Novel beta-glucocerebrosidase chaperone compounds identified from cell-based screening reduce pathologically accumulated glucosylsphingosine in iPS-derived neuronal cells.

The beta-glucocerebrosidase (GBA1) gene encodes the lysosomal beta-glucocerebrosidase (GCase) that metabolizes the lipids glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). Biallelic loss-of-function mutations in GBA1 such as L444P cause Gaucher disease (GD), which is the most prevalent lysosomal storage disease and is histopathologically characterized by abnormal accumulation of the GCase substrates GlcCer and GlcSph. GD with neurological symptoms is associated with severe mutations in the GBA1 gene, most of which cause impairment in the process of GCase trafficking to lysosomes. Given that recombinant GCase protein cannot cross the blood-brain barrier due to its high molecular weight, it is invaluable to develop a brain-penetrant small-molecule pharmacological chaperone as a viable therapeutic strategy to boost GCase activity in the central nervous system. Despite considerable efforts to screen potent GCase activators/chaperones, cell-free assays using recombinant GCase protein have yielded compounds with only marginal efficacy and micromolar EC50 that would not have sufficient clinical efficacy or an acceptable safety margin. Therefore, we utilized a fluorescence-labeled GCase suicide inhibitor, MDW933, to directly monitor lysosomal GCase activity and performed a cell-based screening in fibroblasts from a GD patient with homozygotic L444P mutations. Here, we identified novel compounds that increase the fluorescence signal from labeled GCase with L444P mutations in a dose-dependent manner. Secondary assays using an artificial cell-permeable lysosomal GCase substrate also demonstrated that the identified compounds augment lysosomal GCase L444P in the fibroblast. Moreover, those compounds increased the total GCase L444P protein levels, suggesting the pharmacological chaperone-like mechanism of action. To further elucidate the effect of the compounds on the endogenous GCase substrate GlcSph, we generated iPSC-derived dopaminergic neurons with a GBA1 L444P mutation that exhibit GlcSph accumulation in vitro. Importantly, the identified compounds reduce GlcSph in iPSC-derived dopaminergic neurons with a GBA1 L444P mutation, indicating that the increase in lysosomal GCase resulting from application of the compounds leads to the clearance of pathologically-accumulated GlcSph. Together, our findings pave the way for developing potent and efficacious GCase chaperone compounds as a potential therapeutic approach for neurological GD.

Prolyl-tRNA synthetase inhibition promotes cell death in SK-MEL-2 cells through GCN2-ATF4 pathway activation.

Protein translation is highly activated in cancer tissues through oncogenic mutations and amplifications, and this can support survival and aberrant proliferation. Therefore, blocking translation could be a promising way to block cancer progression. The process of charging a cognate amino acid to tRNA, a crucial step in protein synthesis, is mediated by tRNA synthetases such as prolyl tRNA synthetase (PRS). Interestingly, unlike pan-translation inhibitors, we demonstrated that a novel small molecule PRS inhibitor (T-3861174) induced cell death in several tumor cell lines including SK-MEL-2 without complete suppression of translation. Additionally, our findings indicated that T-3861174-induced cell death was caused by activation of the GCN2-ATF4 pathway. Furthermore, the PRS inhibitor exhibited significant anti-tumor activity in several xenograft models without severe body weight losses. These results indicate that PRS is a druggable target, and suggest that T-3861174 is a potential therapeutic agent for cancer therapy.

Contributions of interfacial residues of human Interleukin15 to the specificity and affinity for its private alpha-receptor.

Human interleukin 15 (hIL15) is a soluble cytokine that plays a key role in the maintenance of long-lasting responses against pathogens and a valuable target for the treatment of autoimmune diseases. In this study, we sought to elucidate the thermodynamic basis of the recognition mechanism for its private alpha-receptor (hIL15Ralpha), considered the first step of the interleukin's activation pathway. Binding of wild-type hIL15 to its alpha-receptor is characterized by a very slow dissociation rate constant and driven by a favorable enthalpy change. We further studied the kinetic and energetic consequences of substituting residues of hIL15 located at the contact interface by means of the surface plasmon resonance technique. Replacement of negatively charged residues with Ala indicates that the energetics of interaction is primarily driven by electrostatic forces, manifested by a dramatic acceleration of the dissociation step and a reduction of favorable binding enthalpy. Our analyses also unveiled a novel and critical role for residue Tyr26 in the interaction, which facilitates desolvation of key charged residues during the assembly of the complex. These results were rationalized in terms of a previously reported structure of hIL15.hIL15alpha, demonstrating that the binding energetics is dominated by interactions occurring at three hot spots whose spatial locations coincide with a previously proposed structural division of the contact interface in three regions. Specifically, Region 1 is the main contributor to the binding energy of the complex by establishing very favorable electrostatic interactions with the receptor; Region 2 is also dominated by electrostatic forces, although of a lesser intensity; and Region 3 confers specificity to the association by means of high shape complementarity and by bringing additional stabilization energy to the complex. The biological impact of hIL15 mutations with the most effect on alpha-receptor binding was evaluated in a cell-based proliferation assay, validating the conclusions of our thermodynamic analyses and highlighting the functional importance of molecular contacts that promote prolonged binding of the interleukin to the alpha-receptor. In closing, the thermodynamics and physicochemical nature of the interactions observed in IL15h.IL15Ralpha complex, together with interactions in Region 3 of the interleukin, poses a stark contrast with the structurally related and sometimes functionally redundant interleukin 2.

Electron microscopy and computational studies of Ebh, a giant cell-wall-associated protein from Staphylococcus aureus.

Ebh, a giant protein found in staphylococci, contains several domains, including a large central region with 52 imperfect repeats of a domain composed of 126 amino acids. We used electron microscopy to observe the rod-like structure of a partial Ebh protein containing 10 repeating units. This is the first report of the direct observation of an Ebh structure containing a large number of repeating units, although structures containing one, two, or four repeating units have been reported. The observed structure of the partial Ebh protein was distorted and had a length of ca. 520A and a width of ca. 21A. The observed structures were consistent with those deduced from crystal structure analysis, suggesting that the Ebh domains are connected to form a rod-like structure. The crystal structure data revealed distorted, string-like features in the simulated structure of the whole-length Ebh protein. Superposition of fragments of the simulated whole-length structure of the Ebh protein onto each electron micrograph showed a high level of correlation between the observed and calculated structures. These results suggest that Ebh is composed of highly flexible filate molecules. The highly repetitive structure and the associated unique structural flexibility of Ebh support the proposed function of this protein, i.e. binding to sugars in the cell wall. This binding might result in intra-cell-wall cross-linking that contributes to the rigidity of bacterial cells.

A helical string of alternately connected three-helix bundles for the cell wall-associated adhesion protein Ebh from Staphylococcus aureus.

The 1.1 MDa cell-wall-associated adhesion protein of staphylococci, Ebh, consists of several distinct regions, including a large central region with 52 imperfect repeats of 126 amino acid residues. We investigated the structure of this giant molecule by X-ray crystallography, circular dichroism (CD) spectrometry, and small-angle X-ray scattering (SAXS). The crystal structure of two repeats showed that each repeat consists of two distinct three-helix bundles, and two such repeats are connected along the long axis, resulting in a rod-like structure that is 120 A in length. CD and SAXS analyses of the samples with longer repeats suggested that each repeat has an identical structure, and that such repeats are connected tandemly to form a rod-like structure in solution, the length of which increased proportionately with the number of repeating units. On the basis of these results, it was proposed that Ebh is a 320 nm rod-like molecule with some plasticity at module junctions.