Protein-RNA interactions mediated by silvestrol-insight into a unique molecular clamp.

Molecular staples or interfacial inhibitors are small molecules that exert their activity through co-association with macromolecules leading to various effects on target functions. Some molecules inhibit target activity, while others generate gain-of-function complexes. We and others have previously identified two structurally distinct classes of molecular staples, pateamine A and rocaglates.

Structural Analysis of Mammalian Sialic Acid Esterase.

Sialic acid esterase (SIAE) catalyzes the removal of O-acetyl groups from sialic acids found on cell surface glycoproteins to regulate cellular processes such as B cell receptor signalling and apoptosis. Loss-of-function mutations in SIAE are associated with several common autoimmune diseases including Crohn's, ulcerative colitis, and arthritis. To gain a better understanding of the function and regulation of this protein, we determined crystal structures of SIAE from three mammalian homologs, including an acetate bound structure.

PPM1G dephosphorylates eIF4E in control of mRNA translation and cell proliferation.

The mRNA 5'cap-binding eukaryotic translation initiation factor 4E (eIF4E) plays a critical role in the control of mRNA translation in health and disease. One mechanism of regulation of eIF4E activity is via phosphorylation of eIF4E by MNK kinases, which promotes the translation of a subset of mRNAs encoding pro-tumorigenic proteins. Work on eIF4E phosphatases has been paltry.

Isolation of short RNAs with homogeneous 3'-ends using quaternary-amine anion exchange chromatography.

Visualizing RNA-protein interactions through structural approaches requires the use of RNA molecules purified to homogeneity. We describe here a simple and effective method, free of acrylamide contamination and without using UV radiation, to separate synthesized, heterogeneous RNA transcripts (up to ∼15 nucleotides) at single-nucleotide resolution by quaternary-amine anion exchange chromatography. The quality of short RNAs isolated through this method is validated by gel electrophoresis, mass spectrometry, and crystallization with a protein-binding partner.

Secondary deficiency of neuraminidase 1 contributes to CNS pathology in neurological mucopolysaccharidoses via hypersialylation of brain glycoproteins.

Mucopolysaccharidoses (MPS) are lysosomal storage diseases caused by defects in catabolism of glycosaminoglycans. MPS I, II, III and VII are associated with lysosomal accumulation of heparan sulphate and manifest with neurological deterioration. Most of these neurological MPS currently lack effective treatments.

Structure of the immunoregulatory sialidase NEU1.

Sialic acids linked to glycoproteins and glycolipids are important mediators of cell and protein recognition events. These sugar residues are removed by neuraminidases (sialidases). Neuraminidase-1 (sialidase-1 or NEU1) is a ubiquitously expressed mammalian sialidase located in lysosomes and on the cell membrane.

Deubiquitinase catalytic activity of MYSM1 is essential in vivo for hematopoiesis and immune cell development.

Myb-like SWIRM and MPN domains 1 (MYSM1) is a chromatin binding protein with deubiquitinase (DUB) catalytic activity. Rare MYSM1 mutations in human patients result in an inherited bone marrow failure syndrome, highlighting the biomedical significance of MYSM1 in the hematopoietic system. We and others characterized Mysm1-knockout mice as a model of this disorder and established that MYSM1 regulates hematopoietic function and leukocyte development in such models through different mechanisms.

Structures of the mannose-6-phosphate pathway enzyme, GlcNAc-1-phosphotransferase.

The mannose-6-phosphate (M6P) pathway is responsible for the transport of hydrolytic enzymes to lysosomes. N-acetylglucosamine-1-phosphotransferase (GNPT) catalyzes the first step of tagging these hydrolases with M6P, which when recognized by receptors in the Golgi diverts them to lysosomes. Genetic defects in the GNPT subunits, GNPTAB and GNPTG, cause the lysosomal storage diseases mucolipidosis types II and III.

Targeting DEAD-box RNA helicases: The emergence of molecular staples.

RNA helicases constitute a large family of proteins that play critical roles in mediating RNA function. They have been implicated in all facets of gene expression pathways involving RNA, from transcription to processing, transport and translation, and storage and decay. There is significant interest in developing small molecule inhibitors to RNA helicases as some family members have been documented to be dysregulated in neurological and neurodevelopment disorders, as well as in cancers.

Structure of the murine lysosomal multienzyme complex core.

The enzymes β-galactosidase (GLB1) and neuraminidase 1 (NEU1; sialidase 1) participate in the degradation of glycoproteins and glycolipids in the lysosome. To remain active and stable, they associate with PPCA [protective protein cathepsin A (CTSA)] into a high-molecular weight lysosomal multienzyme complex (LMC), of which several forms exist. Genetic defects in these three proteins cause the lysosomal storage diseases GM1-gangliosidosis/mucopolysaccharidosis IV type B, sialidosis, and galactosialidosis, respectively.

Functional mimicry revealed by the crystal structure of an eIF4A:RNA complex bound to the interfacial inhibitor, desmethyl pateamine A.

Interfacial inhibitors exert their biological effects through co-association with two macromolecules. The pateamine A (PatA) class of molecules function by stabilizing eukaryotic initiation factor (eIF) 4A RNA helicase onto RNA, resulting in translation initiation inhibition. Here, we present the crystal structure of an eIF4A1:RNA complex bound to an analog of the marine sponge-derived natural product PatA, C5-desmethyl PatA (DMPatA).

The mTORC1/S6K/PDCD4/eIF4A Axis Determines Outcome of Mitotic Arrest.

mTOR is a serine/threonine kinase and a master regulator of cell growth and proliferation. Raptor, a scaffolding protein that recruits substrates to mTOR complex 1 (mTORC1), is known to be phosphorylated during mitosis, but the significance of this phosphorylation remains largely unknown. Here we show that raptor expression and mTORC1 activity are dramatically reduced in cells arrested in mitosis.

Crystal structure of the nucleotide-metabolizing enzyme NTPDase4.

The ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) are a family of enzymes found on the cell surface and in the lumen of certain organelles, that are major regulators of purinergic signaling. Their intracellular roles, however, have not been clearly defined. NTPDase4 (UDPase, ENTPD4) is a Golgi protein potentially involved in nucleotide recycling as part of protein glycosylation, and is also found in lysosomes, where its purpose is unknown.

Crystal Structure of the Mannose-6-Phosphate Uncovering Enzyme.

Most lysosomal hydrolytic enzymes reach their destination via the mannose-6-phosphate (M6P) pathway. The enzyme N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase (NAGPA, or "uncovering enzyme") catalyzes the second step in the M6P tag formation, namely the removal of the masking N-acetylglucosamine (GlcNAc) portion. Defects in this protein are associated with non-syndromic stuttering.

Double-Stranded Biotinylated Donor Enhances Homology-Directed Repair in Combination with Cas9 Monoavidin in Mammalian Cells.

Homology-directed repair (HDR) induced by site specific DNA double-strand breaks with CRISPR-Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non-homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered a Cas9 protein fused to a monoavidin domain to bind biotinylated donor DNA. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9-donor DNA complexes into cells.

Identification of Allosteric Inhibitors against Active Caspase-6.

Caspase-6 is a cysteine protease that plays essential roles in programmed cell death, axonal degeneration, and development. The excess neuronal activity of Caspase-6 is associated with Alzheimer disease neuropathology and age-dependent cognitive impairment. Caspase-6 inhibition is a promising strategy to stop early stage neurodegenerative events, yet finding potent and selective Caspase-6 inhibitors has been a challenging task due to the overlapping structural and functional similarities between caspase family members.

The structure of mammalian β-mannosidase provides insight into β-mannosidosis and nystagmus.

β-Mannosidase is a lysosomal enzyme from the glycosyl hydrolase family 2 that cleaves the single β(1-4)-linked mannose at the nonreducing end of N-glycosylated proteins, and plays an important role in the polysaccharide degradation pathway. Mutations in the MANBA gene, which encodes the β-mannosidase, can lead to the lysosomal storage disease β-mannosidosis, as well as nystagmus, an eye condition characterized by involuntary eye movements. Here, we present the first structures of a mammalian β-mannosidase in both the apo- and mannose-bound forms.

Molecular Mechanism of Inhibition of Acid Ceramidase by Carmofur.

Human acid ceramidase (AC) is a lysosomal cysteine amidase, which has received a great deal of interest in recent years as a potential target for the development of new therapeutics against melanoma and glioblastoma tumors. Despite the strong interest in obtaining structural information, only the structures of the apo-AC enzyme in its zymogen and activated conformations are available. In this work, the crystal structure of AC in complex with the covalent carmofur inhibitor is presented.

Molecular mechanism of activation of the immunoregulatory amidase NAAA.

Palmitoylethanolamide is a bioactive lipid that strongly alleviates pain and inflammation in animal models and in humans. Its signaling activity is terminated through degradation by -acylethanolamine acid amidase (NAAA), a cysteine hydrolase expressed at high levels in immune cells. Pharmacological inhibitors of NAAA activity exert profound analgesic and antiinflammatory effects in rodent models, pointing to this protein as a potential target for therapeutic drug discovery.

Crystal structure of saposin D in an open conformation.

Saposins are accessory proteins that aid in the degradation of sphingolipids by hydrolytic enzymes. Their structure usually comprises four α-helices arranged in various conformations including an open, V-shaped form that is generally associated with the ability to interact with membranes and/or enzymes to accentuate activity. Saposin D is required by the lysosomal hydrolase, acid ceramidase, which breaks down ceramide into sphingosine and free fatty acid, to display optimal activity.

Structural Analysis of the Bacterial Effector AvrA Identifies a Critical Helix Involved in Substrate Recognition.

Bacterial effector proteins are essential for the infection and proliferation of pathogenic bacteria through manipulation of host immune response pathways. AvrA is a Salmonella effector that belongs to the YopJ family of acetyltransferases, which suppresses c-JUN N-terminal kinase (JNK) signaling in mammals through acetylation of mitogen-activated receptor kinase kinases 4 and 7 (MKK4/7). Interestingly, there are two paralogues of AvrA that differ by only a single internal leucine residue, which when absent (AvrA) abrogates the ability to suppress JNK signaling.

Structural basis for nucleotide recognition by the ectoenzyme CD203c.

Unlabelled: The ecto-nucleotide pyrophosphatase/phosphodiesterase (NPP) enzyme family modulates purinergic signaling by degrading extracellular nucleotides. CD203c (NPP3, ENPP3) regulates the inflammatory response of basophils via ATP hydrolysis and is a marker for allergen sensitivity on the surface of these cells. Multiple other roles and substrates have also been proposed for this protein.

Structural basis for the activation of acid ceramidase.

Acid ceramidase (aCDase, ASAH1) hydrolyzes lysosomal membrane ceramide into sphingosine, the backbone of all sphingolipids, to regulate many cellular processes. Abnormal function of aCDase leads to Farber disease, spinal muscular atrophy with progressive myoclonic epilepsy, and is associated with Alzheimer's, diabetes, and cancer. Here, we present crystal structures of mammalian aCDases in both proenzyme and autocleaved forms.

Crystal structure of the mammalian lipopolysaccharide detoxifier.

LPS is a potent bacterial endotoxin that triggers the innate immune system. Proper recognition of LPS by pattern-recognition receptors requires a full complement of typically six acyl chains in the lipid portion. Acyloxyacyl hydrolase (AOAH) is a host enzyme that removes secondary (acyloxyacyl-linked) fatty acids from LPS, rendering it immunologically inert.