1H, 13C, and 15N resonance assignments and solution structure of the N-terminal divergent calponin homology (NN-CH) domain of human intraflagellar transport protein 54.

The intraflagellar transport (IFT) machinery plays a crucial role in the bidirectional trafficking of components necessary for ciliary signaling, such as the Hedgehog, Wnt/PCR, and cAMP/PKA systems. Defects in some components of the IFT machinery cause dysfunction, leading to a wide range of human diseases and developmental disorders termed ciliopathies, such as nephronophthisis. The IFT machinery comprises three sub-complexes: BBsome, IFT-A, and IFT-B. The IFT protein 54 (IFT54) is an important component of the IFT-B sub-complex. In anterograde movement, IFT54 binds to active kinesin-II, walking along the cilia microtubule axoneme and carrying the dynein-2 complex in an inactive state, which works for retrograde movement. Several mutations in IFT54 are known to cause Senior-Loken syndrome, a ciliopathy. IFT54 possesses a divergent Calponin Homology (CH) domain termed as NN-CH domain at its N-terminus. However, several aspects of the function of the NN-CH domain of IFT54 are still obscure. Here, we report the 1H, 15N, and 13C resonance assignments of the NN-CH domain of human IFT54 and its solution structure. The NN-CH domain of human IFT54 adopts essentially the α1-α2-α3-α4-α5 topology as that of mouse IFT54, whose structure was determined by X-ray crystallographic study. The structural information and assignments obtained in this study shed light on the molecular function of the NN-CH domain in IFT54.

Highly flexible PEG-LifeAct constructs act as tunable biomimetic actin crosslinkers.

In vitro studies of actin filament networks crosslinked with dynamic actin binding proteins provide critical insights into cytoskeletal mechanics as well as inspiration for new adaptive materials design. However, discontinuous variance in the physiochemical properties of actin binding proteins impedes holistic relationships between crosslinker molecular parameters, network structure, and mechanics. Bio-synthetic constructs composed of synthetic polymer backbones and actin binding motifs would enable crosslinkers with engineered physiochemical properties to directly target the desired structure-property relationships. As a proof of concept, bio-synthetic crosslinkers composed of highly flexible polyethylene glycol (PEG) polymers functionalized with the actin binding peptide LifeAct, are explored as actin crosslinkers. Using bulk rheology and fluorescence microscopy, these constructs are shown to modulate actin filament network structure and mechanics in a contour length dependent manner, while maintaining the stress-stiffening behavior inherent to actin filament networks. These results encourage the design of more diverse and complex peptide-polymer crosslinkers to interrogate and control semi-flexible polymer networks.

Fascin - F-actin interaction studied by molecular dynamics simulation and protein network analysis.

Actin bundles are an important component of cellular cytoskeleton and participate in the movement of cells. The formation of actin bundles requires the participation of many actin binding proteins (ABPs). Fascin is a member of ABPs, which plays a key role in bundling filamentous actin (F-actin) to bundles. However, the detailed interactions between fascin and F-actin are unclear. In this study, we construct an atomic-level structure of fascin - F-actin complex based on a rather poor cryo-EM data with resolution of 20 nm. We first optimized the geometries of the complex by molecular dynamics (MD) simulation and analyzed the binding site and pose of fascin which bundles two F-actin chains. Next, binding free energy of fascin was calculated by MM/GBSA method. Finally, protein structure network analysis (PSNs) was performed to analyze the key residues for fascin binding. Our results show that residues of K22, E27, E29, K41, K43, R110, R149, K358, R408 and K471 on fascin are important for its bundling, which are in good agreement with the experimental data. On the other hand, the consistent results indicate that the atomic-level model of fascin - F-actin complex is reliable. In short, this model can be used to understand the detailed interactions between fascin and F-actin, and to develop novel potential drugs targeting fascin.Communicated by Ramaswamy H. Sarma.

A Candida auris-specific adhesin, Scf1, governs surface association, colonization, and virulence.

Candida auris is an emerging fungal pathogen responsible for health care-associated outbreaks that arise from persistent surface and skin colonization. We characterized the arsenal of adhesins used by C. auris and discovered an uncharacterized adhesin, Surface Colonization Factor (Scf1), and a conserved adhesin, Iff4109, that are essential for the colonization of inert surfaces and mammalian hosts. SCF1 is apparently specific to C. auris, and its expression mediates adhesion to inert and biological surfaces across isolates from all five clades. Unlike canonical fungal adhesins, which function through hydrophobic interactions, Scf1 relies on exposed cationic residues for surface association. SCF1 is required for C. auris biofilm formation, skin colonization, virulence in systemic infection, and colonization of inserted medical devices.

Nanoscale Structural Comparison of Fibrillin-1 Microfibrils Isolated from Marfan and Non-Marfan Syndrome Human Aorta.

Fibrillin-1 microfibrils are essential elements of the extracellular matrix serving as a scaffold for the deposition of elastin and endowing connective tissues with tensile strength and elasticity. Mutations in the fibrillin-1 gene (FBN1) are linked to Marfan syndrome (MFS), a systemic connective tissue disorder that, besides other heterogeneous symptoms, usually manifests in life-threatening aortic complications. The aortic involvement may be explained by a dysregulation of microfibrillar function and, conceivably, alterations in the microfibrils' supramolecular structure. Here, we present a nanoscale structural characterization of fibrillin-1 microfibrils isolated from two human aortic samples with different FBN1 gene mutations by using atomic force microscopy, and their comparison with microfibrillar assemblies purified from four non-MFS human aortic samples. Fibrillin-1 microfibrils displayed a characteristic "beads-on-a-string" appearance. The microfibrillar assemblies were investigated for bead geometry (height, length, and width), interbead region height, and periodicity. MFS fibrillin-1 microfibrils had a slightly higher mean bead height, but the bead length and width, as well as the interbead height, were significantly smaller in the MFS group. The mean periodicity varied around 50-52 nm among samples. The data suggest an overall thinner and presumably more frail structure for the MFS fibrillin-1 microfibrils, which may play a role in the development of MFS-related aortic symptomatology.

Revealing the biotoxicity of phosphorene oxide nanosheets based on the villin headpiece.

Phosphorene, a novel member of the two-dimensional nanomaterial family, has demonstrated great potential in biomedical applications, such as photothermal therapy, drug delivery and antibacterial. However, phosphorene is unstable and easily oxidized in an aerobic environment. In this paper, using larger-scale molecular dynamics simulations, we investigated the disruption of phosphorene oxide (PO) to the structure of a model protein, villin headpiece subdomain (HP35). It shows that the disruption of PO nanosheets to the protein structure is enhanced with increasing oxidation concentration of PO, while PO's oxidation mode has very little effect on the PO-HP35 interaction. PO with a low oxidation concentration has certain biocompatibility to HP35. Oxygen atoms filling into the groove region in the puckered surface of phosphorene enhance the dispersion interaction between phosphorene and HP35, which enhances the disruption of phosphorene to the structure of HP35. Compared with the dispersion interaction, the electrostatic interaction between PO and the protein has a negligible effect on the structural damage of HP35. These findings might shed light on the biological toxicity of PO nanosheets and would be helpful for future potential biomedical applications of PO nanosheets, such as nanodrugs and antibacterial agents.

The Actin Crosslinker Fascin Regulates Cell Chirality.

The left-right (L-R) asymmetry of the cells, or cell chirality, is a well-known intrinsic property derived from the dynamic organization of the actin cytoskeleton. Cell chirality can be regulated by actin-binding proteins such as α-actinin-1 and can also be mediated by certain signaling pathways, such as protein kinase C (PKC) signaling. Fascin, an actin crosslinker known to mediate parallel bundling of actin filaments, appears as a prominent candidate in cell chirality regulation, given its role in facilitating cell migration as an important PKC substrate. Here, it is shown that the chirality of NIH/3T3 cells can be altered by PKC activation and fascin manipulation. With either small-molecule drug inhibition or genetic knockdown of fascin, the chirality of 3T3 cells is reversed from a clockwise (CW) bias to a counterclockwise (CCW) bias on ring-shaped micropatterns, accompanied by the reversal in cell directional migration. The Ser-39 fascin-actin binding sites are further explored in cell chirality regulation. The findings of this study reveal the critical role of fascin as an important intermediator in cell chirality, shedding novel insights into the mechanisms of L-R asymmetric cell migration and multicellular morphogenesis.

Parallel actin monomers in the 8S complex of actin-INF2.

Polymerization and depolymerization of actin play an essential role in eukaryotic cells. Actin exists in cells in both monomeric (G-actin) and filamentous (polymer, F-actin) forms. Actin binding proteins (ABPs) facilitate the transition between these two states, and their interactions with these two states of actin are critical for actin-based cellular processes. Rapid depolymerization of actin is assisted in the brain and/or other cells by its oxidation by the enzyme Mical (yielding Mox-actin), and/or by the binding of Inverted Formin 2 (INF2) - which can also accelerate filaments formation. At their stoichiometric molar ratio INF2 and actin yield the 8S complex (consisting of 4 actin monomers: 2 INF2 dimer molecules). Using biochemical and biophysical methods, we investigate the structural arrangement of actin in the 8S particles and the interaction of INF2 with actin and Mox-actin. To that end, we show 2 D class averages of 8S particles obtained by negative staining electron microscopy. We also show that: (i) 8S particles can seed rapid actin assembly; (ii) Mox-actin and INF2 form 8S particles at proteins ratios similar to those of unoxidized actin; (iii) chemical crosslinkings suggest that actin monomers are in a parallel orientation in the 8S particles of both actin and Mox-actin; and (iv) INF2 accelerates the disassembly of Mox-F-actin. Our results provide better understanding of actin's arrangement in the 8S particles formed during actin depolymerization and in the early polymerization stages of both actin and Mox-actin.Communicated by Ramaswamy H. Sarma.

Structural mechanism for bidirectional actin cross-linking by T-plastin.

To orchestrate cell mechanics, trafficking, and motility, cytoskeletal filaments must assemble into higher-order networks whose local subcellular architecture and composition specify their functions. Cross-linking proteins bridge filaments at the nanoscale to control a network's µm-scale geometry, thereby conferring its mechanical properties and functional dynamics. While these interfilament linkages are key determinants of cytoskeletal function, their structural mechanisms remain poorly understood. Plastins/fimbrins are an evolutionarily ancient family of tandem calponin-homology domain (CHD) proteins required to construct multiple classes of actin networks, which feature diverse geometries specialized to power cytokinesis, microvilli and stereocilia biogenesis, and persistent cell migration. Here, we focus on the structural basis of actin network assembly by human T-plastin, a ubiquitously expressed isoform necessary for the maintenance of stable cellular protrusions generated by actin polymerization forces. By implementing a machine-learning-enabled cryo-electron microscopy pipeline for visualizing cross-linkers bridging multiple filaments, we uncover a sequential bundling mechanism enabling T-plastin to bridge pairs of actin filaments in both parallel and antiparallel orientations. T-plastin populates distinct structural landscapes in these two bridging orientations that are selectively compatible with actin networks featuring divergent architectures and functions. Our structural, biochemical, and cell biological data highlight inter-CHD linkers as key structural elements underlying flexible but stable cross-linking that are likely to be disrupted by T-plastin mutations that cause hereditary bone diseases.

The role of Actopaxin in tumor metastasis.

Actopaxin is a newly discovered focal adhesions (FAs) protein, actin-binding protein and pseudopodia-enriched molecule. It can not only bind to a variety of FAs proteins (such as Paxillin, ILK and PINCH) and non-FAs proteins (such as TESK1, CdGAP, ß2-adaptin, G3BP2, ADAR1 and CD29), but also participates in multiple signaling pathways. Thus, it plays a crucial role in regulating important processes of tumor metastasis, including matrix degradation, migration, and invasion, etc. This review covers the latest progress in the structure and function of Actopaxin, its interaction with other proteins as well as its involvement in regulating tumor development and metastasis. Additionally, the current limitations for Actopaxin related studies and the possible research directions on it in the future are also discussed. It is hoped that this review can assist relevant researchers to obtain a deep understanding of the role that Actopaxin plays in tumor progression, and also enlighten further research and development of therapeutic approaches for the treatment of tumor metastasis.

A fine balance of hydrophobic-electrostatic communication pathways in a pH-switching protein.

Allostery is the phenomenon of coupling between distal binding sites in a protein. Such coupling is at the crux of protein function and regulation in a myriad of scenarios, yet determining the molecular mechanisms of coupling networks in proteins remains a major challenge. Here, we report mechanisms governing pH-dependent myristoyl switching in monomeric hisactophilin, whereby the myristoyl moves between a sequestered state, i.e., buried within the core of the protein, to an accessible state, in which the myristoyl has increased accessibility for membrane binding. Measurements of the pH and temperature dependence of amide chemical shifts reveal protein local structural stability and conformational heterogeneity that accompany switching. An analysis of these measurements using a thermodynamic cycle framework shows that myristoyl-proton coupling at the single-residue level exists in a fine balance and extends throughout the protein. Strikingly, small changes in the stereochemistry or size of core and surface hydrophobic residues by point mutations readily break, restore, or tune myristoyl switch energetics. Synthesizing the experimental results with those of molecular dynamics simulations illuminates atomistic details of coupling throughout the protein, featuring a large network of hydrophobic interactions that work in concert with key electrostatic interactions. The simulations were critical for discerning which of the many ionizable residues in hisactophilin are important for switching and identifying the contributions of nonnative interactions in switching. The strategy of using temperature-dependent NMR presented here offers a powerful, widely applicable way to elucidate the molecular mechanisms of allostery in proteins at high resolution.

ATP allosterically stabilizes integrin-linked kinase for efficient force generation.

SignificanceThe pseudokinase integrin-linked kinase (ILK) is a central component of focal adhesions, cytoplasmic multiprotein complexes that integrate and transduce biochemical and mechanical signals from the extracellular environment into the cell and vice versa. However, the precise molecular functions, particularly the mechanosensory properties of ILK and the significance of retained adenosine triphosphate (ATP) binding, are still unclear. Combining molecular-dynamics simulations with cell biology, we establish a role for ATP binding to pseudokinases. We find that ATP promotes the structural stability of ILK, allosterically influences the interaction between ILK and its binding partner parvin at adhesions, and enhances the mechanoresistance of this complex. On the cellular level, ATP binding facilitates efficient traction force buildup, focal adhesion stabilization, and efficient cell migration.

Topological data analysis gives two folding paths in HP35(nle-nle), double mutant of villin headpiece subdomain.

The folding dynamics of proteins is a primary area of interest in protein science. We carried out topological data analysis (TDA) of the folding process of HP35(nle-nle), a double-mutant of the villin headpiece subdomain. Using persistent homology and non-negative matrix factorization, we reduced the dimension of protein structure and investigated the flow in the reduced space. We found this protein has two folding paths, distinguished by the pairings of inter-helix residues. Our analysis showed the excellent performance of TDA in capturing the formation of tertiary structure.

Fibrillin-1 gene mutations in a Chinese cohort with congenital ectopia lentis: spectrum and genotype-phenotype analysis.

AIMS: To identify the mutation spectrum and genotype-phenotype correlations of fibrillin-1 (FBN1) mutations in a Chinese cohort with congenital ectopia lentis (EL). METHODS: Patients clinically suspected of congenital zonulopathy were screened using panel-based next-generation sequencing followed by multiplex ligation-dependent probe amplification. All the probands were subjected to thorough ocular examinations. Molecular and clinical data were integrated in pursuit of genotype-phenotype correlation. RESULTS: A total of 131 probands of FBN1 mutations from unrelated families were recruited. Around 65% of the probands were children younger than 9 years old. Overall, 110 distinct FBN1 mutations were identified, including 39 novel ones. The most at-risk regions were exons 13, 2, 6, 15, 24 and 33 in descending order of mutation frequency. The most prevalent mutation was c.184C>T (seven, 5.34%) in the coding sequence and c.5788+5G>A (three, 2.29%) in introns. Missense mutations were the most frequent type (103, 78.63%); half of which were distributed in the N-terminal regions (53, 51.46%). The majority of missense mutations were detected in one of the calcium-binding epidermal growth factor-like domains (62, 60.19%), and 39 (62.90%) of them were substitutions of conserved cysteine residues. Microspherophakia (MSP) was found in 15 patients (11.45%). Mutations in the middle region (exons 22-42), especially exon 26, had higher risks of combined MSP (OR, 5.51 (95% CI 1.364 to 22.274), p=0.017). CONCLUSIONS: This study extended the knowledge of the FBN1 mutation spectrum and provided novel insights into its clinical correlation regarding EL and MSP in the Chinese population.

Progress in deep Markov state modeling: Coarse graining and experimental data restraints.

Recent advances in deep learning frameworks have established valuable tools for analyzing the long-timescale behavior of complex systems, such as proteins. In particular, the inclusion of physical constraints, e.g., time-reversibility, was a crucial step to make the methods applicable to biophysical systems. Furthermore, we advance the method by incorporating experimental observables into the model estimation showing that biases in simulation data can be compensated for. We further develop a new neural network layer in order to build a hierarchical model allowing for different levels of details to be studied. Finally, we propose an attention mechanism, which highlights important residues for the classification into different states. We demonstrate the new methodology on an ultralong molecular dynamics simulation of the Villin headpiece miniprotein.

Biallelic SYNE2 Missense Mutations Leading to Nesprin-2 Giant Hypo-Expression Are Associated with Intellectual Disability and Autism.

Autism spectrum disorder (ASD) is a group of neurological and developmental disabilities characterised by clinical and genetic heterogeneity. The current study aimed to expand ASD genotyping by investigating potential associations with SYNE2 mutations. Specifically, the disease-causing variants of SYNE2 in 410 trios manifesting neurodevelopmental disorders using whole-exome sequencing were explored. The consequences of the identified variants were studied at the transcript level using quantitative polymerase chain reaction (qPCR). For validation, immunofluorescence and immunoblotting were performed to analyse mutational effects at the protein level. The compound heterozygous variants of SYNE2 (NM_182914.3:c.2483T>G; p.(Val828Gly) and NM_182914.3:c.2362G>A; p.(Glu788Lys)) were identified in a 4.5-year-old male, clinically diagnosed with autism spectrum disorder, developmental delay and intellectual disability. Both variants reside within the nesprin-2 giant spectrin repeat (SR5) domain and are predicted to be highly damaging using in silico tools. Specifically, a significant reduction of nesprin-2 giant protein levels is revealed in patient cells. SYNE2 transcription and the nuclear envelope localisation of the mutant proteins was however unaffected as compared to parental control cells. Collectively, these data provide novel insights into the cardinal role of the nesprin-2 giant in neurodevelopment and suggest that the biallelic hypomorphic SYNE2 mutations may be a new cause of intellectual disability and ASD.

Targeting Pin1 renders pancreatic cancer eradicable by synergizing with immunochemotherapy.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by notorious resistance to current therapies attributed to inherent tumor heterogeneity and highly desmoplastic and immunosuppressive tumor microenvironment (TME). Unique proline isomerase Pin1 regulates multiple cancer pathways, but its role in the TME and cancer immunotherapy is unknown. Here, we find that Pin1 is overexpressed both in cancer cells and cancer-associated fibroblasts (CAFs) and correlates with poor survival in PDAC patients. Targeting Pin1 using clinically available drugs induces complete elimination or sustained remissions of aggressive PDAC by synergizing with anti-PD-1 and gemcitabine in diverse model systems. Mechanistically, Pin1 drives the desmoplastic and immunosuppressive TME by acting on CAFs and induces lysosomal degradation of the PD-1 ligand PD-L1 and the gemcitabine transporter ENT1 in cancer cells, besides activating multiple cancer pathways. Thus, Pin1 inhibition simultaneously blocks multiple cancer pathways, disrupts the desmoplastic and immunosuppressive TME, and upregulates PD-L1 and ENT1, rendering PDAC eradicable by immunochemotherapy.

Fascin-induced actin protrusions are suppressed by dendritic networks in giant unilamellar vesicles.

The interactions between actin networks and cell membrane are immensely important for eukaryotic cell functions including cell shape changes, motility, polarity establishment, and adhesion. Actin-binding proteins are known to compete and cooperate using a finite amount of actin monomers to form distinct actin networks. How actin-bundling protein fascin and actin-branching protein Arp2/3 complex compete to remodel membranes is not entirely clear. To investigate fascin- and Arp2/3-mediated actin network remodeling, we applied a reconstitution approach encapsulating bundled and dendritic actin networks inside giant unilamellar vesicles (GUVs). Independently reconstituted, membrane-bound Arp2/3 nucleation forms an actin cortex in GUVs, whereas fascin mediates formation of actin bundles that protrude out of GUVs. Coencapsulating both fascin and Arp2/3 complex leads to polarized dendritic aggregates and significantly reduces membrane protrusions, irrespective of whether the dendritic network is membrane bound or not. However, reducing Arp2/3 complex while increasing fascin restores membrane protrusion. Such changes in network assembly and the subsequent interplay with membrane can be attributed to competition between fascin and Arp2/3 complex to utilize a finite pool of actin.

MICAL-L1 is required for cargo protein delivery to the cell surface.

Secreted proteins are transported along intracellular route from the endoplasmic reticulum through the Golgi before reaching the plasma membrane. Small GTPase Rab and their effectors play a key role in membrane trafficking. Using confocal microscopy, we showed that MICAL-L1 was associated with tubulo-vesicular structures and exhibited a significant colocalization with markers of the Golgi apparatus and recycling endosomes. Super resolution STORM microscopy suggested at the molecular level, a very close association of MICAL-L1 and microdomains in the Golgi cisternae. Using a synchronized secretion assay, we report that the shRNA-mediated depletion of MICAL-L1 impaired the delivery of a subset of cargo proteins to the cell surface. The process of membrane tubulation was monitored in vitro, and we observe that recombinant MICAL-L1-RBD domain may contribute to promote PACSINs-mediated membrane tubulation. Interestingly, two hydrophobic residues at the C-terminus of MICAL-L1 appeared to be important for phosphatidic acid binding, and for association with membrane tubules. Our results reveal a new role for MICAL-L1 in cargo delivery to the plasma membrane.

Cochlear supporting cells require GAS2 for cytoskeletal architecture and hearing.

In mammals, sound is detected by mechanosensory hair cells that are activated in response to vibrations at frequency-dependent positions along the cochlear duct. We demonstrate that inner ear supporting cells provide a structural framework for transmitting sound energy through the cochlear partition. Humans and mice with mutations in GAS2, encoding a cytoskeletal regulatory protein, exhibit hearing loss due to disorganization and destabilization of microtubule bundles in pillar and Deiters' cells, two types of inner ear supporting cells with unique cytoskeletal specializations. Failure to maintain microtubule bundle integrity reduced supporting cell stiffness, which in turn altered cochlear micromechanics in Gas2 mutants. Vibratory responses to sound were measured in cochleae from live mice, revealing defects in the propagation and amplification of the traveling wave in Gas2 mutants. We propose that the microtubule bundling activity of GAS2 imparts supporting cells with mechanical properties for transmitting sound energy through the cochlea.