Human Saposin B Ligand Binding and Presentation to α-Galactosidase A.

Sphingolipid activator protein B (saposin B; SapB) is an essential activator of globotriaosylceramide (Gb3) catabolism by α-galactosidase A. However, the manner by which SapB stimulates α-galactosidase A activity remains unknown. To uncover the molecular mechanism of SapB presenting Gb3 to α-galactosidase A, we subjected the fluorescent substrate globotriaosylceramide-nitrobenzoxidazole (Gb3-NBD) to a series of biochemical and structural assays involving SapB.

A domain-swapped CaMKII conformation facilitates linker-mediated allosteric regulation.

Ca signaling plays a key role in physiological processes such as memory formation and cardiac function. Ca/calmodulin-dependent protein kinase II (CaMKII) is the primary kinase that responds to Ca inputs in these cells. There are four CaMKII paralogs in mammals which are alternatively spliced in the variable linker region to create upwards of 70 different variants.

Ca2+/CaM dependent protein kinase II (CaMKII)α and CaMKIIβ hub domains adopt distinct oligomeric states and stabilities.

Ca /calmodulin-dependent protein kinase II (CaMKII) is a multidomain serine/threonine kinase that plays important roles in the brain, heart, muscle tissue, and eggs/sperm. The N-terminal kinase and regulatory domain is connected by a flexible linker to the C-terminal hub domain. The hub domain drives the oligomeric organization of CaMKII, assembling the kinase domains into high local concentration.

CaMKII binds both substrates and activators at the active site.

Ca/calmodulin-dependent protein kinase II (CaMKII) is a signaling protein required for long-term memory. When activated by Ca/CaM, it sustains activity even after the Ca dissipates. In addition to the well-known autophosphorylation-mediated mechanism, interaction with specific binding partners also persistently activates CaMKII.

Malectin/Malectin-like domain-containing proteins: A repertoire of cell surface molecules with broad functional potential.

Cell walls are at the front line of interactions between walled-organisms and their environment. They support cell expansion, ensure cell integrity and, for multicellular organisms such as plants, they provide cell adherence, support cell shape morphogenesis and mediate cell-cell communication. Wall-sensing, detecting perturbations in the wall and signaling the cell to respond accordingly, is crucial for growth and survival.

Characterization of CaMKIIα holoenzyme stability.

Ca /calmodulin-dependent protein kinase II (CaMKII) is a Ser/Thr kinase necessary for long-term memory formation and other Ca -dependent signaling cascades such as fertilization. Here, we investigated the stability of CaMKIIα using a combination of differential scanning calorimetry (DSC), X-ray crystallography, and mass photometry (MP). The kinase domain has a low thermal stability (apparent T = 36°C), which is slightly stabilized by ATP/MgCl binding (apparent T = 40°C) and significantly stabilized by regulatory segment binding (apparent T = 60°C).

Ligand-promoted protein folding by biased kinetic partitioning.

Protein folding in cells occurs in the presence of high concentrations of endogenous binding partners, and exogenous binding partners have been exploited as pharmacological chaperones. A combined mathematical modeling and experimental approach shows that a ligand improves the folding of a destabilized protein by biasing the kinetic partitioning between folding and alternative fates (aggregation or degradation). Computationally predicted inhibition of test protein aggregation and degradation as a function of ligand concentration are validated by experiments in two disparate cellular systems.

Human acid sphingomyelinase structures provide insight to molecular basis of Niemann-Pick disease.

Acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and phosphocholine, essential components of myelin in neurons. Genetic alterations in ASM lead to ASM deficiency (ASMD) and have been linked to Niemann-Pick disease types A and B. Olipudase alfa, a recombinant form of human ASM, is being developed as enzyme replacement therapy to treat the non-neurological manifestations of ASMD.

Substrate Promotes Productive Gas Binding in the α-Ketoglutarate-Dependent Oxygenase FIH.

The Fe(2+)/α-ketoglutarate (αKG)-dependent oxygenases use molecular oxygen to conduct a wide variety of reactions with important biological implications, such as DNA base excision repair, histone demethylation, and the cellular hypoxia response. These enzymes follow a sequential mechanism in which O2 binds and reacts after the primary substrate binds, making those structural factors that promote productive O2 binding central to their chemistry. A large challenge in this field is to identify strategies that engender productive turnover.

Variations in the GLA gene correlate with globotriaosylceramide and globotriaosylsphingosine analog levels in urine and plasma.

Recent data have shown that lyso-Gb3, the deacylated derivative of globotriaosylceramide (Gb3), is possibly involved in the pathogenesis of Fabry disease (FD) and might be a clinically useful biomarker of its metabolic load. To test this hypothesis, we assayed Gb3 and lyso-Gb3 and related analogs in plasma and/or urine samples of 12 clinically well-characterized subjects carrying several different GLA variant alleles associated with a wide range of residual α-galactosidase A activities. Urinary Gb3 was measured by HPLC-MS/MS; plasma and urinary lyso-Gb3 and related analogs were measured by UPLC-MS/MS.

Nickel superoxide dismutase: structural and functional roles of His1 and its H-bonding network.

Crystal structures of nickel-dependent superoxide dismutases (NiSODs) reveal the presence of a H-bonding network formed between the NH group of the apical imidazole ligand from His1 and the Glu17 carboxylate from a neighboring subunit in the hexameric enzyme. This interaction is supported by another intrasubunit H-bond between Glu17 and Arg47. In this study, four mutant NiSOD proteins were produced to experimentally evaluate the roles of this H-bonding network and compare the results with prior predictions from density functional theory calculations.

The alpha-galactosidase A p.Arg118Cys variant does not cause a Fabry disease phenotype: data from individual patients and family studies.

Lysosomal α-galactosidase A (α-Gal) is the enzyme deficient in Fabry disease (FD), an X-linked glycosphingolipidosis caused by pathogenic mutations affecting the GLA gene. The early-onset, multi-systemic FD classical phenotype is associated with absent or severe enzyme deficiency, as measured by in vitro assays, but patients with higher levels of residual α-Gal activity may have later-onset, more organ-restricted clinical presentations. A change in the codon 118 of the wild-type α-Gal sequence, replacing basic arginine by a potentially sulfhydryl-binding cysteine residue - GLA p.

Unlocking a caged lysosomal protein from a polymeric nanogel with a pH trigger.

A polymeric nanogel has been used to sequester and turn off a lysosomal protein, acid α-glucosidase (GAA). The nanogel contains a β-thiopropionate cross-linker, which endows the nanogel with pH-sensitivity. While encapsulation of the enzyme fully turns off its activity, approximately 75% of the activity is recovered upon reducing the pH to 5.

Proteolytic activation of human cathepsin A.

Galactosialidosis is a human lysosomal storage disease caused by deficiency in the multifunctional lysosomal protease cathepsin A (also known as protective protein/cathepsin A, PPCA, catA, HPP, and CTSA; EC 3.4.16.

Electrostatic origin of in vitro aggregation of human γ-crystallin.

The proteins α-, β-, and γ-crystallins are the major components of the lens in the human eye. Using dynamic light scattering method, we have performed in vitro investigations of protein-protein interactions in dilute solutions of human γ-crystallin and α-crystallin. We find that γ-crystallin spontaneously aggregates into finite-sized clusters in phosphate buffer solutions.

Pharmacological chaperones for human α-N-acetylgalactosaminidase.

Schindler/Kanzaki disease is an inherited metabolic disease with no current treatment options. This neurologic disease results from a defect in the lysosomal α-N-acetylgalactosaminidase (α-NAGAL) enzyme. In this report, we show evidence that the iminosugar DGJNAc can inhibit, stabilize, and chaperone human α-NAGAL both in vitro and in vivo.

The structure of human GALNS reveals the molecular basis for mucopolysaccharidosis IV A.

Lysosomal enzymes catalyze the breakdown of macromolecules in the cell. In humans, loss of activity of a lysosomal enzyme leads to an inherited metabolic defect known as a lysosomal storage disorder. The human lysosomal enzyme galactosamine-6-sulfatase (GALNS, also known as N-acetylgalactosamine-6-sulfatase and GalN6S; E.

Adaptor-dependent degradation of a cell-cycle regulator uses a unique substrate architecture.

In Caulobacter crescentus, the ClpXP protease degrades several crucial cell-cycle regulators, including the phosphodiesterase PdeA. Degradation of PdeA requires the response regulator CpdR and signals a morphological transition in concert with initiation of DNA replication. Here, we report the structure of a Per-Arnt-Sim (PAS) domain of PdeA and show that it is necessary for CpdR-dependent degradation in vivo and in vitro.

The molecular basis of pharmacological chaperoning in human α-galactosidase.

Fabry disease patients show a deficiency in the activity of the lysosomal enzyme α-galactosidase (α-GAL or α-Gal A). One proposed treatment for Fabry disease is pharmacological chaperone therapy, where a small molecule stabilizes the α-GAL protein, leading to increased enzymatic activity. Using enzyme kinetics, tryptophan fluorescence, circular dichroism, and proteolysis assays, we show that the pharmacological chaperones 1-deoxygalactonojirimycin (DGJ) and galactose stabilize the human α-GAL glycoprotein.

GM1 gangliosidosis and Morquio B disease: an update on genetic alterations and clinical findings.

GM1 gangliosidosis and Morquio B syndrome, both arising from beta-galactosidase (GLB1) deficiency, are very rare lysosomal storage diseases with an incidence of about 1:100,000-1:200,000 live births worldwide. Here we report the beta-galactosidase gene (GLB1) mutation analysis of 21 unrelated GM1 gangliosidosis patients, and of 4 Morquio B patients, of whom two are brothers. Clinical features of the patients were collected and compared with those in literature.

Interconversion of the specificities of human lysosomal enzymes associated with Fabry and Schindler diseases.

The human lysosomal enzymes alpha-galactosidase (alpha-GAL, EC 3.2.1.

Catalytic mechanism of human alpha-galactosidase.

The enzyme alpha-galactosidase (alpha-GAL, also known as alpha-GAL A; E.C. 3.

The 1.9 a structure of human alpha-N-acetylgalactosaminidase: The molecular basis of Schindler and Kanzaki diseases.

alpha-N-acetylgalactosaminidase (alpha-NAGAL; E.C. 3.

Role of conserved tyrosine residues in NiSOD catalysis: a case of convergent evolution.

Superoxide dismutases rely on protein structural elements to adjust the redox potential of the metallocenter to an optimum value near 300 mV (vs NHE), to provide a source of protons for catalysis, and to control the access of anions to the active site. These aspects of the catalytic mechanism are examined herein for recombinant preparations of the nickel-dependent SOD (NiSOD) from Streptomyces coelicolor and for a series of mutants that affect a key tyrosine residue, Tyr9 (Y9F-, Y62F-, Y9F/Y62F-, and D3A-NiSOD). Structural aspects of the nickel sites are examined by a combination of EPR and X-ray absorption spectroscopies, and by single-crystal X-ray diffraction at approximately 1.