Characterization of the catalytic flexible loop in the dihydroorotase domain of the human multi-enzymatic protein CAD.

The dihydroorotase (DHOase) domain of the multifunctional protein carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (CAD) catalyzes the third step in the de novo biosynthesis of pyrimidine nucleotides in animals. The crystal structure of the DHOase domain of human CAD (huDHOase) revealed that, despite evolutionary divergence, its active site components are highly conserved with those in bacterial DHOases, encoded as monofunctional enzymes. An important element for catalysis, conserved from Escherichia coli to humans, is a flexible loop that closes as a lid over the active site. Here, we combined mutagenic, structural, biochemical, and molecular dynamics analyses to characterize the function of the flexible loop in the activity of CAD's DHOase domain. A huDHOase chimera bearing the E. coli DHOase flexible loop was inactive, suggesting the presence of distinctive elements in the flexible loop of huDHOase that cannot be replaced by the bacterial sequence. We pinpointed Phe-1563, a residue absolutely conserved at the tip of the flexible loop in CAD's DHOase domain, as a critical element for the conformational equilibrium between the two catalytic states of the protein. Substitutions of Phe-1563 with Ala, Leu, or Thr prevented the closure of the flexible loop and inactivated the protein, whereas substitution with Tyr enhanced the interactions of the loop in the closed position and reduced fluctuations and the reaction rate. Our results confirm the importance of the flexible loop in CAD's DHOase domain and explain the key role of Phe-1563 in configuring the active site and in promoting substrate strain and catalysis.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the aspartate transcarbamoylase domain of human CAD.

Aspartate transcarbamoylase (ATCase) catalyzes the synthesis of N-carbamoyl-L-aspartate from carbamoyl phosphate and aspartate in the second step of the de novo biosynthesis of pyrimidines. In prokaryotes, the first three activities of the pathway, namely carbamoyl phosphate synthetase (CPSase), ATCase and dihydroorotase (DHOase), are encoded as distinct proteins that function independently or in noncovalent association. In animals, CPSase, ATCase and DHOase are part of a 243 kDa multifunctional polypeptide named CAD. Up-regulation of CAD is essential for normal and tumour cell proliferation. Although the structures of numerous prokaryotic ATCases have been determined, there is no structural information about any eukaryotic ATCase. In fact, the only detailed structural information about CAD is that it self-assembles into hexamers and trimers through interactions of the ATCase domains. Here, the expression, purification and crystallization of the ATCase domain of human CAD is reported. The recombinant protein, which was expressed in bacteria and purified with good yield, formed homotrimers in solution. Crystallization experiments both in the absence and in the presence of the inhibitor PALA yielded small crystals that diffracted X-rays to 2.1 Å resolution using synchrotron radiation. The crystals appeared to belong to the hexagonal space group P6(3)22, and Matthews coefficient calculation indicated the presence of one ATCase subunit per asymmetric unit, with a solvent content of 48%. However, analysis of the intensity statistics suggests a special case of the P21 lattice with pseudo-symmetry and possibly twinning.

Phospho-caveolin-1 mediates integrin-regulated membrane domain internalization.

Growth of normal cells is anchorage dependent because signalling through multiple pathways including Erk, phosphatidylinositol-3-OH kinase (PI(3)K) and Rac requires integrin-mediated cell adhesion. Components of these pathways localize to low-density, cholesterol-rich domains in the plasma membrane named 'lipid rafts' or 'cholesterol-enriched membrane microdomains' (CEMM). We previously reported that integrin-mediated adhesion regulates CEMM transport such that cell detachment from the extracellular matrix triggers CEMM internalization and clearance from the plasma membrane. We now report that this internalization is mediated by dynamin-2 and caveolin-1. Internalization requires phosphorylation of caveolin-1 on Tyr 14. A shift in localization of phospho-caveolin-1 from focal adhesions to caveolae induces CEMM internalization upon cell detachment, which mediates inhibition of Erk, PI(3)K and Rac. These data define a novel molecular mechanism for growth and tumour suppression by caveolin-1.

Caveolin-1 regulates cell polarization and directional migration through Src kinase and Rho GTPases.

Development, angiogenesis, wound healing, and metastasis all involve the movement of cells in response to changes in the extracellular environment. To determine whether caveolin-1 plays a role in cell migration, we have used fibroblasts from knockout mice. Caveolin-1-deficient cells lose normal cell polarity, exhibit impaired wound healing, and have decreased Rho and increased Rac and Cdc42 GTPase activities. Directional persistency of migration is lost, and the cells show an impaired response to external directional stimuli. Both Src inactivation and p190RhoGAP knockdown restore the wild-type phenotype to caveolin-1-deficient cells, suggesting that caveolin-1 stimulates normal Rho GTP loading through inactivation of the Src-p190RhoGAP pathway. These findings highlight the importance of caveolin-1 in the establishment of cell polarity during directional migration through coordination of the signaling of Src kinase and Rho GTPases.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the dihydroorotase domain of human CAD.

CAD is a 243 kDa eukaryotic multifunctional polypeptide that catalyzes the first three reactions of de novo pyrimidine biosynthesis: glutamine-dependent carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase (DHO). In prokaryotes, these activities are associated with monofunctional proteins, for which crystal structures are available. However, there is no detailed structural information on the full-length CAD protein or any of its functional domains apart from that it associates to form a homohexamer of ∼1.5 MDa. Here, the expression, purification and crystallization of the DHO domain of human CAD are reported. The DHO domain forms homodimers in solution. Crystallization experiments yielded small crystals that were suitable for X-ray diffraction studies. A diffraction data set was collected to 1.75 Šresolution using synchrotron radiation at the SLS, Villigen, Switzerland. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a=82.1, b=159.3, c=61.5 Å. The Matthews coefficient calculation suggested the presence of one protein molecule per asymmetric unit, with a solvent content of 48%.

Caveolae couple mechanical stress to integrin recycling and activation.

Cells are subjected to multiple mechanical inputs throughout their lives. Their ability to detect these environmental cues is called mechanosensing, a process in which integrins play an important role. During cellular mechanosensing, plasma membrane (PM) tension is adjusted to mechanical stress through the buffering action of caveolae; however, little is known about the role of caveolae in early integrin mechanosensing regulation. Here, we show that Cav1KO fibroblasts increase adhesion to FN-coated beads when pulled with magnetic tweezers, as compared to wild type fibroblasts. This phenotype is Rho-independent and mainly derived from increased active ß1-integrin content on the surface of Cav1KO fibroblasts. Florescence recovery after photobleaching analysis and endocytosis/recycling assays revealed that active ß1-integrin is mostly endocytosed through the clathrin independent carrier/glycosylphosphatidyl inositol (GPI)-enriched endocytic compartment pathway and is more rapidly recycled to the PM in Cav1KO fibroblasts, in a Rab4 and PM tension-dependent manner. Moreover, the threshold for PM tension-driven ß1-integrin activation is lower in Cav1KO mouse embryonic fibroblasts (MEFs) than in wild type MEFs, through a mechanism dependent on talin activity. Our findings suggest that caveolae couple mechanical stress to integrin cycling and activation, thereby regulating the early steps of the cellular mechanosensing response.

Cells can physically sense their immediate environment by pulling and pushing through integrins, a type of proteins which connects the inside and outside of a cell by being studded through the cellular membrane. This sensing role can only be performed when integrins are in an active state. Two main mechanisms regulate the relative amount of active integrins: one controls the activation of the proteins already at the cell surface; the other, known as recycling, impacts how many new integrins are delivered to the membrane. Both processes are affected by changes in cell membrane tension, which is itself controlled by dimples (or 'caveolae' ­ little caves in Latin) present in the cell surface. Caveolae limit acute changes in tension by taking in (pinching off the dimples) or releasing (dimples flattening) segments of the membrane. However, it is still unclear how integrins and caveolae mechanically interact to regulate the ability for a cell to read its environment. To understand this process, Lolo et al. focused on mouse cells genetically manipulated to not build caveolae on their surfaces, and which cannot properly sense mechanical changes in their surroundings. These were exposed to beads covered in an integrin-binding protein and manipulated using magnetic tweezers. The manipulation showed that mutated cells bound to the beads more strongly than non-modified cells, indicating that they had more active integrins on their surface. This change was due to both an accelerated recycling mechanism (which resulted in more integrin being brought at the surface) and an increase in integrin activation (which was triggered by a higher membrane tension). Caveolae therefore couple mechanical inputs to integrin recycling and activation. Healthy tissues rely on cells correctly sensing physical changes in their environment so they can mount an appropriate response. This ability, for example, is altered in cancerous cells which start to form tumours. The findings by Lolo et al. bring together physics and biology to provide new insights into the potential mechanisms causing such impairments.

Caveolin-1 in cell polarization and directional migration.

Migration is a complex process in which cells move in a given direction either in response to changes in the extracellular environment or as a consequence of an intrinsic propensity for directional movement. Migration plays key roles in many physiological and pathological processes, including development, angiogenesis, tissue regeneration and metastasis. An important role in migration is played by caveolin-1 and caveolae. Caveolae compartmentalize intracellular signalling pathways to orchestrate cell migration. Caveolin-1 presents a polarized distribution in migrating cells and is linked to the cytoskeleton, and changes in its expression modulate migration. Although there are some discrepancies regarding the regulatory effect of caveolin-1, most studies show that it promotes cell movement and polarity. The importance of caveolin-1 has recently been reinforced by studies with Cav1(-/-) cells, which indicate that it establishes polarity during directional migration by coordinating Src kinase and Rho GTPase signalling.

Structural Insight into the Core of CAD, the Multifunctional Protein Leading De Novo Pyrimidine Biosynthesis.

CAD, the multifunctional protein initiating and controlling de novo biosynthesis of pyrimidines in animals, self-assembles into ∼1.5 MDa hexamers. The structures of the dihydroorotase (DHO) and aspartate transcarbamoylase (ATC) domains of human CAD have been previously determined, but we lack information on how these domains associate and interact with the rest of CAD forming a multienzymatic unit. Here, we prove that a construct covering human DHO and ATC oligomerizes as a dimer of trimers and that this arrangement is conserved in CAD-like from fungi, which holds an inactive DHO-like domain. The crystal structures of the ATC trimer and DHO-like dimer from the fungus Chaetomium thermophilum confirm the similarity with the human CAD homologs. These results demonstrate that, despite being inactive, the fungal DHO-like domain has a conserved structural function. We propose a model that sets the DHO and ATC complex as the central element in the architecture of CAD.

Structure and Functional Characterization of Human Aspartate Transcarbamoylase, the Target of the Anti-tumoral Drug PALA.

CAD, the multienzymatic protein that initiates and controls de novo synthesis of pyrimidines in animals, associates through its aspartate transcarbamoylase (ATCase) domain into particles of 1.5 MDa. Despite numerous structures of prokaryotic ATCases, we lack structural information on the ATCase domain of CAD. Here, we report the structure and functional characterization of human ATCase, confirming the overall similarity with bacterial homologs. Unexpectedly, human ATCase exhibits cooperativity effects that reduce the affinity for the anti-tumoral drug PALA. Combining structural, mutagenic, and biochemical analysis, we identified key elements for the necessary regulation and transmission of conformational changes leading to cooperativity between subunits. Mutation of one of these elements, R2024, was recently found to cause the first non-lethal CAD deficit. We reproduced this mutation in human ATCase and measured its effect, demonstrating that this arginine is part of a molecular switch that regulates the equilibrium between low- and high-affinity states for the ligands.

Structure, functional characterization, and evolution of the dihydroorotase domain of human CAD.

Upregulation of CAD, the multifunctional protein that initiates and controls the de novo biosynthesis of pyrimidines in animals, is essential for cell proliferation. Deciphering the architecture and functioning of CAD is of interest for its potential usage as an antitumoral target. However, there is no detailed structural information about CAD other than that it self-assembles into hexamers of ∼1.5 MDa. Here we report the crystal structure and functional characterization of the dihydroorotase domain of human CAD. Contradicting all assumptions, the structure reveals an active site enclosed by a flexible loop with two Zn²âº ions bridged by a carboxylated lysine and a third Zn coordinating a rare histidinate ion. Site-directed mutagenesis and functional assays prove the involvement of the Zn and flexible loop in catalysis. Comparison with homologous bacterial enzymes supports a reclassification of the DHOase family and provides strong evidence against current models of the architecture of CAD.

Mechanotransduction and YAP-dependent matrix remodelling is required for the generation and maintenance of cancer-associated fibroblasts.

To learn more about cancer-associated fibroblasts (CAFs), we have isolated fibroblasts from different stages of breast cancer progression and analysed their function and gene expression. These analyses reveal that activation of the YAP transcription factor is a signature feature of CAFs. YAP function is required for CAFs to promote matrix stiffening, cancer cell invasion and angiogenesis. Remodelling of the ECM and promotion of cancer cell invasion requires the actomyosin cytoskeleton. YAP regulates the expression of several cytoskeletal regulators, including ANLN and DIAPH3, and controls the protein levels of MYL9 (also known as MLC2). Matrix stiffening further enhances YAP activation, thus establishing a feed-forward self-reinforcing loop that helps to maintain the CAF phenotype. Actomyosin contractility and Src function are required for YAP activation by stiff matrices. Further, transient ROCK inhibition is able to disrupt the feed-forward loop, leading to a long-lasting reversion of the CAF phenotype.

MT1-MMP is required for myeloid cell fusion via regulation of Rac1 signaling.

Cell fusion is essential for fertilization, myotube formation, and inflammation. Macrophages fuse under various circumstances, but the molecular signals involved in the distinct steps of their fusion are not fully characterized. Using null mice and derived cells, we show that the protease MT1-MMP is necessary for macrophage fusion during osteoclast and giant-cell formation in vitro and in vivo. Specifically, MT1-MMP is required for lamellipodia formation and for proper cell morphology and motility of bone marrow myeloid progenitors prior to membrane fusion. These functions of MT1-MMP do not depend on MT1-MMP catalytic activity or downstream pro-MMP-2 activation. Instead, MT1-MMP null cells show a decreased Rac1 activity and reduced membrane targeting of Rac1 and the adaptor protein p130Cas. Retroviral rescue experiments and protein binding assays delineate a signaling pathway in which MT1-MMP, via its cytosolic tail, contributes to macrophage migration and fusion by regulating Rac1 activity through an association with p130Cas.