Christian Hedberg

A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis

The microRNA pathway has been implicated in the regulation of synaptic protein synthesis and ultimately in dendritic spine morphogenesis, a phenomenon associated with long-lasting forms of memory. However, the particular microRNAs (miRNAs) involved are largely unknown. Here we identify specific miRNAs that function at synapses to control dendritic spine structure by performing a functional screen. One of the identified miRNAs, miR-138, is highly enriched in the brain, localized within dendrites and negatively regulates the size of dendritic spines in rat hippocampal neurons. miR-138 controls the expression of acyl protein thioesterase 1 (APT1), an enzyme regulating the palmitoylation status of proteins that are known to function at the synapse, including the α13 subunits of G proteins (Gα13). RNA-interference-mediated knockdown of APT1 and the expression of membrane-localized Gα13 both suppress spine enlargement caused by inhibition of miR-138, suggesting that APT1-regulated depalmitoylation of Gα13 might be an important downstream event of miR-138 function. Our results uncover a previously unknown miRNA-dependent mechanism in neurons and demonstrate a previously unrecognized complexity of miRNA-dependent control of dendritic spine morphogenesis.

Target Identification for Small Bioactive Molecules: Finding the Needle in the Haystack

Identification and confirmation of bioactive small-molecule targets is a crucial, often decisive step both in academic and pharmaceutical research. Through the development and availability of several new experimental techniques, target identification is, in principle, feasible, and the number of successful examples steadily grows. However, a generic methodology that can successfully be applied in the majority of the cases has not yet been established. Herein we summarize current methods for target identification of small molecules, primarily for a chemistry audience but also the biological community, for example, the chemist or biologist attempting to identify the target of a given bioactive compound. We describe the most frequently employed experimental approaches for target identification and provide several representative examples illustrating the state-of-the-art. Among the techniques currently available, protein affinity isolation using suitable small-molecule probes (pulldown) and subsequent mass spectrometric analysis of the isolated proteins appears to be most powerful and most frequently applied. To provide guidance for rapid entry into the field and based on our own experience we propose a typical workflow for target identification, which centers on the application of chemical proteomics as the key step to generate hypotheses for potential target proteins.

Small-molecule inhibition of APT1 affects Ras localization and signaling

Cycles of depalmitoylation and repalmitoylation critically control the steady-state localization and function of various peripheral membrane proteins, such as Ras proto-oncogene products. Interference with acylation using small molecules is a strategy to modulate cellular localization--and thereby unregulated signaling--caused by palmitoylated Ras proteins. We present the knowledge-based development and characterization of a potent inhibitor of acyl protein thioesterase 1 (APT1), a bona fide depalmitoylating enzyme that is, so far, poorly characterized in cells. The inhibitor, palmostatin B, perturbs the cellular acylation cycle at the level of depalmitoylation and thereby causes a loss of the precise steady-state localization of palmitoylated Ras. As a consequence, palmostatin B induces partial phenotypic reversion in oncogenic HRasG12V-transformed fibroblasts. We identify APT1 as one of the thioesterases in the acylation cycle and show that this protein is a cellular target of the inhibitor.

The GDI-like solubilizing factor PDEδ sustains the spatial organization and signalling of Ras family proteins

We identify a role for the GDI-like solubilizing factor (GSF) PDEδ in modulating signalling through Ras family G proteins by sustaining their dynamic distribution in cellular membranes. We show that the GDI-like pocket of PDEδ binds and solubilizes farnesylated Ras proteins, thereby enhancing their diffusion in the cytoplasm. This mechanism allows more effective trapping of depalmitoylated Ras proteins at the Golgi and polycationic Ras proteins at the plasma membrane to counter the entropic tendency to distribute these proteins over all intracellular membranes. Thus, PDEδ activity augments K/Hras signalling by enriching Ras at the plasma membrane; conversely, PDEδ down-modulation randomizes Ras distributions to all membranes in the cell and suppresses regulated signalling through wild-type Ras and also constitutive oncogenic Ras signalling in cancer cells. Our findings link the activity of PDEδ in determining Ras protein topography to Ras-dependent signalling.

Identification of Acyl Protein Thioesterases 1 and 2 as the Cellular Targets of the Ras-Signaling Modulators Palmostatin B and M

Finding the target: activity-based proteomic profiling probes based on the depalmitoylation inhibitors palmostatin B and M have been synthesized and were found to target acyl protein thioesterase 1 (APT1) and 2 (APT2) in cells.

Choline-releasing glycerophosphodiesterase EDI3 drives tumor cell migration and metastasis

Metastasis from primary tumors remains a major problem for tumor therapy. In the search for markers of metastasis and more effective therapies, the tumor metabolome is relevant because of its importance to the malignant phenotype and metastatic capacity of tumor cells. Altered choline metabolism is a hallmark of cancer. More specifically, a decreased glycerophosphocholine (GPC) to phosphocholine (PC) ratio was reported in breast, ovarian, and prostate cancers. Improved strategies to exploit this altered choline metabolism are therefore required. However, the critical enzyme cleaving GPC to produce choline, the initial step in the pathway controlling the GPC/PC ratio, remained unknown. In the present work, we have identified the enzyme, here named EDI3 (endometrial differential 3). Purified recombinant EDI3 protein cleaves GPC to form glycerol-3-phosphate and choline. Silencing EDI3 in MCF-7 cells decreased this enzymatic activity, increased the intracellular GPC/PC ratio, and decreased downstream lipid metabolites. Downregulating EDI3 activity inhibited cell migration via disruption of the PKCα signaling pathway, with stable overexpression of EDI3 showing the opposite effect. EDI3 was originally identified in our screening study comparing mRNA levels in metastasizing and nonmetastasizing endometrial carcinomas. Both Kaplan–Meier and multivariate analyses revealed a negative association between high EDI3 expression and relapse-free survival time in both endometrial (P < 0.001) and ovarian (P = 0.029) cancers. Overall, we have identified EDI3, a key enzyme controlling GPC and choline metabolism. Because inhibition of EDI3 activity corrects the GPC/PC ratio and decreases the migration capacity of tumor cells, it represents a possible target for therapeutic intervention.

Development of Highly Potent Inhibitors of the Ras‐Targeting Human Acyl Protein Thioesterases Based on Substrate Similarity Design

A matter of common sense: a common recognition motif consisting of a negatively charged group five to six bonds away (red) from the (thio)ester functionality (green) and a positively charged tail group ten to twelve bonds away (blue) was identified in two native acyl protein thioesterase 1 (APT1) substrates. This similarity led to the design of potent inhibitors of the Ras-depalmitoylating enzyme APT1.

Inhibiting the palmitoylation/depalmitoylation cycle selectively reduces the growth of hematopoietic cells expressing oncogenic Nras

The palmitoylation/depalmitoylation cycle of posttranslational processing is a potential therapeutic target for selectively inhibiting the growth of hematologic cancers with somatic NRAS mutations. To investigate this question at the single-cell level, we constructed murine stem cell virus vectors and assayed the growth of myeloid progenitors. Whereas cells expressing oncogenic N-Ras(G12D) formed cytokine-independent colonies and were hypersensitive to GM-CSF, mutations within the N-Ras hypervariable region induced N-Ras mislocalization and attenuated aberrant progenitor growth. Exposing transduced hematopoietic cells and bone marrow from Nras and Kras mutant mice to the acyl protein thioesterase inhibitor palmostatin B had similar effects on protein localization and colony growth. Importantly, palmostatin B-mediated inhibition was selective for Nras mutant cells, and we mapped this activity to the hypervariable region. These data support the clinical development of depalmitoylation inhibitors as a novel class of rational therapeutics in hematologic malignancies with NRAS mutations.

The mechanism of caseinolytic protease (ClpP) inhibition.

Maintaining homeostasis at the protein level is an important prerequisite for cellular viability for which prokaryotes exhibit several proteolytic machineries, including ClpXP. In 2008, we reported the first small-molecule inhibitor for the proteolytic subunit ClpP and demonstrated that the inhibition of the enzyme in living bacteria significantly attenuates their capability to produce virulence factors, such as life-threatening toxins. Although ClpP has been extensively studied by biochemical and structural methods, the mechanism of small-molecule inhibition of this enzyme is currently poorly understood. Because chemical inhibition may lead to a novel antibacterial therapy, it is important to systematically analyze the binding site, the mechanism of inhibition, the stereogenic preference of the enzyme for inhibitors, the chemical space of putative inhibitors, and how other members of the ClpP family can be inhibited. One major step towards these aims was accomplished by the recently solved crystal structure of homotetradecameric ClpP from Staphylococcus aureus (SaClpP) in its active conformation. With the structural data at hand, we herein report an in-depth mechanistic analysis of S. aureus ClpP inhibition by b-lactones. A screen of a focused library of enantiopure b-lactones revealed the S,Sstereopreference of the protease, which was rationalized by molecular docking. Docking experiments also gave insight into a hitherto unnoted deep hydrophobic pocket next to the active site that accommodates b-lactone substituents in the aposition to the carbonyl group. The binding hypothesis was verified by binding studies with model compounds, detailed kinetic analysis, and protein mutagenesis studies. Furthermore, the replacement of the b-lactone core by other scaffolds resulted in the loss of inhibitory potency, thereby highlighting the importance of a b-lactone moiety for mechanism-based ClpP inhibition. Taken together, these results open intriguing perspectives in the mechanistic understanding of ClpP inhibition and provide direction for the design of potent and pharmacologically optimized inhibitors. We started by testing 22 enantiopure trans-substituted blactones 1–22 for ClpP inhibition (Supporting Information, Figure S1 A). These molecules share a high structural similarity with our previous b-lactone candidates. They feature a decyl chain as R substituent and structural variations in chain lengths as well as in functional groups at the R position (Figure 1A). For all of the compounds, both trans-configured enantiomers (that is, R,R and S,S) were tested for inhibition of recombinantly expressed SaClpP in an assay monitoring the cleavage of a fluorogenic substrate. Almost all of the compounds inhibited SaClpP at 100 mm concentration (100-fold excess over enzyme) after 15 min incubation at 32 8C (Supporting Information, Figure S1 A). By lowering the inhibitor concentration to 10 mm, we were able to differentiate the compounds tested. While most S,Sconfigured lactones lead to inhibition below 10% residual activity, R,R-configured lactones showed essentially no inhibition (Figure 1 B). Incubation of SaClpP with 1.3-fold molar excess of the most potent compound, 2, led to modification of all 14 subunits as revealed by intact-protein mass spectrometry (Figure 1 C). To investigate if the potent in vitro inhibition correlates with ClpP binding in living cells we applied the structurally related alkynylated probe 23 with S,S-configuration for an

The Autodepalmitoylating Activity of APT Maintains the Spatial Organization of Palmitoylated Membrane Proteins

The localization and signaling of S-palmitoylated peripheral membrane proteins is sustained by an acylation cycle in which acyl protein thioesterases (APTs) depalmitoylate mislocalized palmitoylated proteins on endomembranes. However, the APTs are themselves reversibly S-palmitoylated, which localizes thioesterase activity to the site of the antagonistc palmitoylation activity on the Golgi. Here, we resolve this conundrum by showing that palmitoylation of APTs is labile due to autodepalmitoylation, creating two interconverting thioesterase pools: palmitoylated APT on the Golgi and depalmitoylated APT in the cytoplasm, with distinct functionality. By imaging APT-substrate catalytic intermediates, we show that it is the depalmitoylated soluble APT pool that depalmitoylates substrates on all membranes in the cell, thereby establishing its function as release factor of mislocalized palmitoylated proteins in the acylation cycle. The autodepalmitoylating activity on the Golgi constitutes a homeostatic regulation mechanism of APT levels at the Golgi that ensures robust partitioning of APT substrates between the plasma membrane and the Golgi.