Cryo-EM reveals that iRhom2 restrains ADAM17 protease activity to control the release of growth factor and inflammatory signals.

A disintegrin and metalloprotease 17 (ADAM17) is a membrane-tethered protease that triggers multiple signaling pathways. It releases active forms of the primary inflammatory cytokine tumor necrosis factor (TNF) and cancer-implicated epidermal growth factor (EGF) family growth factors. iRhom2, a rhomboid-like, membrane-embedded pseudoprotease, is an essential cofactor of ADAM17. Here, we present cryoelectron microscopy (cryo-EM) structures of the human ADAM17/iRhom2 complex in both inactive and active states. These reveal three regulatory mechanisms. First, exploiting the rhomboid-like hallmark of TMD recognition, iRhom2 interacts with the ADAM17 TMD to promote ADAM17 trafficking and enzyme maturation. Second, a unique iRhom2 extracellular domain unexpectedly retains the cleaved ADAM17 inhibitory prodomain, safeguarding against premature activation and dysregulated proteolysis. Finally, loss of the prodomain from the complex mobilizes the ADAM17 protease domain, contributing to its ability to engage substrates. Our results reveal how a rhomboid-like pseudoprotease has been repurposed during evolution to regulate a potent membrane-tethered enzyme, ADAM17, ensuring the fidelity of inflammatory and growth factor signaling.

Photoactuators Based on Plastically Flexible α-Cyanostilbene Molecular Crystals Driven by the Solid-State [2+2] Cycloaddition Reaction.

Organic photomechanical molecular crystals are promising candidates for photoactuators, which have potential applications as smart materials in various fields. However, it is still challenging to fabricate photomechanical molecular crystals with flexibility because most of the molecular crystals are brittle and the mechanism of flexible crystals remains controversial. Here, a plastically flexible α-cyanostilbene crystal has been synthesized that can undergo solid-state [2+2] cycloaddition reaction under violet or UV irradiation and exhibits excellent photomechanical bending properties. A hook-shaped crystal can lift 0.7 mg object upward by 1.5 cm, which proves its potential for application as photoactuators. When complex with the agarose polymer, the molecules will be in the form of macroscopic crystals, which can drive the composite films to exhibit excellent photomechanical bending performance. Upon irradiation with UV light, the composite film can quickly lift 18.0 mg object upward by 0.3 cm. The results of this work may facilitate the application of macroscale crystals as photoactuators.

Structural and functional insights into Spns2-mediated transport of sphingosine-1-phosphate.

Sphingosine-1-phosphate (S1P) is an important signaling sphingolipid that regulates the immune system, angiogenesis, auditory function, and epithelial and endothelial barrier integrity. Spinster homolog 2 (Spns2) is an S1P transporter that exports S1P to initiate lipid signaling cascades. Modulating Spns2 activity can be beneficial in treatments of cancer, inflammation, and immune diseases. However, the transport mechanism of Spns2 and its inhibition remain unclear. Here, we present six cryo-EM structures of human Spns2 in lipid nanodiscs, including two functionally relevant intermediate conformations that link the inward- and outward-facing states, to reveal the structural basis of the S1P transport cycle. Functional analyses suggest that Spns2 exports S1P via facilitated diffusion, a mechanism distinct from other MFS lipid transporters. Finally, we show that the Spns2 inhibitor 16d attenuates the transport activity by locking Spns2 in the inward-facing state. Our work sheds light on Spns2-mediated S1P transport and aids the development of advanced Spns2 inhibitors.

Organic Crystals with Response to Multiple Stimuli: Mechanical Bending, Acid-Induced Bending and Heating-Induced Jumping.

Multiple stimuli-responsive molecular crystals are attracting extensive attentions due to their potential as smart materials, such as molecular machines, actuators, and sensors. However, the task of giving a single crystal multiple stimuli-responsive properties remains extremely challenging. Herein, we found two polymorphs (Form O and Form R) of a Schiff base compound, which could respond to multiple stimuli (external force, acid, heat). Form O and Form R have different elastic deformability, which can be attributed to the differences in the molecular conformation, structural packing and intermolecular interactions. Moreover, both polymorphs exhibit reversible bending driven by volatile acid vapor, which we hypothesize is caused by reversible protonation reaction of imines with formic acid. In addition, jumping can be triggered by heating due to the significant anisotropic expansion. The integration of reversible bending and jumping into one single crystal expands the application scope of stimuli-responsive crystalline materials.

Molecular mechanism of substrate recognition by folate transporter SLC19A1.

Folate (vitamin B9) is the coenzyme involved in one-carbon transfer biochemical reactions essential for cell survival and proliferation, with its inadequacy causing developmental defects or severe diseases. Notably, mammalian cells lack the ability to de novo synthesize folate but instead rely on its intake from extracellular sources via specific transporters or receptors, among which SLC19A1 is the ubiquitously expressed one in tissues. However, the mechanism of substrate recognition by SLC19A1 remains unclear. Here we report the cryo-EM structures of human SLC19A1 and its complex with 5-methyltetrahydrofolate at 3.5-3.6 Å resolution and elucidate the critical residues for substrate recognition. In particular, we reveal that two variant residues among SLC19 subfamily members designate the specificity for folate. Moreover, we identify intracellular thiamine pyrophosphate as the favorite coupled substrate for folate transport by SLC19A1. Together, this work establishes the molecular basis of substrate recognition by this central folate transporter.

The hemolysin A secretion system is a multi-engine pump containing three ABC transporters.

Type 1 secretion systems (T1SSs) are widespread in pathogenic Gram-negative bacteria, extruding protein substrates following synthesis of the entire polypeptide. The Escherichia coli hemolysin A secretion system has long been considered a prototype in structural and mechanistic studies of T1SSs. Three membrane proteins-an inner membrane ABC transporter HlyB, an adaptor protein HlyD, and an outer membrane porin TolC-are required for secretion. However, the stoichiometry and structure of the complex are unknown. Here, cryo-electron microscopy (cryo-EM) structures determined in two conformations reveal that the inner membrane complex is a hetero-dodecameric assembly comprising three HlyB homodimers and six HlyD subunits. Functional studies indicate that oligomerization of HlyB and HlyD is essential for protein secretion and that polypeptides translocate through a canonical ABC transporter pathway in HlyB. Our data suggest that T1SSs entail three ABC transporters, one that functions as a protein channel and two that allosterically power the translocation process.

Activation and closed-state inactivation mechanisms of the human voltage-gated KV4 channel complexes.

The voltage-gated ion channel activity depends on both activation (transition from the resting state to the open state) and inactivation. Inactivation is a self-restraint mechanism to limit ion conduction and is as crucial to membrane excitability as activation. Inactivation can occur when the channel is open or closed. Although open-state inactivation is well understood, the molecular basis of closed-state inactivation has remained elusive. We report cryo-EM structures of human KV4.2 channel complexes in inactivated, open, and closed states. Closed-state inactivation of KV4 involves an unprecedented symmetry breakdown for pore closure by only two of the four S4-S5 linkers, distinct from known mechanisms of open-state inactivation. We further capture KV4 in a putative resting state, revealing how voltage sensor movements control the pore. Moreover, our structures provide insights regarding channel modulation by KChIP2 and DPP6 auxiliary subunits. Our findings elucidate mechanisms of closed-state inactivation and voltage-dependent activation of the KV4 channel.

lncRNA MSC-AS1 Aggravates Diabetic Nephropathy by Regulating the miR-325/CCNG1 Axis.

BACKGROUND: Diabetic nephropathy (DN) is the most common microvascular complication of diabetes and has become the second leading cause of end-stage renal disease in the world. This study aims to clarify the regulatory mechanism of the lncRNA MSC-AS1/miR-325/cyclin G1 (CCNG1) axis in DN. METHODS: The regulatory mechanism of lncRNA MSC-AS1/miR-325/CCNG1 was evaluated by RT-qPCR, CCK-8 assay, flow cytometry assay, RNA pull-down assay, ELISA, and western blot assay. RESULTS: Upregulation of lncRNA MSC-AS1 was detected in DN patients and HRMC cells treated with high glucose (HG). Knockdown of lncRNA MSC-AS1 reduced the proliferation, fibrosis, and inflammation of HRMC cells induced by HG. In addition, lncRNA MSC-AS1 acts as a miR-325 sponge in the DN. CCNG1 is the direct target of miR-325, which can be positively regulated by lncRNA MSC-AS1 in DN. More importantly, downregulation of miR-325 and upregulation of CCNG1 can attenuate the protective effect of lncRNA MSC-AS1 knockdown on DN. CONCLUSION: lncRNA MSC-AS1 aggravates DN by downregulating miR-325 and upregulating CCNG1.

Target Tracking Algorithm for Table Tennis Using Machine Vision.

The current table tennis robot system has two common problems. One is the table tennis ball speed, which moves fast, and it is difficult for the robot to react in a short time. The second is that the robot cannot recognize the type of the ball's movement, i.e., rotation, top rotation, no rotation, wait, etc. It is impossible to judge whether the ball is rotating and the direction of rotation, resulting in a single return strategy of the robot with poor adaptability. In this paper, these problems are solved by proposing a target trajectory tracking algorithm for table tennis using machine vision combined with Scaled Conjugate Gradient (SCG). Real human-machine game's data are obtained in the proposed algorithm by extracting ten continuous position information and speed information frames for feature selection. These features are used as input data for the deep neural network and then are normalized to create a deep neural network algorithm model. The model is trained by the position information of the successive 20 frames. During the initial sets of experiments, we found the shortcomings of the original SCG algorithm. By setting the accuracy threshold and offline learning of historical data and saving the hidden layer weight matrix, the SCG algorithm was improved. Finally, experiments verify the improved algorithm's feasibility and applicability and show that the proposed algorithm is more suitable for table tennis robots.

Structural insights into the regulation of human serine palmitoyltransferase complexes.

Sphingolipids are essential lipids in eukaryotic membranes. In humans, the first and rate-limiting step of sphingolipid synthesis is catalyzed by the serine palmitoyltransferase holocomplex, which consists of catalytic components (SPTLC1 and SPTLC2) and regulatory components (ssSPTa and ORMDL3). However, the assembly, substrate processing and regulation of the complex are unclear. Here, we present 8 cryo-electron microscopy structures of the human serine palmitoyltransferase holocomplex in various functional states at resolutions of 2.6-3.4 Å. The structures reveal not only how catalytic components recognize the substrate, but also how regulatory components modulate the substrate-binding tunnel to control enzyme activity: ssSPTa engages SPTLC2 and shapes the tunnel to determine substrate specificity. ORMDL3 blocks the tunnel and competes with substrate binding through its amino terminus. These findings provide mechanistic insights into sphingolipid biogenesis governed by the serine palmitoyltransferase complex.

The Machine Vision Measurement Module of the Modularized Flexible Precision Assembly Station for Assembly of Micro- and Meso-Sized Parts.

The machine vision measurement module is indispensable for the Modularized Flexible Precision Assembly Station (MFPAS), which is a fully automatic assembly system being developed at Dalian University of Technology (DUT). MFPAS consists of basic and additional modules, and are expected to be flexible, expandable, and re-configurable to adapt to a variety of parts with a large size range, requiring the machine vision measurement module to be able to achieve accurate measurement of position, as well as orientation of the parts with different size scale. An automatic zooming vision system was set up for evaluation and final integration in MFPAS. Pixel equivalent, principal point and orientation deviation of images were analyzed and experimentally studied using different magnifications of the lens. A new template with circular patterns of different diameters was designed for zoom-lens calibration. The experiments show that the measurement error caused by the variation of the pixel equivalent, principal point and orientation is estimated under 10 µm without online calibration. When high accuracy is required, online calibration can be employed during assembly. The evaluation results of the vision system with or without on-line calibration were given for a better trade-off between accuracy and efficiency during assembly.

Crystal structure of Cas1 in complex with branched DNA.

Cas1 is a key component of the CRISPR adaptation complex, which captures and integrates foreign DNA into the CRISPR array, resulting in the generation of new spacers. We have determined crystal structures of Thermus thermophilus Cas1 involved in new spacer acquisition both in complex with branched DNA and in the free state. Cas1 forms an asymmetric dimer without DNA. Conversely, two asymmetrical dimers bound to two branched DNAs result in the formation of a DNA-mediated tetramer, dimer of structurally asymmetrical dimers, in which the two subunits markedly present different conformations. In the DNA binding complex, the N-terminal domain adopts different orientations with respect to the C-terminal domain in the two monomers that form the dimer. Substrate binding triggers a conformational change in the loop 164-177 segment. This loop is also involved in the 3' fork arm and 5' fork arm strand recognition in monomer A and B, respectively. This study provides important insights into the molecular mechanism of new spacer adaptation.

Structure/cleavage-based insights into helical perturbations at bulge sites within T. thermophilus Argonaute silencing complexes.

We have undertaken a systematic structural study of Thermus thermophilus Argonaute (TtAgo) ternary complexes containing single-base bulges positioned either within the seed segment of the guide or target strands and at the cleavage site. Our studies establish that single-base bulges 7T8, 5A6 and 4A5 on the guide strand are stacked-into the duplex, with conformational changes localized to the bulge site, thereby having minimal impact on the cleavage site. By contrast, single-base bulges 6'U7' and 6'A7' on the target strand are looped-out of the duplex, with the resulting conformational transitions shifting the cleavable phosphate by one step. We observe a stable alignment for the looped-out 6'N7' bulge base, which stacks on the unpaired first base of the guide strand, with the looped-out alignment facilitated by weakened Watson-Crick and reversed non-canonical flanking pairs. These structural studies are complemented by cleavage assays that independently monitor the impact of bulges on TtAgo-mediated cleavage reaction.

Autonomous Generation and Loading of DNA Guides by Bacterial Argonaute.

Several prokaryotic Argonaute proteins (pAgos) utilize small DNA guides to mediate host defense by targeting invading DNA complementary to the DNA guide. It is unknown how these DNA guides are being generated and loaded onto pAgo. Here, we demonstrate that guide-free Argonaute from Thermus thermophilus (TtAgo) can degrade double-stranded DNA (dsDNA), thereby generating small dsDNA fragments that subsequently are loaded onto TtAgo. Combining single-molecule fluorescence, molecular dynamic simulations, and structural studies, we show that TtAgo loads dsDNA molecules with a preference toward a deoxyguanosine on the passenger strand at the position opposite to the 5' end of the guide strand. This explains why in vivo TtAgo is preferentially loaded with guides with a 5' end deoxycytidine. Our data demonstrate that TtAgo can independently generate and selectively load functional DNA guides.

Structural and Mechanistic Basis of PAM-Dependent Spacer Acquisition in CRISPR-Cas Systems.

Bacteria acquire memory of viral invaders by incorporating invasive DNA sequence elements into the host CRISPR locus, generating a new spacer within the CRISPR array. We report on the structures of Cas1-Cas2-dual-forked DNA complexes in an effort toward understanding how the protospacer is sampled prior to insertion into the CRISPR locus. Our study reveals a protospacer DNA comprising a 23-bp duplex bracketed by tyrosine residues, together with anchored flanking 3' overhang segments. The PAM-complementary sequence in the 3' overhang is recognized by the Cas1a catalytic subunits in a base-specific manner, and subsequent cleavage at positions 5 nt from the duplex boundary generates a 33-nt DNA intermediate that is incorporated into the CRISPR array via a cut-and-paste mechanism. Upon protospacer binding, Cas1-Cas2 undergoes a significant conformational change, generating a flat surface conducive to proper protospacer recognition. Here, our study provides important structure-based mechanistic insights into PAM-dependent spacer acquisition.

[Optimization and application of chemically defined medium for 13C metabolic flux analysis of Streptomyces rimosus M4018].

The aim of this study is to develop a synthetic medium suitable for 13C metabolic flux analysis (13C-MFA) of Streptomyces rimosus. The cell growth rate and oxytetracycline production by S. rimosus M4018 were compared when M4018 cells were growth on the optimized chemically defined media with organic nitrogen sources or inorganic nitrogen sources. First, a synthetic medium contained KNO3 as the main nitrogen source was screened, then optimized by a response surface method. Using this new medium, the oxytetracycline yield was increased from 75.2 to 145.6 mg/L. Furthermore, based on the 13C-MFA, we identified that Entner-Doudoroff pathway does not exist in S. rimosus cells cultured in a chemically defined medium with feed of 100% 1-13C labeled glucose. This study is helpful for subsequent 13C-MFA application of S. rimosus.

Crystal structure of the RNA-guided immune surveillance Cascade complex in Escherichia coli.

Clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-associated (Cas) proteins form the CRISPR/Cas system to defend against foreign nucleic acids of bacterial and archaeal origin. In the I-E subtype CRISPR/Cas system, eleven subunits from five Cas proteins (CasA1B2C6D1E1) assemble along a CRISPR RNA (crRNA) to form the Cascade complex. Here we report on the 3.05 Å crystal structure of the 405-kilodalton Escherichia coli Cascade complex that provides molecular details beyond those available from earlier lower-resolution cryo-electron microscopy structures. The bound 61-nucleotide crRNA spans the entire 11-protein subunit-containing complex, where it interacts with all six CasC subunits (named CasC1-6), with its 5' and 3' terminal repeats anchored by CasD and CasE, respectively. The crRNA spacer region is positioned along a continuous groove on the concave surface generated by the aligned CasC1-6 subunits. The five long ß-hairpins that project from individual CasC2-6 subunits extend across the crRNA, with each ß-hairpin inserting into the gap between the last stacked base and its adjacent splayed counterpart, and positioned within the groove of the preceding CasC subunit. Therefore, instead of continuously stacking, the crRNA spacer region is divided into five equal fragments, with each fragment containing five stacked bases flanked by one flipped-out base. Each of those crRNA spacer fragments interacts with CasC in a similar fashion. Furthermore, our structure explains why the seed sequence, with its outward-directed bases, has a critical role in target DNA recognition. In conclusion, our structure of the Cascade complex provides novel molecular details of protein-protein and protein-RNA alignments and interactions required for generation of a complex mediating RNA-guided immune surveillance.

Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage.

We report on crystal structures of ternary Thermus thermophilus Argonaute (TtAgo) complexes with 5'-phosphorylated guide DNA and a series of DNA targets. These ternary complex structures of cleavage-incompatible, cleavage-compatible, and postcleavage states solved at improved resolution up to 2.2 Å have provided molecular insights into the orchestrated positioning of catalytic residues, a pair of Mg(2+) cations, and the putative water nucleophile positioned for in-line attack on the cleavable phosphate for TtAgo-mediated target cleavage by a RNase H-type mechanism. In addition, these ternary complex structures have provided insights into protein and DNA conformational changes that facilitate transition between cleavage-incompatible and cleavage-compatible states, including the role of a Glu finger in generating a cleavage-competent catalytic Asp-Glu-Asp-Asp tetrad. Following cleavage, the seed segment forms a stable duplex with the complementary segment of the target strand.