Opposing activities of IFITM proteins in SARS-CoV-2 infection.

Interferon-induced transmembrane proteins (IFITMs) restrict infections by many viruses, but a subset of IFITMs enhance infections by specific coronaviruses through currently unknown mechanisms. We show that SARS-CoV-2 Spike-pseudotyped virus and genuine SARS-CoV-2 infections are generally restricted by human and mouse IFITM1, IFITM2, and IFITM3, using gain- and loss-of-function approaches. Mechanistically, SARS-CoV-2 restriction occurred independently of IFITM3 S-palmitoylation, indicating a restrictive capacity distinct from reported inhibition of other viruses. In contrast, the IFITM3 amphipathic helix and its amphipathic properties were required for virus restriction. Mutation of residues within the IFITM3 endocytosis-promoting YxxФ motif converted human IFITM3 into an enhancer of SARS-CoV-2 infection, and cell-to-cell fusion assays confirmed the ability of endocytic mutants to enhance Spike-mediated fusion with the plasma membrane. Overexpression of TMPRSS2, which increases plasma membrane fusion versus endosome fusion of SARS-CoV-2, attenuated IFITM3 restriction and converted amphipathic helix mutants into infection enhancers. In sum, we uncover new pro- and anti-viral mechanisms of IFITM3, with clear distinctions drawn between enhancement of viral infection at the plasma membrane and amphipathicity-based mechanisms used for endosomal SARS-CoV-2 restriction.

Membrane-dependent actin polymerization mediated by the Legionella pneumophila effector protein MavH.

L. pneumophila propagates in eukaryotic cells within a specialized niche, the Legionella-containing vacuole (LCV). The infection process is controlled by over 330 effector proteins delivered through the type IV secretion system. In this study, we report that the Legionella MavH effector localizes to endosomes and remodels host actin cytoskeleton in a phosphatidylinositol 3-phosphate (PI(3)P) dependent manner when ectopically expressed. We show that MavH recruits host actin capping protein (CP) and actin to the endosome via its CP-interacting (CPI) motif and WH2-like actin-binding domain, respectively. In vitro assays revealed that MavH stimulates actin assembly on PI(3)P-containing liposomes causing their tubulation. In addition, the recruitment of CP by MavH negatively regulates F-actin density at the membrane. We further show that, in L. pneumophila-infected cells, MavH appears around the LCV at the very early stage of infection and facilitates bacterium entry into the host. Together, our results reveal a novel mechanism of membrane tubulation induced by membrane-dependent actin polymerization catalyzed by MavH that contributes to the early stage of L. pneumophila infection by regulating host actin dynamics.

Human defensins facilitate local unfolding of thermodynamically unstable regions of bacterial protein toxins.

Defensins are short cationic, amphiphilic, cysteine-rich peptides that constitute the front-line immune defense against various pathogens. In addition to exerting direct antibacterial activities, defensins inactivate several classes of unrelated bacterial exotoxins. To date, no coherent mechanism has been proposed to explain defensins' enigmatic efficiency toward various toxins. In this study, we showed that binding of neutrophil ?-defensin HNP1 to affected bacterial toxins caused their local unfolding, potentiated their thermal melting and precipitation, exposed new regions for proteolysis, and increased susceptibility to collisional quenchers without causing similar effects on tested mammalian structural and enzymatic proteins. Enteric ?-defensin HD5 and ?-defensin hBD2 shared similar toxin-unfolding effects with HNP1, albeit to different degrees. We propose that protein susceptibility to inactivation by defensins is contingent to their thermolability and conformational plasticity and that defensin-induced unfolding is a key element in the general mechanism of toxin inactivation by human defensins.

Intein-mediated cytoplasmic reconstitution of a split toxin enables selective cell ablation in mixed populations and tumor xenografts.

The application of proteinaceous toxins for cell ablation is limited by their high on- and off-target toxicity, severe side effects, and a narrow therapeutic window. The selectivity of targeting can be improved by intein-based toxin reconstitution from two dysfunctional fragments provided their cytoplasmic delivery via independent, selective pathways. While the reconstitution of proteins from genetically encoded elements has been explored, exploiting cell-surface receptors for boosting selectivity has not been attained. We designed a robust splitting algorithm and achieved reliable cytoplasmic reconstitution of functional diphtheria toxin from engineered intein-flanked fragments upon receptor-mediated delivery of one of them to the cells expressing the counterpart. Retargeting the delivery machinery toward different receptors overexpressed in cancer cells enables selective ablation of specific subpopulations in mixed cell cultures. In a mouse model, the transmembrane delivery of a split-toxin construct potently inhibits the growth of xenograft tumors expressing the split counterpart. Receptor-mediated delivery of engineered split proteins provides a platform for precise therapeutic and experimental ablation of tumors or desired cell populations while also greatly expanding the applicability of the intein-based protein transsplicing.

Photorhabdus luminescens TccC3 Toxin Targets the Dynamic Population of F-Actin and Impairs Cell Cortex Integrity.

Due to its essential role in cellular processes, actin is a common target for bacterial toxins. One such toxin, TccC3, is an effector domain of the ABC-toxin produced by entomopathogenic bacteria of Photorhabdus spp. Unlike other actin-targeting toxins, TccC3 uniquely ADP-ribosylates actin at Thr-148, resulting in the formation of actin aggregates and inhibition of phagocytosis. It has been shown that the fully modified F-actin is resistant to depolymerization by cofilin and gelsolin, but their effects on partially modified actin were not explored. We found that only F-actin unprotected by tropomyosin is the physiological TccC3 substrate. Yet, ADP-ribosylated G-actin can be produced upon cofilin-accelerated F-actin depolymerization, which was only mildly inhibited in partially modified actin. The affinity of TccC3-ADP-ribosylated G-actin for profilin and thymosin-ß4 was weakened moderately but sufficiently to potentiate spontaneous polymerization in their presence. Interestingly, the Arp2/3-mediated nucleation was also potentiated by T148-ADP-ribosylation. Notably, even partially modified actin showed reduced bundling by plastins and α-actinin. In agreement with the role of these and other tandem calponin-homology domain actin organizers in the assembly of the cortical actin network, TccC3 induced intense membrane blebbing in cultured cells. Overall, our data suggest that TccC3 imposes a complex action on the cytoskeleton by affecting F-actin nucleation, recycling, and interaction with actin-binding proteins involved in the integration of actin filaments with each other and cellular elements.

Rounding Out the Understanding of ACD Toxicity with the Discovery of Cyclic Forms of Actin Oligomers.

Actin is an essential element of both innate and adaptive immune systems and can aid in motility and translocation of bacterial pathogens, making it an attractive target for bacterial toxins. Pathogenic Vibrio and Aeromonas genera deliver actin cross-linking domain (ACD) toxin into the cytoplasm of the host cell to poison actin regulation and promptly induce cell rounding. At early stages of toxicity, ACD covalently cross-links actin monomers into oligomers (AOs) that bind through multivalent interactions and potently inhibit several families of actin assembly proteins. At advanced toxicity stages, we found that the terminal protomers of linear AOs can get linked together by ACD to produce cyclic AOs. When tested against formins and Ena/VASP, linear and cyclic AOs exhibit similar inhibitory potential, which for the cyclic AOs is reduced in the presence of profilin. In coarse-grained molecular dynamics simulations, profilin and WH2-motif binding sites on actin subunits remain exposed in modeled AOs of both geometries. We speculate, therefore, that the reduced toxicity of cyclic AOs is due to their reduced configurational entropy. A characteristic feature of cyclic AOs is that, in contrast to the linear forms, they cannot be straightened to form filaments (e.g., through stabilization by cofilin), which makes them less susceptible to neutralization by the host cell.

DeActs: genetically encoded tools for perturbing the actin cytoskeleton in single cells.

The actin cytoskeleton is essential for many fundamental biological processes, but tools for directly manipulating actin dynamics are limited to cell-permeable drugs that preclude single-cell perturbations. Here we describe DeActs, genetically encoded actin-modifying polypeptides, which effectively induce actin disassembly in eukaryotic cells. We demonstrate that DeActs are universal tools for studying the actin cytoskeleton in single cells in culture, tissues, and multicellular organisms including various neurodevelopmental model systems.

Oligomerization Affects the Ability of Human Cyclase-Associated Proteins 1 and 2 to Promote Actin Severing by Cofilins.

Actin-depolymerizing factor (ADF)/cofilins accelerate actin turnover by severing aged actin filaments and promoting the dissociation of actin subunits. In the cell, ADF/cofilins are assisted by other proteins, among which cyclase-associated proteins 1 and 2 (CAP1,2) are particularly important. The N-terminal half of CAP has been shown to promote actin filament dynamics by enhancing ADF-/cofilin-mediated actin severing, while the central and C-terminal domains are involved in recharging the depolymerized ADP-G-actin/cofilin complexes with ATP and profilin. We analyzed the ability of the N-terminal fragments of human CAP1 and CAP2 to assist human isoforms of "muscle" (CFL2) and "non-muscle" (CFL1) cofilins in accelerating actin dynamics. By conducting bulk actin depolymerization assays and monitoring single-filament severing by total internal reflection fluorescence (TIRF) microscopy, we found that the N-terminal domains of both isoforms enhanced cofilin-mediated severing and depolymerization at similar rates. According to our analytical sedimentation and native mass spectrometry data, the N-terminal recombinant fragments of both human CAP isoforms form tetramers. Replacement of the original oligomerization domain of CAPs with artificial coiled-coil sequences of known oligomerization patterns showed that the activity of the proteins is directly proportional to the stoichiometry of their oligomerization; i.e., tetramers and trimers are more potent than dimers, which are more effective than monomers. Along with higher binding affinities of the higher-order oligomers to actin, this observation suggests that the mechanism of actin severing and depolymerization involves simultaneous or consequent and coordinated binding of more than one N-CAP domain to F-actin/cofilin complexes.

Persistent nuclear actin filaments inhibit transcription by RNA polymerase II.

Actin is abundant in the nucleus and it is clear that nuclear actin has important functions. However, mystery surrounds the absence of classical actin filaments in the nucleus. To address this question, we investigated how polymerizing nuclear actin into persistent nuclear actin filaments affected transcription by RNA polymerase II. Nuclear filaments impaired nuclear actin dynamics by polymerizing and sequestering nuclear actin. Polymerizing actin into stable nuclear filaments disrupted the interaction of actin with RNA polymerase II and correlated with impaired RNA polymerase II localization, dynamics, gene recruitment, and reduced global transcription and cell proliferation. Polymerizing and crosslinking nuclear actin in vitro similarly disrupted the actin-RNA-polymerase-II interaction and inhibited transcription. These data rationalize the general absence of stable actin filaments in mammalian somatic nuclei. They also suggest a dynamic pool of nuclear actin is required for the proper localization and activity of RNA polymerase II.

Pathogenic Mechanisms of Actin Cross-Linking Toxins: Peeling Away the Layers.

Actin cross-linking toxins are produced by Gram-negative bacteria from Vibrio and Aeromonas genera. The toxins were named actin cross-linking domains (ACD), since the first and most of the subsequently discovered ACDs were found as effector domains in larger MARTX and VgrG toxins. Among recognized human pathogens, ACD is produced by Vibrio cholerae, Vibrio vulnificus, and Aeromonas hydrophila. Upon delivery to the cytoplasm of a host cell, ACD covalently cross-links actin monomers into non-polymerizable actin oligomers of various lengths. Provided sufficient doses of toxin are delivered, most or all actin can be promptly cross-linked into non-functional oligomers, leading to cell rounding, detachment from the substrate and, in many cases, cell death. Recently, a deeper layer of ACD toxicity with a less obvious but more potent mechanism was discovered. According to this finding, low doses of the ACD-produced actin oligomers can actively disrupt the actin cytoskeleton by potently inhibiting essential actin assembly proteins, formins. The first layer of toxicity is direct (as actin is the immediate and the only target), passive (since ACD-cross-linked actin oligomers are toxic only because they are non-functional), and less potent (as bulk quantities of one of the most abundant cytoplasmic proteins, actin, have to be modified). The second mechanism is indirect (as major targets, formins, are not affected by ACD directly), active (because actin oligomers act as "secondary" toxins), and highly potent [as it affects scarce and essential actin-binding proteins (ABPs)].

Targeting and inactivation of bacterial toxins by human defensins.

Defensins, as a prominent family of antimicrobial peptides (AMP), are major effectors of the innate immunity with a broad range of immune modulatory and antimicrobial activities. In particular, defensins are the only recognized fast-response molecules that can neutralize a broad range of bacterial toxins, many of which are among the deadliest compounds on the planet. For a decade, the mystery of how a small and structurally conserved group of peptides can neutralize a heterogeneous group of toxins with little to no sequential and structural similarity remained unresolved. Recently, it was found that defensins recognize and target structural plasticity/thermodynamic instability, fundamental physicochemical properties that unite many bacterial toxins and distinguish them from the majority of host proteins. Binding of human defensins promotes local unfolding of the affected toxins, destabilizes their secondary and tertiary structures, increases susceptibility to proteolysis, and leads to their precipitation. While the details of toxin destabilization by defensins remain obscure, here we briefly review properties and activities of bacterial toxins known to be affected by or resilient to defensins, and discuss how recognized features of defensins correlate with the observed inactivation.

Calcium binding is essential for plastin 3 function in Smn-deficient motoneurons.

The actin-binding and bundling protein, plastin 3 (PLS3), was identified as a protective modifier of spinal muscular atrophy (SMA) in some patient populations and as a disease modifier in animal models of SMA. How it functions in this process, however, is not known. Because PLS3 is an actin-binding/bundling protein, we hypothesized it would likely act via modification of the actin cytoskeleton in axons and neuromuscular junctions to protect motoneurons in SMA. To test this, we examined the ability of other known actin cytoskeleton organizing proteins to modify motor axon outgrowth phenotypes in an smn morphant zebrafish model of SMA. While PLS3 can fully compensate for low levels of smn, cofilin 1, profilin 2 and α-actinin 1 did not affect smn morphant motor axon outgrowth. To determine how PLS3 functions in SMA, we generated deletion constructs of conserved PLS3 structural domains. The EF hands were essential for PLS3 rescue of smn morphant phenotypes, and mutation of the Ca(2+)-binding residues within the EF hands resulted in a complete loss of PLS3 rescue. These results indicate that Ca(2+) regulation is essential for the function of PLS3 in motor axons. Remarkably, PLS3 mutants lacking both actin-binding domains were still able to rescue motor axons in smn morphants, although not as well as full-length PLS3. Therefore, PLS3 function in this process may have an actin-independent component.

Retrocyclins neutralize bacterial toxins by potentiating their unfolding.

Defensins are a class of immune peptides with a broad range of activities against bacterial, fungal and viral pathogens. Besides exerting direct anti-microbial activity via dis-organization of bacterial membranes, defensins are also able to neutralize various unrelated bacterial toxins. Recently, we have demonstrated that in the case of human α- and ß-defensins, this later ability is achieved through exploiting toxins' marginal thermodynamic stability, i.e. defensins act as molecular anti-chaperones unfolding toxin molecules and exposing their hydrophobic regions and thus promoting toxin precipitation and inactivation [Kudryashova et al. (2014) Immunity 41, 709-721]. Retrocyclins (RCs) are humanized synthetic θ-defensin peptides that possess unique cyclic structure, differentiating them from α- and ß-defensins. Importantly, RCs are more potent against some bacterial and viral pathogens and more stable than their linear counterparts. However, the mechanism of bacterial toxin inactivation by RCs is not known. In the present study, we demonstrate that RCs facilitate unfolding of bacterial toxins. Using differential scanning fluorimetry (DSF), limited proteolysis and collisional quenching of internal tryptophan fluorescence, we show that hydrophobic regions of toxins normally buried in the molecule interior become more exposed to solvents and accessible to proteolytic cleavage in the presence of RCs. The RC-induced unfolding of toxins led to their precipitation and abrogated activity. Toxin inactivation by RCs was strongly diminished under reducing conditions, but preserved at physiological salt and serum concentrations. Therefore, despite significant structural diversity, α-, ß- and θ-defensins employ similar mechanisms of toxin inactivation, which may be shared by anti-microbial peptides from other families.

Thermodynamic properties of the effector domains of MARTX toxins suggest their unfolding for translocation across the host membrane.

MARTX (multifunctional autoprocessing repeats-in-toxin) family toxins are produced by Vibrio cholerae, Vibrio vulnificus, Aeromonas hydrophila and other Gram-negative bacteria. Effector domains of MARTX toxins cross the cytoplasmic membrane of a host cell through a putative pore formed by the toxin's glycine-rich repeats. The structure of the pore is unknown and the translocation mechanism of the effector domains is poorly understood. We examined the thermodynamic stability of the effector domains of V. cholerae and A. hydrophila MARTX toxins to elucidate the mechanism of their translocation. We found that all but one domain in each toxin are thermodynamically unstable and several acquire a molten globule state near human physiological temperatures. Fusion of the most stable cysteine protease domain to the adjacent effector domain reduces its thermodynamic stability ∼ 1.4-fold (from D G H 2 O 21.8 to 16.1 kJ mol(-1) ). Precipitation of several individual domains due to thermal denaturation is reduced upon their fusion into multi-domain constructs. We speculate that low thermostability of the MARTX effector domains correlates with that of many other membrane-penetrating toxins and implies their unfolding for cell entry. This study extends the list of thermolabile bacterial toxins, suggesting that this quality is essential and could be susceptible for selective targeting of pathogenic toxins.

Remodeling of actin filaments by ADF/cofilin proteins.

Cofilin/ADF proteins play key roles in the dynamics of actin, one of the most abundant and highly conserved eukaryotic proteins. We used cryoelectron microscopy to generate a 9-Å resolution three-dimensional reconstruction of cofilin-decorated actin filaments, the highest resolution achieved for a complex of F-actin with an actin-binding protein. We show that the cofilin-induced change in the filament twist is due to a unique conformation of the actin molecule unrelated to any previously observed state. The changes between the actin protomer in naked F-actin and in the actin-cofilin filament are greater than the conformational changes between G- and F-actin. Our results show the structural plasticity of actin, suggest that other actin-binding proteins may also induce large but different conformational changes, and show that F-actin cannot be described by a single molecular model.

Stabilization of F-actin by Salmonella effector SipA resembles the structural effects of inorganic phosphate and phalloidin.

Entry of Salmonella into host enterocytes relies on its pathogenicity island 1 effector SipA. We found that SipA binds to F-actin in a 1:2 stoichiometry with sub-nanomolar affinity. A cryo-EM reconstruction revealed that SipA's globular core binds at the groove between actin strands, whereas the extended C-terminal arm penetrates deeply into the inter-strand space, stabilizing F-actin from within. The unusually strong binding of SipA is achieved by a combination of fast association via the core and very slow dissociation dictated by the arm. Similar to Pi, BeF3, and phalloidin, SipA potently inhibited actin depolymerization by actin depolymerizing factor (ADF)/cofilin, which correlated with increased filament stiffness, supporting the hypothesis that F-actin's mechanical properties contribute to the recognition of its nucleotide state by protein partners. The remarkably strong binding to F-actin maximizes the toxin's effects at the injection site while minimizing global influence on the cytoskeleton and preventing pathogen detection by the host cell.

ATP and ADP actin states.

This minireview is dedicated to the memory of Henryk Eisenberg and honors his major contributions to many areas of biophysics and to the analysis of macromolecular states and interactions in particular. This work reviews the ATP and ADP states of a ubiquitous protein, actins, and considers the present evidence for and against unique, nucleotide-dependent conformations of this protein. The effects of ATP and ADP on specific structural elements of actins, its loops and clefts, as revealed by mutational, crosslinking, spectroscopic, and EPR methods are discussed. It is concluded that the existing evidence points to dynamic equilibria of these structural elements among various conformational states in both ATP- and ADP-actins, with the nucleotides impacting the equilibria distributions.

Actin Bundles Dynamics and Architecture.

Cells use the actin cytoskeleton for many of their functions, including their division, adhesion, mechanosensing, endo- and phagocytosis, migration, and invasion. Actin bundles are the main constituent of actin-rich structures involved in these processes. An ever-increasing number of proteins that crosslink actin into bundles or regulate their morphology is being identified in cells. With recent advances in high-resolution microscopy and imaging techniques, the complex process of bundles formation and the multiple forms of physiological bundles are beginning to be better understood. Here, we review the physiochemical and biological properties of four families of highly conserved and abundant actin-bundling proteins, namely, α-actinin, fimbrin/plastin, fascin, and espin. We describe the similarities and differences between these proteins, their role in the formation of physiological actin bundles, and their properties-both related and unrelated to their bundling abilities. We also review some aspects of the general mechanism of actin bundles formation, which are known from the available information on the activity of the key actin partners involved in this process.

Stabilization of F-actin by Salmonella effector SipA resembles the structural effects of inorganic phosphate and phalloidin.

Entry of Salmonella into host enterocytes strictly relies on its pathogenicity island 1 effector SipA. We found that SipA binds to F-actin in a unique mode in a 1:2 stoichiometry with picomolar affinity. A cryo-EM reconstruction revealed that SipA's globular core binds at the grove between actin strands, whereas the extended C-terminal arm penetrates deeply into the inter-strand space, stabilizing F-actin from within. The unusually strong binding of SipA is achieved via a combination of fast association via the core and very slow dissociation dictated by the arm. Similarly to Pi, BeF3, and phalloidin, SipA potently inhibited actin depolymerization by ADF/cofilin, which correlated with the increased filament stiffness, supporting the hypothesis that F-actin's mechanical properties contribute to the recognition of its nucleotide state by protein partners. The remarkably strong binding to F-actin maximizes the toxin's effects at the injection site while minimizing global influence on the cytoskeleton and preventing pathogen detection by the host cell.

Structural states and dynamics of the D-loop in actin.

Conformational changes induced by ATP hydrolysis on actin are involved in the regulation of complex actin networks. Previous structural and biochemical data implicate the DNase I binding loop (D-loop) of actin in such nucleotide-dependent changes. Here, we investigated the structural and conformational states of the D-loop (in solution) using cysteine scanning mutagenesis and site-directed labeling. The reactivity of D-loop cysteine mutants toward acrylodan and the mobility of spin labels on these mutants do not show patterns of an α-helical structure in monomeric and filamentous actin, irrespective of the bound nucleotide. Upon transition from monomeric to filamentous actin, acrylodan emission spectra and electron paramagnetic resonance line shapes of labeled mutants are blue-shifted and more immobilized, respectively, with the central residues (residues 43-47) showing the most drastic changes. Moreover, complex electron paramagnetic resonance line shapes of spin-labeled mutants suggest several conformational states of the D-loop. Together with a new (to our knowledge) actin crystal structure that reveals the D-loop in a unique hairpin conformation, our data suggest that the D-loop equilibrates in F-actin among different conformational states irrespective of the nucleotide state of actin.