-SNX coat complex on the membrane.

Enzymatic Insertion of Lipids Increases Membrane Tension for Inhibiting Drug Resistant Cancer Cells.

Although lipids contribute to cancer drug resistance, it is challenging to target diverse range of lipids. Here, we show enzymatically inserting exceedingly simple synthetic lipids into membranes for increasing membrane tension and selectively inhibiting drug resistant cancer cells. The lipid, formed by conjugating dodecylamine to d-phosphotyrosine, self-assembles to form micelles. Enzymatic dephosphorylation of the micelles inserts the lipids into membranes and increases membrane tension. The micelles effectively inhibit a drug resistant glioblastoma cell (T98G) or a triple-negative breast cancer cell (HCC1937), without inducing acquired drug resistance. Moreover, the enzymatic reaction of the micelles promotes the accumulation of the lipids in the membranes of subcellular organelles (e.g., endoplasmic reticulum (ER), Golgi, and mitochondria), thus activating multiple regulated cell death pathways. This work, in which for the first time membrane tension is increased to inhibit cancer cells, illustrates a new and powerful supramolecular approach for antagonizing difficult drug targets.

Structure-Activity Relationship of Peptide-Conjugated Chloramphenicol for Inhibiting Escherichia coli.

Intravenous administration of a prodrug, chloramphenicol succinate (CLsu), is ineffective. Recently, we have shown that conjugation of diglycine of CLsu (CLsuGG) not only increases the antibiotic efficacy against Escherichia coli but also reduces adverse drug effects against bone marrow stromal cells. Here, we report the synthesis of structural analogues of CLsuGG and their activities against E. coli. These analogues reveal several trends: (i) except the water-insoluble analogues, the attachment of peptides to CLsu enhances the efficacy of the prodrugs; (ii) negative charges, high steric hindrance in the side chains, or a rigid diester decreases the activities of prodrugs in comparison to CLsuGG; (iii) dipeptides apparently increase the efficacy of the prodrugs most effectively; and so forth. This work suggests that conjugating peptides to CLsu effectively modulates the properties of prodrugs. The structure-activity relationship of these new conjugates may provide useful insights for expanding the pool of antibiotics.