Monomer Sequestration Research Articles

Cannabidiol-Mediated Sequestration of Alzheimer’s Amyloid-<i>β</i> Peptides in ADDL Oligomers

Cannabidiol (CBD), one of the most studied phytocannabinoids, is non-psychotropic and can induce protective effects on the central nervous system against acute and chronic brain injury. Interestingly, CBD inhibits processes relating to amyloid beta (Aβ)-induced neurotoxicity in mouse models of Alzheimer’s disease, though the detailed molecular mechanism underlying the CBD neurotoxicity modulation is not fully understood. In this study, using atomic force microscopy, we find that CBD promotes the aggregation of Aβ peptides, enhancing the formation of Aβ oligomers, also known as Aβ-derived diffusible ligands (ADDLs). The CBD-mediated sequestration of Aβ monomers in soluble ADDLs could reduce neurotoxicity. This study highlights a possible role of CBD in modulating the formation of ADDL aggregates and provides insight into potentially neuroprotective properties of CBD in Alzheimer’s disease.

Perphenazine-Macrocycle Conjugates Rapidly Sequester the Aβ42 Monomer and Prevent Formation of Toxic Oligomers and Amyloid.

Alzheimer's disease is imposing a growing social and economic burden worldwide, and effective therapies are urgently required. One possible approach to modulation of the disease outcome is to use small molecules to limit the conversion of monomeric amyloid (Aβ42) to cytotoxic amyloid oligomers and fibrils. We have synthesized modulators of amyloid assembly that are unlike others studied to date: these compounds act primarily by sequestering the Aβ42 monomer. We provide kinetic and nuclear magnetic resonance data showing that these perphenazine conjugates divert the Aβ42 monomer into amorphous aggregates that are not cytotoxic. Rapid monomer sequestration by the compounds reduces fibril assembly, even in the presence of pre-formed fibrillar seeds. The compounds are therefore also able to disrupt monomer-dependent secondary nucleation, the autocatalytic process that generates the majority of toxic oligomers. The inhibitors have a modular design that is easily varied, aiding future exploration and use of these tools to probe the impact of distinct Aβ42 species populated during amyloid assembly.

The binding of the small heat-shock protein αB-crystallin to fibrils of α-synuclein is driven by entropic forces

Molecular chaperones are key components of the cellular proteostasis network whose role includes the suppression of the formation and proliferation of pathogenic aggregates associated with neurodegenerative diseases. The molecular principles that allow chaperones to recognize misfolded and aggregated proteins remain, however, incompletely understood. To address this challenge, here we probe the thermodynamics and kinetics of the interactions between chaperones and protein aggregates under native solution conditions using a microfluidic platform. We focus on the binding between amyloid fibrils of α-synuclein, associated with Parkinson's disease, to the small heat-shock protein αB-crystallin, a chaperone widely involved in the cellular stress response. We find that αB-crystallin binds to α-synuclein fibrils with high nanomolar affinity and that the binding is driven by entropy rather than enthalpy. Measurements of the change in heat capacity indicate significant entropic gain originates from the disassembly of the oligomeric chaperones that function as an entropic buffer system. These results shed light on the functional roles of chaperone oligomerization and show that chaperones are stored as inactive complexes which are capable of releasing active subunits to target aberrant misfolded species.

Nucleation limits the lengths of actin filaments assembled by formin

Formins stimulate actin polymerization by promoting both filament nucleation and elongation. Because nucleation and elongation draw upon a common pool of actin monomers, the rate at which each reaction proceeds influences the other. This interdependent mechanism determines the number of filaments assembled over the course of a polymerization reaction, as well as their equilibrium lengths. In this study, we used kinetic modeling and in vitro polymerization reactions to dissect the contributions of filament nucleation and elongation to the process of formin-mediated actin assembly. We found that the rates of nucleation and elongation evolve over the course of a polymerization reaction. The period over which each process occurs is a key determinant of the total number of filaments that are assembled, as well as their average lengths at equilibrium. Inclusion of formin in polymerization reactions speeds filament nucleation, thus increasing the number and shortening the lengths of filaments that are assembled over the course of the reaction. Modulation of the elongation rate produces modest changes in the equilibrium lengths of formin-bound filaments. However, the dependence of filament length on the elongation rate is limited by the number of filament ends generated via formin’s nucleation activity. Sustained elongation of small numbers of formin-bound filaments, therefore, requires inhibition of nucleation via monomer sequestration and a low concentration of activated formin. Our results underscore the mechanistic advantage for keeping formin’s nucleation efficiency relatively low in cells, where unregulated actin assembly would produce deleterious effects on cytoskeletal dynamics. Under these conditions, differences in the elongation rates mediated by formin isoforms are most likely to impact the kinetics of actin assembly.

Insights into the evolution of regulated actin dynamics via characterization of primitive gelsolin/cofilin proteins from Asgard archaea

Asgard archaea genomes contain potential eukaryotic-like genes that provide intriguing insight for the evolution of eukaryotes. The eukaryotic actin polymerization/depolymerization cycle is critical for providing force and structure in many processes, including membrane remodeling. In general, Asgard genomes encode two classes of actin-regulating proteins from sequence analysis, profilins and gelsolins. Asgard profilins were demonstrated to regulate actin filament nucleation. Here, we identify actin filament severing, capping, annealing and bundling, and monomer sequestration activities by gelsolin proteins from Thorarchaeota (Thor), which complete a eukaryotic-like actin depolymerization cycle, and indicate complex actin cytoskeleton regulation in Asgard organisms. Thor gelsolins have homologs in other Asgard archaea and comprise one or two copies of the prototypical gelsolin domain. This appears to be a record of an initial preeukaryotic gene duplication event, since eukaryotic gelsolins are generally comprise three to six domains. X-ray structures of these proteins in complex with mammalian actin revealed similar interactions to the first domain of human gelsolin or cofilin with actin. Asgard two-domain, but not one-domain, gelsolins contain calcium-binding sites, which is manifested in calcium-controlled activities. Expression of two-domain gelsolins in mammalian cells enhanced actin filament disassembly on ionomycin-triggered calcium release. This functional demonstration, at the cellular level, provides evidence for a calcium-controlled Asgard actin cytoskeleton, indicating that the calcium-regulated actin cytoskeleton predates eukaryotes. In eukaryotes, dynamic bundled actin filaments are responsible for shaping filopodia and microvilli. By correlation, we hypothesize that the formation of the protrusions observed from Lokiarchaeota cell bodies may involve the gelsolin-regulated actin structures.

Tuning the Length of Cooperative Supramolecular Polymers under Thermodynamic Control.

In the field of supramolecular (co)polymerizations, the ability to predict and control the composition and length of the supramolecular (co)polymers is a topic of great interest. In this work, we elucidate the mechanism that controls the polymer length in a two-component cooperative supramolecular polymerization and unveil the role of the second component in the system. We focus on the supramolecular copolymerization between two derivatives of benzene-1,3,5-tricarboxamide (BTA) monomers: a-BTA and Nle-BTA. As a single component, a-BTA cooperatively polymerizes into long supramolecular polymers, whereas Nle-BTA only forms dimers. By mixing a-BTA and Nle-BTA in different ratios, two-component systems are obtained, which are analyzed in-depth by combining spectroscopy and light-scattering techniques with theoretical modeling. The results show that the length of the supramolecular polymers formed by a-BTA is controlled by competitive sequestration of a-BTA monomers by Nle-BTA, while the obvious alternative Nle-BTA acts as a chain-capper is not operative. This sequestration of a-BTA leads to short, stable species coexisting with long cooperative aggregates. The analysis of the experimental data by theoretical modeling elucidates the thermodynamic parameters of the copolymerization, the distributions of the various species, and the composition and length of the supramolecular polymers at various mixing ratios of a-BTA and Nle-BTA. Moreover, the model was used to generalize our results and to predict the impact of adding a chain-capper or a competitor on the length of the cooperative supramolecular polymers under thermodynamic control. Overall, this work unveils comprehensive guidelines to master the nature of supramolecular (co)polymers and brings the field one step closer to applications.

Erratum to: Hypoxia Augments Increased HIF-1a and Reduced Survival Protein p-Akt in Gelsolin (GSN)-Dependent Cardiomyoblast Cell Apoptosis.

Cytoskeleton filaments play an important role in cellular functions such as maintaining cell shape, cell motility, intracellular transport, and cell division. Actin-binding proteins (ABPs) have numerous functions including regulation of actin filament nucleation, elongation, severing, capping, cross linking, and actin monomer sequestration. Gelsolin (GSN) is one of the actin-binding proteins. Gelsolin (GSN) is one of the actin-binding proteins that regulate cell morphology, differentiation, movement, and apoptosis. GSN also regulates cell morphology, differentiation, movement, and apoptosis. In this study, we have used H9c2 cardiomyoblast cell and H9c2-GSN stable clones to understand the roles and mechanisms of GSN overexpression in hypoxia-induced cardiomyoblast cell death. The data show that hypoxia or GSN overexpression induces HIF-1α expression and reduces the expression of survival markers p-Akt and Bcl-2 in H9c2 cardiomyoblast cells. Under hypoxic conditions, GSN overexpression further reduces p-Akt expression and elevates total as well as cleaved GSN levels and HIF-1α levels. In addition, GSN overexpression enhances apoptosis in cardiomyoblasts under hypoxia. Hypoxic challenge further induced activated caspase-3 and cell death that was attenuated after GSN knock down, which implies that GSN is a critical therapeutic target against hypoxia-induced cardiomyoblast cell death.

Distinct mechanisms of phenotypic effects of inactivation and prionization of Swi1 protein in Saccharomyces cerevisiae

Prions are proteins that under the same conditions can exist in two or more conformations, and at least one of the conformations has infectious properties. The prionization of a protein is typically accompanied by its functional inactivation due to sequestration of monomers by the prion aggregates. The most of prions has been identified in the yeast Saccharomyces cerevisiae. One of them is [SWI+], a prion isoform of the Swi1 protein, which is a component of the evolutionarily conserved chromatin remodeling complex SWI/SNF. Earlier, it was shown that the prionization of [SWI+] induces a nonsense suppression, which leads to weak growth of the [SWI+] strains containing mutant variants of the SUP35 gene and the nonsense allele ade1-14UGA on selective medium without adenine. This effect occurs because of [SWI+] induction that causes a decrease in the amount of the SUP45 mRNA. Strains carrying the SWI1 deletion exhibit significantly higher suppression of the ade1-14UGA nonsense mutation than the [SWI+] strains. In the present study, we identified genes whose expression is altered in the background of the SWI1 deletion using RNA sequencing. We found that the ade1-14UGA suppression in the swi1Δ strains is caused by an increase in the expression of this mutant allele of the ADE1 gene. At the same time, the SUP45 expression level in the swi1Δ strains does not significantly differ from the expression level of this gene in the [swi-] strains. Thus, we have shown that the phenotypic effects of Swi1 prionization and deletion are mediated by different molecular mechanisms. Based on these data, we have concluded that the prionization of proteins is not only unequal to their inactivation, but also can lead to the acquisition of novel phenotypic effects and functions.

Hypoxia Augments Increased HIF-1α and Reduced Survival Protein p-Akt in Gelsolin (GSN)-Dependent Cardiomyoblast Cell Apoptosis.

Cytoskeleton filaments play an important role in cellular functions such as maintaining cell shape, cell motility, intracellular transport, and cell division. Actin-binding proteins (ABPs) have numerous functions including regulation of actin filament nucleation, elongation, severing, capping, cross linking, and actin monomer sequestration. Gelsolin (GSN) is one of the actin-binding proteins. Gelsolin (GSN) is one of the actin-binding proteins that regulate cell morphology, differentiation, movement, and apoptosis. GSN also regulates cell morphology, differentiation, movement, and apoptosis. In this study, we have used H9c2 cardiomyoblast cell and H9c2-GSN stable clones to understand the roles and mechanisms of GSN overexpression in hypoxia-induced cardiomyoblast cell death. The data show that hypoxia or GSN overexpression induces HIF-1α expression and reduces the expression of survival markers p-Akt and Bcl-2 in H9c2 cardiomyoblast cells. Under hypoxic conditions, GSN overexpression further reduces p-Akt expression and elevates total as well as cleaved GSN levels and HIF-1α levels. In addition, GSN overexpression enhances apoptosis in cardiomyoblasts under hypoxia. Hypoxic challenge further induced activated caspase-3 and cell death that was attenuated after GSN knock down, which implies that GSN is a critical therapeutic target against hypoxia-induced cardiomyoblast cell death.

A novel role for WAVE1 in controlling actin network growth rate and architecture.

Branched actin filament networks in cells are assembled through the combined activities of Arp2/3 complex and different WASP/WAVE proteins. Here we used TIRF and electron microscopy to directly compare for the first time the assembly kinetics and architectures of actin filament networks produced by Arp2/3 complex and dimerized VCA regions of WAVE1, WAVE2, or N-WASP. WAVE1 produced strikingly different networks from WAVE2 or N-WASP, which comprised unexpectedly short filaments. Further analysis showed that the WAVE1-specific activity stemmed from an inhibitory effect on filament elongation both in the presence and absence of Arp2/3 complex, which was observed even at low stoichiometries of WAVE1 to actin monomers, precluding an effect from monomer sequestration. Using a series of VCA chimeras, we mapped the elongation inhibitory effects of WAVE1 to its WH2 ("V") domain. Further, mutating a single conserved lysine residue potently disrupted WAVE1's inhibitory effects. Taken together, our results show that WAVE1 has unique activities independent of Arp2/3 complex that can govern both the growth rates and architectures of actin filament networks. Such activities may underlie previously observed differences between the cellular functions of WAVE1 and WAVE2.

P.1.g.001 Neuroprotective efficacy of coenzyme Q10 in rat model of transient focal ischemia

Thymosin β4 (Tβ4) is a developmentally expressed 43-amino acid peptide that inhibits organization of the actin-cytoskeleton by sequestration of G-actin monomers. Tβ4 improves cardiac function after myocardial infarction in adult mice and promotes healing properties in both dermal and corneal wounds. We tested the hypothesis that Tβ4 improves functional neurological outcome in a rat model of embolic stroke. Experimental Procedures: Male Wistar rats (n=18) were subjected to embolic middle cerebral artery occlusion (MCAo). Tβ4 (6 mg/kg, IP) was administered 24 h after MCAo and then every 3 days for four additional doses (n=9). Rats treated with saline were used as a control (n=9). The adhesive-removal test (ART) and modified Neurological Severity Score (mNSS) were performed to measure functional outcome. Rats were sacrificed 56 days after MCAo. Immunostaining was performed with antibodies against NG-2 (chondroitin sulfate proteoglycan), CNPase (2”, 3”-cyclic nucleotide 3'-phosphodiesterase) to detect immature and mature oligodendrocytes. Neurofilament-H (NF-H) antibodies were used to detect axons while myelinated axons were identified with Bielschowsky/Luxol (B/L) Blue staining. EBA (endothelial barrier antigen) was used for detection of mature vessels. Results: Ischemic rats treated with Tβ4 demonstrated a significant overall improvement (P<0.01) in the ART and the mNSS when compared to controls. Significant improvement was observed beginning at 14 and 35 days, respectively. Lesion volumes showed no significant differences between the two groups. Treatment with Tβ4 increased myelinated axons and increased vessel density in the ischemic boundary (P<0.05) and augmented remyelination which was associated with an increase of oligodendrocyte progenitor cells (OPCs) and myelinating oligodendrocytes (P<0.05). Conclusions: The present study suggests that Tβ4 improves neurological functional outcome after embolic stroke in rats. Axonal remodeling from mobilization of OPCs is proposed as contributing to Tβ4 induced functional improvement.

Toxoplasma Gondii Targets the Host Actin Cytoskeleton during Invasion, GO Figure

T. gondii belongs to the phylum apicomplexa, which also includes Plasmodium, the agent of malaria. T. gondii quickly establishes infection as a fast-replicating form, the tachyzoite, which invades numerous cell types throughout the body. The parasite can cause encephalitis and neurologic diseases. Unlike viruses and intracellular bacteria, Toxoplasma actively penetrates the host cell without using typical host-uptake pathways that involve coated pits or actin based engulfment. During invasion, the extracellular parasite triggers the assembly of a unique zoite-cell junction and in less than 20 sec progressively propels its ∼ 2um long body through the junction into the growing vacuole. How the growing PV and its parasite passes through the barrier of the host cell cortical actin remains unknown. Here we provide evidence that Toxofilin, an actin-binding protein secreted by the invading parasites into the host cell cytoplasm at the onset of invasion, targets the host cortical actin cytoskeleton to facilitate vacuole folding and tachytozoites invasion. Indeed, Toxoplasma tachyzoites lacking toxofilin exhibit delayed invasion kinetics. Correlative light and electron tomography allowed tracking toxofilin in action within the host cell and showed that integrity of the host cortical actin cytoskeleton was disrupted at the site of parasite entry, suggesting that toxofilin induces actin depolymerization in close proximity to the zoite apex at the onset of invasion. Quantitative fluorescent speckle microscopy indicates that toxofilin facilitate tachyzoite invasion by upregulating host cortical actin filament turnover, consistent with toxofilin monomer sequestration and barbed end filament-capping capabilities in vitro. The ability of toxofilin to expedite invasion by regulating the turnover of cortical actin filaments in the host cell is likely to confer a selective advantage to the parasite. Delorme-Walker et al, J Cell Sci. 2012 Jul 5. [Epub ahead of print] PubMed

Structure of a Longitudinal Actin Dimer Assembled by Tandem W Domains: Implications for Actin Filament Nucleation

Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin β4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helix of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with intersubunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL cross-linked to actin Cys374 and show it to be nearly identical with non-cross-linked W-Actin structures. This result validates the use of cross-linking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why nucleation-promoting factors of the Arp2/3 complex, which are related to tandem-W-domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of thymosin β4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.

Thymosin β4 improves functional neurological outcome in a rat model of embolic stroke

Thymosin β4 (Tβ4) is a developmentally expressed 43-amino acid peptide that inhibits organization of the actin-cytoskeleton by sequestration of G-actin monomers. Tβ4 improves cardiac function after myocardial infarction in adult mice and promotes healing properties in both dermal and corneal wounds. We tested the hypothesis that Tβ4 improves functional neurological outcome in a rat model of embolic stroke. Experimental Procedures: Male Wistar rats ( n=18) were subjected to embolic middle cerebral artery occlusion (MCAo). Tβ4 (6 mg/kg, IP) was administered 24 h after MCAo and then every 3 days for four additional doses ( n=9). Rats treated with saline were used as a control ( n=9). The adhesive-removal test (ART) and modified Neurological Severity Score (mNSS) were performed to measure functional outcome. Rats were sacrificed 56 days after MCAo. Immunostaining was performed with antibodies against NG-2 (chondroitin sulfate proteoglycan), CNPase (2”, 3”-cyclic nucleotide 3'-phosphodiesterase) to detect immature and mature oligodendrocytes. Neurofilament-H (NF-H) antibodies were used to detect axons while myelinated axons were identified with Bielschowsky/Luxol (B/L) Blue staining. EBA (endothelial barrier antigen) was used for detection of mature vessels. Results: Ischemic rats treated with Tβ4 demonstrated a significant overall improvement ( P<0.01) in the ART and the mNSS when compared to controls. Significant improvement was observed beginning at 14 and 35 days, respectively. Lesion volumes showed no significant differences between the two groups. Treatment with Tβ4 increased myelinated axons and increased vessel density in the ischemic boundary ( P<0.05) and augmented remyelination which was associated with an increase of oligodendrocyte progenitor cells (OPCs) and myelinating oligodendrocytes ( P<0.05). Conclusions: The present study suggests that Tβ4 improves neurological functional outcome after embolic stroke in rats. Axonal remodeling from mobilization of OPCs is proposed as contributing to Tβ4 induced functional improvement.

α-Synuclein and Its A30P Mutant Affect Actin Cytoskeletal Structure and Dynamics

The function of alpha-synuclein, a soluble protein abundant in the brain and concentrated at presynaptic terminals, is still undefined. Yet, alpha-synuclein overexpression and the expression of its A30P mutant are associated with familial Parkinson's disease. Working in cell-free conditions, in two cell lines as well as in primary neurons we demonstrate that alpha-synuclein and its A30P mutant have different effects on actin polymerization. Wild-type alpha-synuclein binds actin, slows down its polymerization and accelerates its depolymerization, probably by monomer sequestration; A30P mutant alpha-synuclein increases the rate of actin polymerization and disrupts the cytoskeleton during reassembly of actin filaments. Consequently, in cells expressing mutant alpha-synuclein, cytoskeleton-dependent processes, such as cell migration, are inhibited, while exo- and endocytic traffic is altered. In hippocampal neurons from mice carrying a deletion of the alpha-synuclein gene, electroporation of wild-type alpha-synuclein increases actin instability during remodeling, with growth of lamellipodia-like structures and apparent cell enlargement, whereas A30P alpha-synuclein induces discrete actin-rich foci during cytoskeleton reassembly. In conclusion, alpha-synuclein appears to play a major role in actin cytoskeletal dynamics and various aspects of microfilament function. Actin cytoskeletal disruption induced by the A30P mutant might alter various cellular processes and thereby play a role in the pathogenesis of neurodegeneration.

Dimer Disruption and Monomer Sequestration by Alkyl Tripeptides Are Successful Strategies for Inhibiting Wild-Type and Multidrug-Resistant Mutated HIV-1 Proteases

Wild-type and drug-resistant mutated HIV-1 proteases are active as dimers. This work describes the inhibition of their dimerization by a new series of alkyl tripeptides that target the four-stranded antiparallel beta-sheet formed by the interdigitation of the N- and C-monomer ends of each monomer. Analytical ultracentrifugation was used to give experimental evidence of their mode of action that is disruption of the active homodimer with formation of inactive monomer-inhibitor complexes. The minimum length of the alkyl chain needed to inhibit dimerization was established. Sequence variations led to a most potent HIV-PR dimerization inhibitor: palmitoyl-Leu-Glu-Tyr (Kid = 0.3 nM). Insertion of d-amino acids at the first two positions of the peptide moiety increased the inhibitor resistance to proteolysis without abolishing the inhibitory effect. Molecular dynamics simulations of the inhibitor series complexed with wild-type and mutated HIV-PR monomers corroborated the kinetic data. They suggested that the lipopeptide peptide moiety replaces the middle strand in the highly conserved intermolecular four-stranded beta-sheet formed by the peptide termini of each monomer, and the alkyl chain is tightly grasped by the active site groove capped by the beta-hairpin flap in a "superclosed" conformation. These new inhibitors were equally active in vitro against both wild-type and drug-resistant multimutated proteases, and the model suggested that the mutations in the monomer did not interfere with the inhibitor.

The β‐Thymosin/WH2 Fold

Remodeling of the actin cytoskeleton in cells is tightly regulated by a vast number of actin-binding proteins (ABPs). These proteins interact with actin via a limited set of conserved folding motifs. One of the most abundant actin-binding motifs is the beta-thymosin fold, represented by the prototypical actin-monomer sequestering protein thymosin-beta4. Among many cytoskeletal proteins, the beta-thymosin fold adopts a shorter form, known as the WASP homology domain 2. Some characteristic features of the beta-thymosin/WH2 fold include its small size (17-43 aa), significant sequence and length variability, frequent occurrence in the form of tandem repeats, and remarkable multifunctionality. This paper discusses the relationship between structure and function of the beta-thymosin/WH2 fold on the basis of four examples: (1) actin monomer sequestration (thymosin-beta4), (2) actin filament nucleation (WASP-Arp2/3 complex, Lmod, and spire), (3) actin filament elongation (Ena/VASP), and (4) cytoskeleton scaffolding (IRSp53 and MIM). Although the core function of the beta-thymosin/WH2 domain in all these proteins is actin binding, specific changes in the sequence of the domain and modular organization of the proteins in which it is found give rise to diverse functions in the regulation of actin cytoskeleton dynamics.

Characterisation of IRTKS, a novel IRSp53/MIM family actin regulator with distinct filament bundling properties

IRSp53 is a scaffold protein that contains an IRSp53/MIM homology domain (IMD) that bundles actin filaments and interacts with the small GTPase Rac. IRSp53 also binds to the small GTPase Cdc42 and to Scar/WAVE and Mena/VASP proteins to regulate the actin cytoskeleton. We have characterised a novel IMD-containing protein, insulin receptor tyrosine kinase substrate (IRTKS), which has widespread tissue distribution, is a substrate for the insulin receptor and binds Rac. Unlike IRSp53, IRTKS does not interact with Cdc42. Expression of IRTKS induces clusters of short actin bundles rather than filopodia-like protrusions. This difference may be attributable to a short carboxyl-terminal (Ct) extension present on IRTKS, which resembles a WASP-homology 2 (WH2) motif. Addition of the Ct extension to IRSp53 causes an apparent shortening of bundles induced by the IMD in vitro, and in cultured cells, suggesting that the Ct extension of IRTKS modulates the organising activity of the IMD. Lastly, we could not detect actin monomer sequestration by the Ct extension of IRTKS as would be expected with a conventional WH2 motif, but it did interact with actin filaments.

Actin-bound structures of Wiskott–Aldrich syndrome protein (WASP)-homology domain 2 and the implications for filament assembly

Wiskott-Aldrich syndrome protein (WASP)-homology domain 2 (WH2) is a small and widespread actin-binding motif. In the WASP family, WH2 plays a role in filament nucleation by Arp2/3 complex. Here we describe the crystal structures of complexes of actin with the WH2 domains of WASP, WASP-family verprolin homologous protein, and WASP-interacting protein. Despite low sequence identity, WH2 shares structural similarity with the N-terminal portion of the actin monomer-sequestering thymosin beta domain (Tbeta). We show that both domains inhibit nucleotide exchange by targeting the cleft between actin subdomains 1 and 3, a common binding site for many unrelated actin-binding proteins. Importantly, WH2 is significantly shorter than Tbeta but binds actin with approximately 10-fold higher affinity. WH2 lacks a C-terminal extension that in Tbeta4 becomes involved in monomer sequestration by interfering with intersubunit contacts in F-actin. Owing to their shorter length, WH2 domains connected in tandem by short linkers can coexist with intersubunit contacts in F-actin and are proposed to function in filament nucleation by lining up actin subunits along a filament strand. The WH2-central region of WASP-family proteins is proposed to function in an analogous way by forming a special class of tandem repeats whose function is to line up actin and Arp2 during Arp2/3 nucleation. The structures also suggest a mechanism for how profilin-binding Pro-rich sequences positioned N-terminal to WH2 could feed actin monomers directly to WH2, thereby playing a role in filament elongation.

Genetic Characterization of Accumulation of Polyhydroxyalkanoate from Styrene in Pseudomonas putida CA-3

Pseudomonas putida CA-3 is capable of accumulating medium-chain-length polyhydroxyalkanoates (MCL-PHAs) when growing on the toxic pollutant styrene as the sole source of carbon and energy. In this study, we report on the molecular characterization of the metabolic pathways involved in this novel bioconversion. With a mini-Tn5 random mutagenesis approach, acetyl-coenzyme A (CoA) was identified as the end product of styrene metabolism in P. putida CA-3. Amplified flanking-region PCR was used to clone functionally expressed phenylacetyl-CoA catabolon genes upstream from the sty operon in P. putida CA-3, previously reported to generate acetyl-CoA moieties from the styrene catabolic intermediate, phenylacetyl-CoA. However, the essential involvement of a (non-phenylacetyl-CoA) catabolon-encoded 3-hydroxyacyl-CoA dehydrogenase is also reported. The link between de novo fatty acid synthesis and PHA monomer accumulation was investigated, and a functionally expressed 3-hydroxyacyl-acyl carrier protein-CoA transacylase (phaG) gene in P. putida CA-3 was identified. The deduced PhaG amino acid sequence shared >99% identity with a transacylase from P. putida KT2440, involved in 3-hydroxyacyl-CoA MCL-PHA monomer sequestration from de novo fatty acid synthesis under inorganic nutrient-limited conditions. Similarly, with P. putida CA-3, maximal phaG expression was observed only under nitrogen limitation, with concomitant PHA accumulation. Thus, beta-oxidation and fatty acid de novo synthesis appear to converge in the generation of MCL-PHA monomers from styrene in P. putida CA-3. Cloning and functional characterization of the pha locus, responsible for PHA polymerization/depolymerization is also reported and the significance and future prospects of this novel bioconversion are discussed.