Effect of the incomplete separation of bound and free ligand on binding measurements

The sensitivity for the determination of lithium by emission in the nitrous oxide-acetylene flame rivals that of graphite furnace atomic absorption and is freer of chemical interference. Details of its application to analysis of organs of small animals forlithium at physiological (normal) levels are presented. Matrix matching and standardization by additions are unnecessary.     

Roles of creatine in the regulation of cardiac protein synthesis

The observation that increased muscular activity leads to muscle hypertrophy is well known, but identification of the biochemical and physiological mechanisms by which this occurs remains an important problem. Experiments have been described (5, 6) which suggest that creatine, an end product of contraction, is involved in the control of contractile protein synthesis in differentiating skeletal muscle cells and may be the chemical signal coupling increased muscular activity and the increased muscular mass. During contraction, the creatine concentration in muscle transiently increases as creatine phosphate is hydrolyzed to regenerate ATP. In isometric contraction in skeletal muscle for example, Edwards and colleagues (3) have found that nearly all of the creatine phosphate is hydrolyzed. In this case, the creatine concentration is increased about twofold, and it is this transient change in creatine concentration which is postulated to lead to increased contractile protein synthesis. If creatine is found in several intracellular compartments, as suggested by Lee and Vissher (7), local changes in concentration may be greater then twofold. A specific effect on contractile protein synthesis seems reasonable in light of the work of Rabinowitz (13) and of Page et al. (11), among others, showing disproportionate accumulation of myofibrillar and mitochondrial proteins in response to work-induced hypertrophy and thyroxin-stimulated growth. Previous experiments (5, 6) have shown that skeletal muscles cells which have differentiated in vitro or in vivo synthesize myosin heavy-chain and actin, the major myofibrillar polypeptides, faster when supplied creatine in vitro. The stimulation is specific for contractile protein synthesis since neither the rate of myosin turnover nor the rates of synthesis of noncontractile protein and DNA are affected by creatine. The experiments reported in this communication were undertaken to test whether creatine selectively stimulates contractile protein synthesis in heart as it does in skeletal muscle.     

Microengineered systems with iPSC-derived cardiac and hepatic cells to evaluate drug adverse effects

Hepatic and cardiac drug adverse effects are among the leading causes of attrition in drug development programs, in part due to predictive failures of current animal or in vitro models. Hepatocytes and cardiomyocytes differentiated from human induced pluripotent stem cells (iPSCs) hold promise for predicting clinical drug effects, given their human-specific properties and their ability to harbor genetically determined characteristics that underlie inter-individual variations in drug response. Currently, the fetal-like properties and heterogeneity of hepatocytes and cardiomyocytes differentiated from iPSCs make them physiologically different from their counterparts isolated from primary tissues and limit their use for predicting clinical drug effects. To address this hurdle, there have been ongoing advances in differentiation and maturation protocols to improve the quality and use of iPSC-differentiated lineages. Among these are in vitro hepatic and cardiac cellular microsystems that can further enhance the physiology of cultured cells, can be used to better predict drug adverse effects, and investigate drug metabolism, pharmacokinetics, and pharmacodynamics to facilitate successful drug development. In this article, we discuss how cellular microsystems can establish microenvironments for these applications and propose how they could be used for potentially controlling the differentiation of hepatocytes or cardiomyocytes. The physiological relevance of cells is enhanced in cellular microsystems by simulating properties of tissue microenvironments, such as structural dimensionality, media flow, microfluidic control of media composition, and co-cultures with interacting cell types. Recent studies demonstrated that these properties also affect iPSC differentiations and we further elaborate on how they could control differentiation efficiency in microengineered devices. In summary, we describe recent advances in the field of cellular microsystems that can control the differentiation and maturation of hepatocytes and cardiomyocytes for drug evaluation. We also propose how future research with iPSCs within engineered microenvironments could enable their differentiation for scalable evaluations of drug effects.     

Structural Basis for the Interaction of the Golgi-Associated Retrograde Protein Complex with the t-SNARE Syntaxin 6

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Altered trafficking of lysosomal proteins in Hermansky-Pudlak syndrome due to mutations in the beta 3A subunit of the AP-3 adaptor

Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defective lysosome-related organelles. Here, we report the identification of two HPS patients with mutations in the beta 3A subunit of the heterotetrameric AP-3 complex. The patients' fibroblasts exhibit drastically reduced levels of AP-3 due to enhanced degradation of mutant beta 3A. The AP-3 deficiency results in increased surface expression of the lysosomal membrane proteins CD63, lamp-1, and lamp-2, but not of nonlysosomal proteins. These differential effects are consistent with the preferential interaction of the AP-3 mu 3A subunit with tyrosine-based signals involved in lysosomal targeting. Our results suggest that AP-3 functions in protein sorting to lysosomes and provide an example of a human disease in which altered trafficking of integral membrane proteins is due to mutations in a component of the sorting machinery.     

Interactions of GGA3 with the ubiquitin sorting machinery

The Golgi-localized, gamma-ear-containing, Arf-binding (GGA) proteins constitute a family of clathrin adaptors that are mainly associated with the trans-Golgi network (TGN) and mediate the sorting of mannose 6-phosphate receptors. This sorting is dependent on the interaction of the VHS domain of the GGAs with acidic-cluster-dileucine signals in the cytosolic tails of the receptors. Here we demonstrate the existence of another population of GGAs that are associated with early endosomes. RNA interference (RNAi) of GGA3 expression results in accumulation of the cation-independent mannose 6-phosphate receptor and internalized epidermal growth factor (EGF) within enlarged early endosomes. This perturbation impairs the degradation of internalized EGF, a process that is normally dependent on the sorting of ubiquitinated EGF receptors (EGFRs) to late endosomes. Protein interaction analyses show that the GGAs bind ubiquitin. The VHS and GAT domains of GGA3 are responsible for this binding, as well as for interactions with TSG101, a component of the ubiquitin-dependent sorting machinery. Thus, GGAs may have additional roles in sorting of ubiquitinated cargo.     

Pallidin is a component of a multi-protein complex involved in the biogenesis of lysosome-related organelles

The Hermansky–Pudlak syndrome defines a group of genetic disorders characterized by defective lysosome-related organelles such as melanosomes and platelet dense bodies. Hermansky–Pudlak syndrome can be caused by mutations of at least four genes in humans and 15 genes in mice. One of these genes is mutated in the pallid mouse strain and encodes a novel protein named pallidin (L. Huang, Y. M. Kuo and J. Gitschier, Nat Genet 1999; 23: 329–332). Pallidin has no homology to any other known protein and no recognizable functional motifs. We have conducted a biochemical characterization of human pallidin using a newly developed polyclonal antibody. We show that pallidin is a ubiquitously expressed ∼ 25 kDa protein found both in the cytosol and peripherally associated to membranes. Sedimentation velocity analyses show that native pallidin has a sedimentation coefficient of ∼ 5.1 S, much larger than expected from the molecular mass of the pallidin polypeptide. In line with this observation, cosedimentation and coprecipitation analyses reveal that pallidin is part of a hetero-oligomeric complex. One of the subunits of this complex is the product of another Hermansky–Pudlak syndrome gene, muted. Fibroblasts derived from the muted mouse strain exhibit reduced levels of pallidin, suggesting that the absence of the muted protein destabilizes pallidin. These observations indicate that pallidin is a subunit of a novel multi-protein complex involved in the biogenesis of lysosome-related organelles.     

Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae

The biosynthetic sorting of hydrolases to the yeast vacuole involves transport along two distinct routes referred to as the carboxypeptidase Y and alkaline phosphatase pathways. To identify genes involved in sorting to the vacuole, we conducted a genome-wide screen of 4653 homozygous diploid gene deletion strains of Saccharomyces cerevisiae for missorting of carboxypeptidase Y. We identified 146 mutant strains that secreted strong-to-moderate levels of carboxypeptidase Y. Of these, only 53 of the corresponding genes had been previously implicated in vacuolar protein sorting, whereas the remaining 93 had either been identified in screens for other cellular processes or were only known as hypothetical open reading frames. Among these 93 were genes encoding: 1) the Ras-like GTP-binding proteins Arl1p and Arl3p, 2) actin-related proteins such as Arp5p and Arp6p, 3) the monensin and brefeldin A hypersensitivity proteins Mon1p and Mon2p, and 4) 15 novel proteins designated Vps61p-Vps75p. Most of the novel gene products were involved only in the carboxypeptidase Y pathway, whereas a few, including Mon1p, Mon2p, Vps61p, and Vps67p, appeared to be involved in both the carboxypeptidase Y and alkaline phosphatase pathways. Mutants lacking some of the novel gene products, including Arp5p, Arp6p, Vps64p, and Vps67p, were severely defective in secretion of mature alpha-factor. Others, such as Vps61p, Vps64p, and Vps67p, displayed defects in the actin cytoskeleton at 30 degrees C. The identification and phenotypic characterization of these novel mutants provide new insights into the mechanisms of vacuolar protein sorting, most notably the probable involvement of the actin cytoskeleton in this process.     

Enthoprotin: a novel clathrin-associated protein identified through subcellular proteomics

Despite numerous advances in the identification of the molecular machinery for clathrin-mediated budding at the plasma membrane, the mechanistic details of this process remain incomplete. Moreover, relatively little is known regarding the regulation of clathrin-mediated budding at other membrane systems. To address these issues, we have utilized the powerful new approach of subcellular proteomics to identify novel proteins present on highly enriched clathrin-coated vesicles (CCVs). Among the ten novel proteins identified is the rat homologue of a predicted gene product from human, mouse, and Drosophila genomics projects, which we named enthoprotin. Enthoprotin is highly enriched on CCVs isolated from rat brain and liver extracts. In cells, enthoprotin demonstrates a punctate staining pattern that is concentrated in a perinuclear compartment where it colocalizes with clathrin and the clathrin adaptor protein (AP)1. Enthoprotin interacts with the clathrin adaptors AP1 and with Golgi-localized, gamma-ear-containing, Arf-binding protein 2. Through its COOH-terminal domain, enthoprotin binds to the terminal domain of the clathrin heavy chain and stimulates clathrin assembly. These data suggest a role for enthoprotin in clathrin-mediated budding on internal membranes. Our study reveals the utility of proteomics in the identification of novel vesicle trafficking proteins.