Enrique M. De La Cruz

The ectonucleotidase ENPP1 is the major extracellular hydrolase of the innate immune system activator 2′3′-cyclic GMP-AMP (cGAMP). Tumors that overexpress ENPP1 and rapidly degrade cGAMP avoid immune surveillance in the tumor microenvironment and are highly resistant to cancer immunotherapy. Inhibition of cGAMP degradation by ENPP1 has emerged as a promising strategy to improve cancer therapies. A direct, real-time assay of ENPP1 enzymatic activity would benefit quantitative evaluation of candidate ENPP1 inhibitors. The nonphysiological substrate p-nitrophenyl 5′-thymidine monophosphate is commonly used for this purpose, as it offers a readily detectable colorimetric readout that can be evaluated in real time. However, compounds that potently inhibit p-nitrophenyl 5′-thymidine monophosphate hydrolysis can weakly inhibit ENPP1 with physiological nucleotide substrates, highlighting the importance of testing ENPP1 inhibitors with native substrates ( i.e. , cGAMP). No direct, real-time assays for cGAMP hydrolysis are established. Here, we present a real-time, spectrophotometric assay to monitor ENPP1-catalyzed cGAMP hydrolysis. The increase in extinction coefficient associated with conversion of substrate(s) to AMP and GMP products is used to convert time courses of absorbance change to rates of product formation. Time courses of GMP product formation generated from the absorbance change superimpose with those generated by the direct measurement of GMP product concentration via chemical quench-flow and HPLC analysis. ENPP1 inhibition by the nonhydrolyzable ATP analog, α,β-methylene-ATP, yields an inhibition constant ( K I ) comparable to the independently determined binding affinity. This spectroscopic assay can be performed using a standard, laboratory UV-vis spectrophotometer and has the potential to be scaled up to a high-throughput, multiwell plate setup.

Arp2/3 complex nucleates branched actin networks that drive cell motility and intracellular trafficking. Coronins, a family of seven proteins in humans, inhibit Arp2/3 complex in vitro and reduce branch density in cells. Coro7, a distant member of this family, features two β-propeller domains (β1β2) and C-terminal Central-Acidic (CA) domains and remains poorly studied. Here, cryo-EM and biochemical data show that CA binds subunit Arp3 of free Arp2/3 complex with ~1 µM affinity, inhibiting polymerization like Arpin, while displacing Arp3's autoinhibitory C-terminal tail and promoting the active, short-pitch conformation, like WASP-family nucleation-promoting factors. Full-length Coro7, however, does not inhibit Arp2/3 complex polymerization but effectively induces debranching, whereas the isolated β1β2 or CA domains do not. In cells, Coro7 depletion disrupts ER-Golgi transport, which is rescued by full-length Coro7 but not by truncated variants. These results suggest that Coro7 functions as an Arp2/3 complex branch disassembly factor implicated in actin-dependent ER-Golgi trafficking.

Ectonucleotide Pyrophosphatase Phosphodiesterase 1 (ENPP1) is a transmembrane glycoprotein enzyme with an extracellular catalytic domain that hydrolyzes ATP into AMP and pyrophosphate (PP i ). The ENPP1 ATPase is the major source of extracellular PP i , a critical physiological regulator of calcium phosphate crystal formation and biomineralization. ENPP1 deficiency lowers systemic PP i levels and induces life-threatening arterial calcifications. Enzyme replacement therapy (ERT) with a soluble ENPP1 biologic restores plasma PP i and eliminates calcification and associated mortality in ongoing clinical trials in patients with ENPP1 deficiency. Despite the significant role of ENPP1 in inhibiting vascular calcification and regulating mammalian biomineralization via extracellular PP i levels, little is known about the molecular mechanism of PP i liberation by ENPP1. Here, we provide a kinetic analysis of the ENPP1 catalytic ATPase cycle. Our analysis shows that ATP cleavage, PP i release, and hydrolysis of the covalent AMP-ENPP1 intermediate are rapid (>1000 sec −1 ) and that AMP product release is slow and rate-limiting. The steady-state Michaelis constant of ATP substrate ( K M,T ) is comparable to physiological serum ATP levels of ∼100 nM, rendering ENPP1 activity sensitive to small changes in serum ATP. AMP binds strongly, with an affinity comparable to K M,T , such that ENPP1 is subject to intrinsic regulatory feedback by AMP under physiological concentrations of ∼100 nM. This product inhibition can attenuate ENPP1 during periods of high PP i liberation, maintaining relatively constant plasma PP i levels. The quantitative parameters of the ENPP1 ATPase cycle provided here allow for predictable outcomes of ENPP1 ERT and provide plausible expectations for other PP i -linked calcification disorders.

Actin is essential for the survival and pathogenicity of the Apicomplexan parasite Toxoplasma gondii , where it plays essential functions in cargo transport, invasion, egress, and organelle inheritance. Recent work has shown that, unlike vertebrate skeletal muscle actin, purified T . gondii actin filaments (TgAct1) can undergo rapid treadmilling, due to large differences in the barbed- and pointed-end critical concentrations, rapid subunit dissociation from filament ends, and a rapid nucleotide exchange rate constant from free monomers. Previous structural analysis suggested that the unique assembly properties of TgAct1 filaments may be a functional consequence of reduced contacts between the DNAse-1–binding loop (D-loop) of a filament subunit and its adjacent, long-axis subunit neighbor. Because the D-loop makes stabilizing interactions between neighboring subunits, it has been implicated in regulating the mechanical properties of actin filaments. In this study, we measured the bending persistence length ( L B ) of TgAct1 filaments and the filament length distribution. We found that despite compromised intersubunit D-loop contacts, TgAct1 filaments have similar bending stiffness and thermodynamic stability as vertebrate actin filaments. Analysis of published cryo-EM image density maps indicates that TgAct1 filaments retain a stabilizing intersubunit salt bridge between E168 and K62 and reveals visible density between Y167 and S61 of adjacent filament subunits, consistent with a conserved cation binding site proximal to the D-loop, as initially identified in vertebrate skeletal muscle actin filaments. These results favor a mechanism in which weak D-loop interactions compromise TgAct1 subunit incorporation at filament ends, while minimally affecting overall subunit interactions within filaments.

Actin filament assembly and the regulation of its mechanical properties are fundamental processes essential for eukaryotic cell function. Residue E167 in vertebrate actins forms an inter-subunit salt bridge with residue K61 of the adjacent subunit. Saccharomyces cerevisiae actin filaments are more flexible than vertebrate filaments and have an alanine at this position (A167). Substitution of this alanine for a glutamic acid (A167E) confers Saccharomyces cerevisiae actin filaments with salt-dependent stiffness similar to vertebrate actins. We developed an optimized cryogenic electron microscopy workflow refining sample preparation and vitrification to obtain near-atomic resolution structures of wild-type and A167E mutant Saccharomyces cerevisiae actin filaments. The difference between these structures allowed us to pinpoint the potential binding site of a filament-associated cation that controls the stiffness of the filaments in vertebrate and A167E Saccharomyces cerevisiae actins. Through an analysis of previously published high-resolution reconstructions of vertebrate actin filaments, along with a newly determined high-resolution vertebrate actin structure in the absence of potassium, we identified a unique peak near residue 167 consistent with the binding of a magnesium ion. Our findings show how magnesium can contribute to filament stiffening by directly bridging actin subunits and allosterically affecting the orientation of the DNase-I binding loop of actin, which plays a regulatory role in modulating actin filament stiffness and interactions with regulatory proteins.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) codes for a type 2 transmembrane glycoprotein which hydrolyzes extracellular phosphoanhydrides into bio-active molecules that regulate, inter alia, ectopic mineralization, bone formation, vascular endothelial proliferation, and the innate immune response. The clinical phenotypes produced by ENPP1 deficiency are disparate, ranging from life-threatening arterial calcifications to cutaneous hypopigmentation. To investigate associations between disease phenotype and enzyme activity we quantified the enzyme velocities of 29 unique ENPP1 pathogenic variants in 41 patients enrolled in an NIH study along with 33 other variants reported in literature. We correlated the relative enzyme velocities with the presenting clinical diagnoses, performing the catalytic velocity measurements simultaneously in triplicate using a high-throughput assay to reduce experimental variation. We found that ENPP1 variants associated with autosomal dominant phenotypes reduced enzyme velocities by 50% or more, whereas variants associated with insulin resistance had non-significant effects on enzyme velocity. In Cole’s disease the catalytic velocities of ENPP1 variants associated with AD forms trended to lower values than those associated with autosomal recessive forms – 8–32% vs. 33% of WT, respectively. Additionally, ENPP1 variants leading to life-threatening vascular calcifications in GACI patients had widely variable enzyme activities, ranging from no significant differences compared to WT to the complete abolishment of enzyme velocity. Finally, disease severity in GACI did not correlate with the mean enzyme velocity of the variants present in affected compound heterozygotes but did correlate with the more severely damaging variant. In summary, correlation of ENPP1 enzyme velocity with disease phenotypes demonstrate that enzyme velocities below 50% of WT levels are likely to occur in the context of autosomal dominant disease (due to a monoallelic variant), and that disease severity in GACI infants correlates with the more severely damaging ENPP1 variant in compound heterozygotes, not the mean velocity of the pathogenic variants present.

In this article the ‘Results’ heading was inadvertently captured as part of the subsequent subheading ‘Cryo-EM structure of a mature Arp2/3 complex branch junction at 2.7 Å resolution’. The original article has been corrected.

Abstract Arp2/3 complex nucleates branched actin filaments for cell and organelle movements. Here we report a 2.7 Å resolution cryo-EM structure of the mature branch junction formed by S. pombe Arp2/3 complex that provides details about interactions with both mother and daughter filaments. We determine a second structure at 3.2 Å resolution with the phosphate analog BeF x bound with ADP to Arp3 and ATP bound to Arp2. In this ADP-BeF x transition state the outer domain of Arp3 is rotated 2° toward the mother filament compared with the ADP state and makes slightly broader contacts with actin in both the mother and daughter filaments. Thus, dissociation of P i from the ADP-P i transition state reduces the interactions of Arp2/3 complex with the actin filaments and may contribute to the lower mechanical stability of mature branch junctions with ADP bound to the Arps. Our structures also reveal that the mother filament in contact with Arp2/3 complex is slightly bent and twisted, consistent with the preference of Arp2/3 complex binding curved actin filaments. The small degree of twisting constrains models of actin filament mechanics.

The cytoskeletal protein actin plays a critical role in the pathogenicity of the intracellular parasite, Toxoplasma gondii, mediating invasion and egress, cargo transport, and organelle inheritance. Advances in live cell imaging have revealed extensive filamentous actin networks in the Apicomplexan parasite, but there are conflicting data regarding the biochemical and biophysical properties of Toxoplasma actin. Here, we imaged the in vitro assembly of individual Toxoplasma actin filaments in real time, showing that native, unstabilized filaments grow tens of microns in length. Unlike skeletal muscle actin, Toxoplasma filaments intrinsically undergo rapid treadmilling due to a high critical concentration, fast monomer dissociation, and rapid nucleotide exchange. Cryo-EM structures of jasplakinolide-stabilized and native (i.e. unstabilized) filaments show an architecture like skeletal actin, with differences in assembly contacts in the D-loop that explain the dynamic nature of the filament, likely a conserved feature of Apicomplexan actin. This work demonstrates that evolutionary changes at assembly interfaces can tune the dynamic properties of actin filaments without disrupting their conserved structure.

Cofilin family proteins have essential roles in remodeling the cytoskeleton through filamentous actin depolymerization and severing. The short, unstructured N-terminal region of cofilin is critical for actin binding and harbors the major site of inhibitory phosphorylation. Atypically for a disordered sequence, the N-terminal region is highly conserved, but specific aspects driving this conservation are unclear. Here, we screen a library of 16,000 human cofilin N-terminal sequence variants for their capacity to support growth in S. cerevisiae in the presence or absence of the upstream regulator LIM kinase. Results from the screen and biochemical analysis of individual variants reveal distinct sequence requirements for actin binding and regulation by LIM kinase. LIM kinase recognition only partly explains sequence constraints on phosphoregulation, which are instead driven to a large extent by the capacity for phosphorylation to inactivate cofilin. We find loose sequence requirements for actin binding and phosphoinhibition, but collectively they restrict the N-terminus to sequences found in natural cofilins. Our results illustrate how a phosphorylation site can balance potentially competing sequence requirements for function and regulation.

The organization of actin filaments (F-actin) into crosslinked networks determines the transmission of mechanical stresses within the cytoskeleton and subsequent changes in cell and tissue shape. Principally mediated by proteins such as α-actinin, F-actin crosslinking increases both network connectivity and rigidity, thereby facilitating stress transmission at low crosslinking yet attenuating transmission at high crosslinker concentration. Here, we engineer a two-dimensional model of the actomyosin cytoskeleton, in which myosin-induced mechanical stresses are controlled by light. We alter the extent of F-actin crosslinking by the introduction of oligomerized cofilin. At pH 6.5, F-actin severing by cofilin is weak, but cofilin bundles and crosslinks filaments. Given its effect of lowering the F-actin bending stiffness, cofilin- crosslinked networks are significantly more flexible and softer in bending than networks crosslinked by α-actinin. Thus, upon local activation of myosin-induced contractile stress, the network bends out-of-plane in contrast to the in-plane compression as observed with networks crosslinked by α-actinin. Here, we demonstrate that local effects on filament mechanics by cofilin introduces novel large-scale network material properties that enable the sculpting of complex shapes in the cell cytoskeleton.

Cooperative ligand binding to linear polymers is fundamental in many scientific disciplines, particularly biological and chemical physics and engineering. Such ligand binding interactions have been widely modeled using infinite one-dimensional (1D) Ising models even in cases where the linear polymers are more complex (e.g. actin filaments and other double-stranded linear polymers). Here, we use sequence-generating and transfer matrix methods to obtain an analytical method for cooperative equilibrium ligand binding to double-stranded Ising lattices. We use this exact solution to evaluate binding properties and features and analyze experimental binding data of cooperative binding of the regulatory protein, cofilin, to actin filaments. This analysis, with additional experimental information about the observed bound cofilin cluster sizes and filament structure, reveals that a bound cofilin promotes cooperative binding to its longitudinal nearest-neighbors but has very modest effects on lateral nearest-neighbors. The bound cofilin cluster sizes calculated from the best fit parameters from the double-stranded model are considerably larger than when calculated with the 1D model, consistent with experimental observations made by electron microscopy and fluorescence imaging. The exact solution obtained and the method for using the solution developed here can be widely used for analysis of variety of multistranded lattice systems.

The shape of cells is the outcome of the balance of inner forces produced by the actomyosin network and the resistive forces produced by cell adhesion to their environment. The specific contributions of contractile, anchoring and friction forces to network deformation rate and orientation are difficult to disentangle in living cells where they influence each other. Here, we reconstituted contractile actomyosin networks in vitro to study specifically the role of the friction forces between the network and its anchoring substrate. To modulate the magnitude and spatial distribution of friction forces, we used glass or lipids surface micropatterning to control the initial shape of the network. We adapted the concentration of Nucleating Promoting Factor on each surface to induce the assembly of actin networks of similar densities and compare the deformation of the network toward the centroid of the pattern shape upon myosin-induced contraction. We found that actin network deformation was faster and more coordinated on lipid bilayers than on glass, showing the resistance of friction to network contraction. To further study the role of the spatial distribution of these friction forces, we designed heterogeneous micropatterns made of glass and lipids. The deformation upon contraction was no longer symmetric but biased toward the region of higher friction. Furthermore, we showed that the pattern of friction could robustly drive network contraction and dominate the contribution of asymmetric distributions of myosins. Therefore, we demonstrate that during contraction, both the active and resistive forces are essential to direct the actin network deformation.

Abstract The cytoskeletal protein actin plays a critical role in the pathogenicity of Toxoplasma gondii , mediating invasion and egress, cargo transport, and organelle inheritance. Advances in live cell imaging have revealed extensive filamentous actin networks in the Apicomplexan parasite, but there is conflicting data regarding the biochemical and biophysical properties of Toxoplasma actin. Here, we imaged the in vitro assembly of individual Toxoplasma actin filaments in real time, showing that native, unstabilized filaments grow tens of microns in length. Unlike skeletal muscle actin, Toxoplasma filaments intrinsically undergo rapid treadmilling due to a high critical concentration, fast monomer dissociation, and rapid nucleotide exchange. Cryo-EM structures of stabilized and unstabilized filaments show an architecture like skeletal actin, with differences in assembly contacts in the D-loop that explain the dynamic nature of the filament, likely a conserved feature of Apicomplexan actin. This work demonstrates that evolutionary changes at assembly interfaces can tune dynamic properties of actin filaments without disrupting their conserved structure.

Summary Cofilin family proteins have essential roles in remodeling the cytoskeleton through filamentous actin depolymerization and severing. The short unstructured N-terminal region of cofilin is critical for actin binding and harbors the major site of inhibitory phosphorylation. Atypically for a disordered sequence, the N-terminal region is highly conserved, but the aspects of cofilin functionality driving this conservation are not clear. Here, we screened a library of 16,000 human cofilin N-terminal sequence variants for their capacity to support growth in S. cerevisiae in the presence or absence of the upstream regulator LIM kinase. Results from the screen and subsequent biochemical analysis of individual variants revealed distinct sequence requirements for actin binding and regulation by LIM kinase. While the presence of a serine, rather than threonine, phosphoacceptor residue was essential for phosphorylation by LIM kinase, the native cofilin N-terminus was otherwise a suboptimal LIM kinase substrate. This circumstance was not due to sequence requirements for actin binding and severing, but rather appeared primarily to maintain the capacity for phosphorylation to inactivate cofilin. Overall, the individual sequence requirements for cofilin function and regulation were remarkably loose when examined separately, but collectively restricted the N-terminus to sequences found in natural cofilins. Our results illustrate how a regulatory phosphorylation site can balance potentially competing sequence requirements for function and regulation.

Cell morphology is faithfully coupled to its identity but the coupling mechanism remains elusive. Using somatic cell reprogramming into pluripotency as a model system, we show that activity of the extracellular signal-regulated kinase (ERK) is tuned by cellular morphomechanic state to direct cell fate. Pluripotent cells and somatic cells reprogramming into pluripotency allocate large amounts of actin into their nucleus, which morphs cells to become taller than 10 μm, a minimal height required for the pluripotent identity. Accumulated nuclear actin binds to TFII-IΔ, an atypical transcription factor that translocates into the nucleus upon signaling. TFII-IΔ also binds to and activates ERK. The binding of TFII-IΔ by nuclear actin reduces ERK activity, in coordination with changes in cell/colony height. The tight coupling between cell height and nuclear actin accumulation necessitates the degree of ERK tuning to be mild. Mild ERK inhibition by chemicals recapitulates the tuning by actin-TFII-IΔ and turns most cells in reprogramming cultures into pluripotency. Thus, we uncover a novel mechanism for how cell morphology couples to its identity via the actin-TFII-IΔ-ERK axis, identifying points of intervention in cell fate manipulation.

Supplementary Fig. S2 from Identification of small-molecule inhibitors of autotaxin that inhibit melanoma cell migration and invasion

Supplementary Fig. S3 from Identification of small-molecule inhibitors of autotaxin that inhibit melanoma cell migration and invasion

Supplementary Fig. S1 from Identification of small-molecule inhibitors of autotaxin that inhibit melanoma cell migration and invasion

<div>Abstract<p>Autotaxin (ATX) is a prometastatic enzyme initially isolated from the conditioned medium of human melanoma cells that stimulates a myriad of biological activities, including angiogenesis and the promotion of cell growth, survival, and differentiation through the production of lysophosphatidic acid (LPA). ATX increases the aggressiveness and invasiveness of transformed cells, and ATX levels directly correlate with tumor stage and grade in several human malignancies. To study the role of ATX in the pathogenesis of malignant melanoma, we developed antibodies and small-molecule inhibitors against recombinant human protein. Immunohistochemistry of paraffin-embedded human tissue shows that ATX levels are markedly increased in human primary and metastatic melanoma relative to benign nevi. Chemical screens identified several small-molecule inhibitors with binding constants ranging from nanomolar to low micromolar. Cell migration and invasion assays with melanoma cell lines show that ATX markedly stimulates melanoma cell migration and invasion, an effect suppressed by ATX inhibitors. The migratory phenotype can be rescued by the addition of the enzymatic product of ATX, LPA, confirming that the observed inhibition is linked to suppression of LPA production by ATX. Chemical analogues of the inhibitors show structure-activity relationships important for ATX inhibition and indicate pathways for their optimization. These studies suggest that ATX is an approachable molecular target for the rational design of chemotherapeutic agents directed against malignant melanoma. [Mol Cancer Ther 2008;7(10):3352–62]</p></div>

<div>Abstract<p>Autotaxin (ATX) is a prometastatic enzyme initially isolated from the conditioned medium of human melanoma cells that stimulates a myriad of biological activities, including angiogenesis and the promotion of cell growth, survival, and differentiation through the production of lysophosphatidic acid (LPA). ATX increases the aggressiveness and invasiveness of transformed cells, and ATX levels directly correlate with tumor stage and grade in several human malignancies. To study the role of ATX in the pathogenesis of malignant melanoma, we developed antibodies and small-molecule inhibitors against recombinant human protein. Immunohistochemistry of paraffin-embedded human tissue shows that ATX levels are markedly increased in human primary and metastatic melanoma relative to benign nevi. Chemical screens identified several small-molecule inhibitors with binding constants ranging from nanomolar to low micromolar. Cell migration and invasion assays with melanoma cell lines show that ATX markedly stimulates melanoma cell migration and invasion, an effect suppressed by ATX inhibitors. The migratory phenotype can be rescued by the addition of the enzymatic product of ATX, LPA, confirming that the observed inhibition is linked to suppression of LPA production by ATX. Chemical analogues of the inhibitors show structure-activity relationships important for ATX inhibition and indicate pathways for their optimization. These studies suggest that ATX is an approachable molecular target for the rational design of chemotherapeutic agents directed against malignant melanoma. [Mol Cancer Ther 2008;7(10):3352–62]</p></div>

Supplementary Fig. S2 from Identification of small-molecule inhibitors of autotaxin that inhibit melanoma cell migration and invasion

Supplementary Fig. S3 from Identification of small-molecule inhibitors of autotaxin that inhibit melanoma cell migration and invasion

Supplementary Fig. S1 from Identification of small-molecule inhibitors of autotaxin that inhibit melanoma cell migration and invasion

Actin cytoskeleton force generation, sensing, and adaptation are dictated by the bending and twisting mechanics of filaments. Here, we use magnetic tweezers and microfluidics to twist and pull individual actin filaments and evaluate their response to applied loads. Twisted filaments bend and dissipate torsional strain by adopting a supercoiled plectoneme. Pulling prevents plectoneme formation, which causes twisted filaments to sever. Analysis over a range of twisting and pulling forces and direct visualization of filament and single subunit twisting fluctuations yield an actin filament torsional persistence length of ~10 µm, similar to the bending persistence length. Filament severing by cofilin is driven by local twist strain at boundaries between bare and decorated segments and is accelerated by low pN pulling forces. This work explains how contractile forces generated by myosin motors accelerate filament severing by cofilin and establishes a role for filament twisting in the regulation of actin filament stability and assembly dynamics.