Drebrin Protects Assembled Actin from INF2-FFC-mediated Severing and Stabilizes Cell Protrusions.

Highly specialized cells, such as neurons and podocytes, have arborized morphologies that serve their specific functions. Actin cytoskeleton and its associated proteins are responsible for the distinctive shapes of cells. The mechanism of their cytoskeleton regulation - contributing to cell shape maintenance - is yet to be fully clarified. Inverted formin 2 (INF2), one of the modulators of the cytoskeleton, is an atypical formin that can both polymerize and depolymerize actin filaments depending on its molar ratio to actin. Prior work has established that INF2 binds to the sides of actin filaments and severs them. Drebrin is another actin-binding protein that also binds filaments laterally and stabilizes them, but the interplay between drebrin and INF2 on actin filament stabilization is not well understood. Here, we have used biochemical assays, electron microscopy, and total internal reflection fluorescence microscopy imaging to show that drebrin protects actin filaments from severing by INF2 without inhibiting its polymerization activity. Notably, truncated drebrin - DrbA1-300 - is sufficient for this protection, though not as effective as the full-length protein. INF2 and drebrin are abundantly expressed in highly specialized cells and are crucial for the temporal regulation of their actin cytoskeleton, consistent with their involvement in peripheral neuropathy.

Disassembly of bundled F-actin and cellular remodeling via an interplay of Mical, cofilin, and F-actin crosslinkers.

Cellular form and function are controlled by the assembly and stability of actin cytoskeletal structures-but disassembling/pruning these structures is equally essential for the plasticity and remodeling that underlie behavioral adaptations. Importantly, the mechanisms of actin assembly have been well-defined-including that it is driven by actin's polymerization into filaments (F-actin) and then often bundling by crosslinking proteins into stable higher-order structures. In contrast, it remains less clear how these stable bundled F-actin structures are rapidly disassembled. We now uncover mechanisms that rapidly and extensively disassemble bundled F-actin. Using biochemical, structural, and imaging assays with purified proteins, we show that F-actin bundled with one of the most prominent crosslinkers, fascin, is extensively disassembled by Mical, the F-actin disassembly enzyme. Furthermore, the product of this Mical effect, Mical-oxidized actin, is poorly bundled by fascin, thereby further amplifying Mical's disassembly effects on bundled F-actin. Moreover, another critical F-actin regulator, cofilin, also affects fascin-bundled filaments, but we find herein that it synergizes with Mical to dramatically amplify its disassembly of bundled F-actin compared to the sum of their individual effects. Genetic and high-resolution cellular assays reveal that Mical also counteracts crosslinking proteins/bundled F-actin in vivo to control cellular extension, axon guidance, and Semaphorin/Plexin cell-cell repulsion. Yet, our results also support the idea that fascin-bundling serves to dampen Mical's F-actin disassembly in vitro and in vivo-and that physiologically relevant cellular remodeling requires a fine-tuned interplay between the factors that build bundled F-actin networks and those that disassemble them.

Difference in Catalytic Loop Repositioning Leads to GMP Variation between Two Human GBP Homologues.

Interferon-gamma-inducible human large GTPases, hGBP1 and hGBP2, have a distinctive feature of hydrolyzing GTP to GDP and GMP through successive phosphate cleavages. In hGBP1, GMP is the major product, which is essential for its anti-pathogenic activities. However, its close homologue hGBP2 produces significantly less GMP, despite having a similar active site architecture. The molecular basis for less GMP formation and catalytic residue(s) in hGBP2 are not fully explored. To address these issues, we performed systematic biochemical, biophysical, and microsecond simulation studies. Our data suggest that the less GMP formation in hGBP2 is due to the lack of H-bond formation between the W79 side-chain (located near the active site) and main-chain carbonyl of K76 (present in the catalytic loop) in the substrate-bound hGBP2. The absence of this H-bond could not redirect the catalytic loop toward the beta phosphate after the cleavage of gamma-phosphate, a step essential for enhanced GMP formation. Furthermore, based on the mutational and structural analyses, this study for the first time indicates that the same residue, T75, mediates both phosphate cleavages in hGBP2 and hGBP1. This suggests the conservation of the catalytic residue in hGBP homologues. These findings emphasize the indispensable role of correct catalytic loop repositioning for efficient beta phosphate cleavage. This led us to propose a new substrate hydrolysis mechanism by hGBP1 and hGBP2, which may also be helpful to understand the GTP hydrolysis in other hGBP homologues. Overall, the study could provide insight into how these two close homologues play crucial roles in host-mediated immunity through different mechanisms.

MICAL-mediated oxidation of actin and its effects on cytoskeletal and cellular dynamics.

Actin and its dynamic structural remodelings are involved in multiple cellular functions, including maintaining cell shape and integrity, cytokinesis, motility, navigation, and muscle contraction. Many actin-binding proteins regulate the cytoskeleton to facilitate these functions. Recently, actin's post-translational modifications (PTMs) and their importance to actin functions have gained increasing recognition. The MICAL family of proteins has emerged as important actin regulatory oxidation-reduction (Redox) enzymes, influencing actin's properties both in vitro and in vivo. MICALs specifically bind to actin filaments and selectively oxidize actin's methionine residues 44 and 47, which perturbs filaments' structure and leads to their disassembly. This review provides an overview of the MICALs and the impact of MICAL-mediated oxidation on actin's properties, including its assembly and disassembly, effects on other actin-binding proteins, and on cells and tissue systems.

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.

The alpha helix of the intermediate region in hGBP-1 acts as a coupler for enhanced GMP formation.

The interferon-gamma inducible large GTPase human guanylate binding protein-1 (hGBP-1) plays a key role in anti-pathogenic and anti-proliferative functions. This protein hydrolyzes GTP to both GDP and GMP (predominant product) through sequential phosphate cleavages, which makes it functionally distinct from other GTPases. Previous study on truncated variants of hGBP-1 suggested that the α-helix present in the intermediate region is essential for dimerization and thus for GMP formation. However, the role of this helix in the full-length protein in GMP formation is not clearly understood. Here, we present that substitution of the helix with a Gly-rich flexible (GGS)3 sequence in the full-length hGBP-1 (termed as linker protein) showed a drastic decrease in GMP formation. Unlike wild-type, the linker protein is not capable of undergoing substrate-induced dimerization and thereby transition state-induced tetramerization, suggesting the importance of the helix in oligomerization. Furthermore, we examined the effect of interactions between this helix and the α2-helix of the globular domain in GMP formation through mutational studies. The L118G mutation in the α2-helix showed a significantly reduced GMP formation. These results indicate that the interactions of the α-helix with the α2-helix are essential for enhanced GMP production. We propose that these interactions help in the oligomerization-assisted proper positioning of the catalytic machinery for efficient second phosphate cleavage. These findings thus provide a better understanding into the regulation of GMP formation in a large GTPase hGBP-1.

Both beta sheet breaker and alpha helix forming pentapeptide inhibits protein fibrillation: Implication for the treatment of amyloid disorders.

For the first time, the effect of two novel designed pentapeptides on amyloid growth of human insulin using combined biophysical, microscopic, cell viability and computational approaches. Collective experimental data from ThT, ANS, and TEM demonstrate that in spite of having contrasting features, both peptides can effectively inhibit amyloid formation by prolonging lag phase, slowing down aggregation rate, and reducing final fibril formation (up to 84.26% and 85.24% by P1 and P7 respectively). Although pure amyloid caused profound cellular toxicity in SH-SY5Y neuronal cells, amyloid formed in the presence of peptides showed much reduced cellular toxicity. Such an inhibitory effect can be attributed to interference with the structural transition of insulin toward ß-sheet structure by peptides. Furthermore, molecular dynamic simulations confirm that peptide preferentially binds to nearby region which is more prone to form aggregates that consequently disrupts self-assembly into amyloid fibrils (P1 and P7 possess inhibition constant value of 0.000183 and 0.000216 nm, respectively), supporting our experimental observations. This study underscores the information about the sequence based inhibition mechanism of peptides that might dictate their inhibition or modulation capacity, which might be helpful in designing anti-amyloid therapeutics.

Understanding the lower GMP formation in large GTPase hGBP-2 and role of its individual domains in regulation of GTP hydrolysis.

The interferon γ-inducible large GTPases, human guanylate-binding protein (hGBP)-1 and hGBP-2, mediate antipathogenic and antiproliferative effects in human cells. Both proteins hydrolyse GTP to GDP and GMP through successive cleavages of phosphate bonds, a property that functionally distinguishes them from other GTPases. However, it is unclear why hGBP-2 yields lower GMP than hGBP-1 despite sharing a high sequence identity (~ 78%). We previously reported that the hGBP-1 tetramer is crucial for enhanced GMP formation. We show here that the hGBP-2 tetramer has no role in GMP formation. Using truncated hGBP-2 variants, we found that its GTP-binding domain alone hydrolyses GTP only to GDP. However, this domain along with the intermediate region enabled dimerization and hydrolysed GTP further to GMP. We observed that unlike in hGBP-1, the helical domain of hGBP-2 has an insignificant role in the regulation of GTP hydrolysis, suggesting that the differences in GMP formation between hGBP-2 and hGBP-1 arise from differences in their GTP-binding domains. A large sequence variation seen in the guanine cap may be responsible for the lower GMP formation in hGBP-2. Moreover, we identified the sites in the hGBP-2 domains that are critical for both dimerization and tetramerization. We also found the existence of hGBP-2 tetramer in mammalian cells, which might have a role in the suppression of the carcinomas. Our study suggests that sequence variation near the active site in these two close homologues leads to differential second phosphate cleavage and highlights the role of individual hGBP-2 domains in the regulation of GTP hydrolysis.

Stabilizing proteins to prevent conformational changes required for amyloid fibril formation.

Amyloid fibrillation is associated with several human maladies, such as Alzheimer's, Parkinson's, Huntington's diseases, prions, amyotrophic lateral sclerosis, and type 2 diabetes diseases. Gaining insights into the mechanism of amyloid fibril formation and exploring novel approaches to fibrillation inhibition are crucial for preventing amyloid diseases. Here, we hypothesized that ligands capable of stabilizing the native state of query proteins might prevent protein unfolding, which, in turn, may reduce the propensity of proteins to form amyloid fibrils. We demonstrated the efficient inhibition of amyloid formation of the human serum albumin (HSA) (up to 85%) and human insulin (up to 80%) by a nonsteroidal anti-inflammatory drug, ibuprofen (IBFN). IBFN significantly increases the conformational stability of both HSA and insulin, as confirmed by differential scanning calorimetry (DSC). Moreover, increasing concentration of IBFN boosts its amyloid inhibitory propensity in a linear fashion by influencing the nucleation phase as assayed by thioflavin T fluorescence, transmission electron microscopy, and dynamic light scattering. Furthermore, circular dichroism analysis supported the DSC results, showing that IBFN binds to the native state of proteins and almost completely prevents their tendency to lose secondary and tertiary structures. Cell toxicity assay confirms that species formed in the presence of IBFN are less toxic to neuronal cells (SH-SY5Y). These results demonstrate the feasibility of using a small molecule to stabilize the native state of proteins, thereby preventing the amyloidogenic conformational changes, which appear to be the common link in several human amyloid diseases.

Tetrameric assembly of hGBP1 is crucial for both stimulated GMP formation and antiviral activity.

Interferon-γ inducible human guanylate binding protein-1 (hGBP1) shows a unique characteristic that hydrolyses GTP to a mixture of GDP and GMP through successive cleavages, with GMP being the major product. Like other large GTPases, hGBP1 undergoes oligomerization upon substrate hydrolysis, which is essential for the stimulation of activity. It also exhibits antiviral activity against many viruses including hepatitis C. However, which oligomeric form is responsible for the stimulated activity leading to enhanced GMP formation and its influence on antiviral activity, are not properly understood. Using mutant and truncated proteins, our data indicate that transition-state-induced tetramerization is associated with higher rate of GMP formation. This is supported by chimaeras that are defective in both tetramerization and enhanced GMP formation. Unlike wild-type protein, chimaeras did not show allosteric interactions, indicating that tetramerization and enhanced GMP formation are allosterically coupled. Hence, we propose that after the cleavage of the first phosphoanhydride bond GDP·Pi-bound protein dimers transiently associate to form a tetramer that acts as an allosteric switch for higher rate of GMP formation. Biochemical and biophysical studies reveal that sequential conformational changes and interdomain communications regulate tetramer formation via dimer. Our studies also show that overexpression of the mutants, defective in tetramer formation in Rep2a cells do not inhibit proliferation of hepatitis C virus, indicating critical role of a tetramer in the antiviral activity. Thus, the present study not only highlights the importance of hGBP1 tetramer in stimulated GMP formation, but also demonstrates its role in the antiviral activity against hepatitis C virus.