Thomas J. Wandless

PI(3,4,5)P3 and PI(4,5)P2 Lipids Target Proteins with Polybasic Clusters to the Plasma Membrane

Many signaling, cytoskeletal, and transport proteins have to be localized to the plasma membrane (PM) in order to carry out their function. We surveyed PM-targeting mechanisms by imaging the subcellular localization of 125 fluorescent protein–conjugated Ras, Rab, Arf, and Rho proteins. Out of 48 proteins that were PM-localized, 37 contained clusters of positively charged amino acids. To test whether these polybasic clusters bind negatively charged phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] lipids, we developed a chemical phosphatase activation method to deplete PM PI(4,5)P2. Unexpectedly, proteins with polybasic clusters dissociated from the PM only when both PI(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] were depleted, arguing that both lipid second messengers jointly regulate PM targeting.

A Rapid, Reversible, and Tunable Method to Regulate Protein Function in Living Cells Using Synthetic Small Molecules

Rapid and reversible methods for perturbing the function of specific proteins are desirable tools for probing complex biological systems. We have developed a general technique to regulate the stability of specific proteins in mammalian cells using cell-permeable, synthetic molecules. We engineered mutants of the human FKBP12 protein that are rapidly and constitutively degraded when expressed in mammalian cells, and this instability is conferred to other proteins fused to these destabilizing domains. Addition of a synthetic ligand that binds to the destabilizing domains shields them from degradation, allowing fused proteins to perform their cellular functions. Genetic fusion of the destabilizing domain to a gene of interest ensures specificity, and the attendant small-molecule control confers speed, reversibility, and dose-dependence to this method. This general strategy for regulating protein stability should enable conditional perturbation of specific proteins with unprecedented control in a variety of experimental settings.

Probing immunosuppressant action with a nonnatural immunophilin ligand.

The immunosuppressants FK506 and rapamycin bind to the same immunophilin, FK506 binding protein (FKBP), and inhibit distinct signal transduction pathways in T lymphocytes. A nonnatural immunophilin ligand, 506BD, which contains only the common structural elements of FK506 and rapamycin, was synthesized and found to be a high-affinity ligand of FKBP and a potent inhibitor of FKBP rotamase activity. Whereas 506BD does not interfere with T cell activation, it does block the immunosuppressive effects of both FK506 and rapamycin. Thus, the common immunophilin binding element of these immunosuppressants, which is responsible for rotamase inhibition, is fused to different effector elements, resulting in the inhibition of different signaling pathways. Inhibition of rotamase activity is an insufficient requirement for mediating these effects.

An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways

We made substantial advances in the implementation of a rapamycin-triggered heterodimerization strategy. Using molecular engineering of different targeting and enzymatic fusion constructs and a new rapamycin analog, Rho GTPases were directly activated or inactivated on a timescale of seconds, which was followed by pronounced cell morphological changes. As signaling processes often occur within minutes, such rapid perturbations provide a powerful tool to investigate the role, selectivity and timing of Rho GTPase–mediated signaling processes.

Solution structure of FKBP, a rotamase enzyme and receptor for FK506 and rapamycin.

Immunophilins, when complexed to immunosuppressive ligands, appear to inhibit signal transduction pathways that result in exocytosis and transcription. The solution structure of one of these, the human FK506 and rapamycin binding protein (FKBP), has been determined by nuclear magnetic resonance (NMR). FKBP has a previously unobserved antiparallel beta-sheet folding topology that results in a novel loop crossing and produces a large cavity lined by a conserved array of aromatic residues; this cavity serves as the rotamase active site and drug-binding pocket. There are other significant structural features (such as a protruding positively charged loop and an apparently flexible loop) that may be involved in the biological activity of FKBP.

A plant-like kinase in Plasmodium falciparum regulates parasite egress from erythrocytes.

Its a Knockout The malaria parasite is one of the most important pathogens of humans. Increasing drug-resistance is an imminent public health disaster, and we urgently need to find new drugs. The recently acquired malarial genomes provide a plethora of targets. However, due to the genetic intractability of the parasite, it has been difficult to identify essential genes in the clinically relevant blood-stage of the parasite. Dvorin et al. (p. 910) investigated the function of a Plasmodium falciparum plant-like calcium-dependent protein kinase, PfCDPK5, which is expressed in the invasive blood-stage forms of the parasite. A system for conditional protein expression allowed the production of a functional knockout in the bloodstream stage of the parasite. PfCDPK5 was required for parasite egress from the human host erythrocyte, an essential step in the parasite life cycle. A calcium-dependent protein kinase is essential for blood-stage proliferation of the human malaria parasite. Clinical malaria is associated with the proliferation of Plasmodium parasites in human erythrocytes. The coordinated processes of parasite egress from and invasion into erythrocytes are rapid and tightly regulated. We have found that the plant-like calcium-dependent protein kinase PfCDPK5, which is expressed in invasive merozoite forms of Plasmodium falciparum, was critical for egress. Parasites deficient in PfCDPK5 arrested as mature schizonts with intact membranes, despite normal maturation of egress proteases and invasion ligands. Merozoites physically released from stalled schizonts were capable of invading new erythrocytes, separating the pathways of egress and invasion. The arrest was downstream of cyclic guanosine monophosphate–dependent protein kinase (PfPKG) function and independent of protease processing. Thus, PfCDPK5 plays an essential role during the blood stage of malaria replication.

SIK1 is a class II HDAC kinase that promotes survival of skeletal myocytes

During physical exercise, increases in motor neuron activity stimulate the expression of muscle-specific genes through the myocyte enhancer factor 2 (MEF2) family of transcription factors. Elevations in intracellular calcium increase MEF2 activity via the phosphorylation-dependent inactivation of class II histone deacetylases (HDACs). In studies to determine the role of the cAMP responsive element binding protein (CREB) in skeletal muscle, we found that mice expressing a dominant-negative CREB transgene (M-ACREB mice) exhibited a dystrophic phenotype along with reduced MEF2 activity. Class II HDAC phosphorylation was decreased in M-ACREB myofibers due to a reduction in amounts of Snf1lk (encoding salt inducible kinase, SIK1), a CREB target gene that functions as a class II HDAC kinase. Inhibiting class II HDAC activity either by viral expression of Snf1lk or by the administration of a small molecule antagonist improved the dystrophic phenotype in M-ACREB mice, pointing to an important role for the SIK1-HDAC pathway in regulating muscle function.

FK506 activates BMPR2, rescues endothelial dysfunction, and reverses pulmonary hypertension

Dysfunctional bone morphogenetic protein receptor-2 (BMPR2) signaling is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We used a transcriptional high-throughput luciferase reporter assay to screen 3,756 FDA-approved drugs and bioactive compounds for induction of BMPR2 signaling. The best response was achieved with FK506 (tacrolimus), via a dual mechanism of action as a calcineurin inhibitor that also binds FK-binding protein-12 (FKBP12), a repressor of BMP signaling. FK506 released FKBP12 from type I receptors activin receptor-like kinase 1 (ALK1), ALK2, and ALK3 and activated downstream SMAD1/5 and MAPK signaling and ID1 gene regulation in a manner superior to the calcineurin inhibitor cyclosporine and the FKBP12 ligand rapamycin. In pulmonary artery endothelial cells (ECs) from patients with idiopathic PAH, low-dose FK506 reversed dysfunctional BMPR2 signaling. In mice with conditional Bmpr2 deletion in ECs, low-dose FK506 prevented exaggerated chronic hypoxic PAH associated with induction of EC targets of BMP signaling, such as apelin. Low-dose FK506 also reversed severe PAH in rats with medial hypertrophy following monocrotaline and in rats with neointima formation following VEGF receptor blockade and chronic hypoxia. Our studies indicate that low-dose FK506 could be useful in the treatment of PAH.

Rapid control of protein level in the apicomplexan Toxoplasma gondii

Analysis of gene function in apicomplexan parasites is limited by the absence of reverse genetic tools that allow easy and rapid modulation of protein levels. The fusion of a ligand-controlled destabilization domain (ddFKBP) to a protein of interest enables rapid and reversible protein stabilization in T. gondii. This allows an efficient functional analysis of proteins that have a dual role during host cell invasion and/or intracellular growth of the parasite.

A general chemical method to regulate protein stability in the mammalian central nervous system

The ability to make specific perturbations to biological molecules in a cell or organism is a central experimental strategy in modern research biology. We have developed a general technique in which the stability of a specific protein is regulated by a cell-permeable small molecule. Mutants of the Escherichia coli dihydrofolate reductase (ecDHFR) were engineered to be degraded, and, when this destabilizing domain is fused to a protein of interest, its instability is conferred to the fused protein resulting in rapid degradation of the entire fusion protein. A small-molecule ligand trimethoprim (TMP) stabilizes the destabilizing domain in a rapid, reversible, and dose-dependent manner, and protein levels in the absence of TMP are barely detectable. The ability of TMP to cross the blood-brain barrier enables the tunable regulation of proteins expressed in the mammalian central nervous system.