Michael J. Hubbard

On target with a new mechanism for the regulation of protein phosphorylation.

There is overwhelming evidence that the reversible phosphorylation of proteins regulates most aspects of cell life. However, the broad specificities displayed by many protein phosphatases and kinases in vitro dictates that their activities be strictly regulated in vivo. Recent evidence indicates that a novel class of proteins, known as targetting subunits, specifies the location, catalytic and regulatory properties of protein phosphatases and kinases, and thereby plays a key role in ensuring the fidelity of protein phosphorylation.

Mitochondrial ATP synthase F1-β-subunit is a calcium-binding protein

Mitochondrial ATP synthase is responsive to changes in cytosolic calcium concentration, but the regulatory mechanisms are unclear. Here we identified a major 52 kDa calcium‐binding protein in rat enamel cells as the mitochondrial ATP synthase F1‐β‐subunit. The F1‐β‐subunit behaved as a low affinity and moderate capacity calcium‐binding protein during comparative 45Ca overlay analyses. Equivalent behavior was shown by the F1‐β‐subunit from rat liver mitochondria, but not by the homologous F1‐α‐subunit, supporting the specificity of calcium binding. Evidence that the catalytic F1‐β‐subunit binds calcium specifically introduces new mechanistic possibilities for regulating ATP synthase, and thereby coordinating ATP production with demand for ATP‐fuelled calcium pump activity.

Calcium transport across the dental enamel epithelium.

Dental enamel is the most highly calcified tissue in mammals, and its formation is an issue of fundamental biomedical importance. The enamel-forming cells must somehow supply calcium in bulk yet avoid the cytotoxic effects of excess calcium. Disrupted calcium transport could contribute to a variety of developmental defects in enamel, and the underlying cellular machinery is a potential target for drugs to improve enamel quality. The mechanisms used to transport calcium remain unclear despite much progress in our understanding of enamel formation. Here, current knowledge of how enamel cells handle calcium is reviewed in the context of findings from other epithelial calcium-transport systems. In the past, most attention has focused on approaches to boost the poor diffusion of calcium in cytosol. Recent biochemical findings led to an alternative proposal that calcium is routed through high-capacity stores associated with the endoplasmic reticulum. Research areas needing further attention and a working model are also discussed. Calcium-handling mechanisms in enamel cells are more generally relevant to the understanding of epithelial calcium transport, biomineralization, and calcium toxicity avoidance.

Molecular cloning of ERp29, a novel and widely expressed resident of the endoplasmic reticulum

We have isolated a full‐length cDNA clone for a novel 29 kDa protein that is highly expressed in rat enamel cells. The clone encodes a 259‐residue protein, here named ERp29, with structural features (signal peptide and a variant endoplasmic reticulum‐retention motif, KEEL) that indicate it is a reticuloplasmin. ERp29 has limited homology with protein disulfide isomerase and its cognates, but lacks their characteristic thioredoxin‐like catalytic moiety and calcium‐binding motifs. ERp29 mRNA was expressed in all rat tissues tested, and a homologous transcript was detected in other animal livers (primate, ruminant, marsupial). In human hepatoma cells, ERp29 mRNA expression was not increased by stresses (tunicamycin, calcium ionophore) that induced other reticuloplasmins. We conclude that ERp29 is a new, highly conserved member of the reticuloplasmin family which is widely expressed. The apparent lack of both calcium binding properties and stress responsiveness distinguish ERp29 from all major reticuloplasmins characterised to date.

ERp29 restricts Connexin43 oligomerization in the endoplasmic reticulum.

Connexin43 (Cx43) is a gap junction protein that forms multimeric channels that enable intercellular communication through the direct transfer of signals and metabolites. Although most multimeric protein complexes form in the endoplasmic reticulum (ER), Cx43 seems to exit from the ER as monomers and subsequently oligomerizes in the Golgi complex. This suggests that one or more protein chaperones inhibit premature Cx43 oligomerization in the ER. Here, we provide evidence that an ER-localized, 29-kDa thioredoxin-family protein (ERp29) regulates Cx43 trafficking and function. Interfering with ERp29 function destabilized monomeric Cx43 oligomerization in the ER, caused increased Cx43 accumulation in the Golgi apparatus, reduced transport of Cx43 to the plasma membrane, and inhibited gap junctional communication. ERp29 also formed a specific complex with monomeric Cx43. Together, this supports a new role for ERp29 as a chaperone that helps stabilize monomeric Cx43 to enable oligomerization to occur in the Golgi apparatus.

Identification of novel candidate genes involved in mineralization of dental enamel by genome-wide transcript profiling.

The gene repertoire regulating vertebrate biomineralization is poorly understood. Dental enamel, the most highly mineralized tissue in mammals, differs from other calcifying systems in that the formative cells (ameloblasts) lack remodeling activity and largely degrade and resorb the initial extracellular matrix. Enamel mineralization requires that ameloblasts undergo a profound functional switch from matrix‐secreting to maturational (calcium transport, protein resorption) roles as mineralization progresses. During the maturation stage, extracellular pH decreases markedly, placing high demands on ameloblasts to regulate acidic environments present around the growing hydroxyapatite crystals. To identify the genetic events driving enamel mineralization, we conducted genome‐wide transcript profiling of the developing enamel organ from rat incisors and highlight over 300 genes differentially expressed during maturation. Using multiple bioinformatics analyses, we identified groups of maturation‐associated genes whose functions are linked to key mineralization processes including pH regulation, calcium handling, and matrix turnover. Subsequent qPCR and Western blot analyses revealed that a number of solute carrier (SLC) gene family members were up‐regulated during maturation, including the novel protein Slc24a4 involved in calcium handling as well as other proteins of similar function (Stim1). By providing the first global overview of the cellular machinery required for enamel maturation, this study provide a strong foundation for improving basic understanding of biomineralization and its practical applications in healthcare. J. Cell. Physiol. 227: 2264–2275, 2012.

Surface Integrity Governs the Proteome of Hypomineralized Enamel

Growing interest in the treatment and prevention of Molar/Incisor Hypomineralization (MIH) warrants investigation into the protein composition of hypomineralized enamel. Hypothesizing abnormality akin to amelogenesis imperfecta, we profiled proteins in hypomineralized enamel from human permanent first molars using a biochemical approach. Hypomineralized enamel was found to have from 3- to 15-fold higher protein content than normal, but a near-normal level of residual amelogenins. This distinguished MIH from hypomaturation defects with high residual amelogenins (amelogenesis imperfecta, fluorosis) and so typified it as a hypocalcification defect. Second, hypomineralized enamel was found to have accumulated various proteins from oral fluid and blood, with differential incorporation depending on integrity of the enamel surface. Pathogenically, these results point to a pre-eruptive disturbance of mineralization involving albumin and, in cases with post-eruptive breakdown, subsequent protein adsorption on the exposed hydroxyapatite matrix. These insights into the pathogenesis and properties of hypomineralized enamel hold significance for prevention and treatment of MIH.

Multisite phosphorylation of the glycogen-binding subunit of protein phosphatase-1G by cyclic AMP-dependent protein kinase and glycogen synthase kinase-3

The glycogen‐binding (G) subunit of protein phosphatase‐1G is phosphorylated stoichiometrically by glycogen synthase kinase‐3 (GSK3), and with a greater catalytic efficiency than glycogen synthase, but only after prior phosphorylation by cyclic AMP‐dependent protein kinase (A‐kinase) at site 1. The residues phosphorylated are the first two serines in the sequence AIFKPGFSPQPSRRGS‐, while the C‐terminal serine (site 1) is one of the two residues phosphorylated by A‐kinase. These findings demonstrate that (i) the G subunit undergoes multisite phosphorylation in vitro; (ii) phosphorylation by GSK3 requires the presence of a C‐terminal phosphoserine residue; (iii) GSK3 can synergise with protein kinases other than casein kinase‐2.

Evidence for the occurrence of calmodulin in the yeasts Candidas albicans and Saccharomyces cerevisiae

Calmodulin was originally characterised as the calcium-dependent activator of brain cyclic nucleotide phosphodiesterase. Subsequently it was shown to be the modulator of a variety of calcium-dependent cellular activities (reviews [l-3]). Calmodulin is now recognised as being a highly conserved and widely distributed protein, having been demonstrated in many vertebrates and invertebrates [3,4], plants and higher fungi [5,6] as well as several unicellular eukaryotes; e.g., Dictyostelium discoideum [7], Blastocladiella emersonii [8], Euglena gracilis and Amoeba proteus [9]. Prokaryotes, in contrast,appear to lack calmodulin [6,7] although (exogenous) calmodulin has been shown to activate the adenylate cyclase of Bordetella pertussis [lo]. The observed distribution of calmodulin has led to the suggestion that it is ubiquitous in eukaryotes [3,5]. However, the inability of several laboratories to detect calmodulin in yeast [6,7,1 l] is in conflict with this assertion. This paper reports the presence of a calmodulln-like protein in extracts of the yeasts Candida albicans and Saccharomyces cerevisiae. The demonstration of this protein appears to be dependent on the use of the protease inhibitor, PMSF.

New Paradigms on the Transport Functions of Maturation-stage Ameloblasts

Fully matured dental enamel is an architecturally and mechanically complex hydroxyapatite-based bioceramic devoid of most of the organic material that was essential in its making. Enamel formation is a staged process principally involving secretory and maturation stages, each associated with major changes in gene expression and cellular function. Cellular activities that define the maturation stage of amelogenesis include ion (e.g., calcium and phosphate) transport and storage, control of intracellular and extracellular pH (e.g., bicarbonate and hydrogen ion movements), and endocytosis. Recent studies on rodent amelogenesis have identified a multitude of gene products that appear to be linked to these cellular activities. This review describes the main cellular activities of these genes during the maturation stage of amelogenesis.