Biomedical subjects

J J Hsuan

Publications and source records attributed to J J Hsuan.

11 recordsLinked to original sources

PubMed1992

Apolipoprotein AI mutation Arg-60 causes autosomal dominant amyloidosis.

A mutation in the gene for apolipoprotein AI (apoAI) was identified in an English family with autosomal dominant non-neuropathic systemic amyloidosis. The plasma of all affected individuals contained a variant apoAI with one additional charge, as well as normal apoAI. The propositus was heterozygous; the coding region of his apoAI gene contained both the normal sequence and a single-base substitution changing the codon for residue 60 of the mature protein from CTG (leucine) to CGG (arginine). Allele-specific oligonucleotide hybridization showed that the other affected individuals were also heterozygotes and that there was concordance of the mutant allele with the presence of variant plasma apoAI. Amyloid fibrils isolated from the spleen of the propositus consisted of proteins that ran as a doublet with an apparent mass of approximately 10 kDa in SDS/PAGE and a trace band at 28 kDa. Electrospray mass spectrometry of the purified 10-kDa material revealed components with mass corresponding to the N-terminal 88, 92, 93, and 94 residues of apoAI each with substitution of arginine for leucine. These observations were confirmed by direct protein sequencing and laser desorption time-of-flight mass analysis. No material with the normal apoAI sequence was detected. The trace band at 28 kDa yielded the N-terminal sequence of mature apoAI, indicating that intact or minimally degraded apoAI was also present in the fibril preparation. Discovery of this mutation and the detailed characterization of the apoAI fragments that form the amyloid fibrils open additional avenues for investigation of amyloidogenesis.

Amino Acid Sequence↗

PubMed1992

Casein kinase II phosphorylation increases the rate of serum response factor-binding site exchange.

Recombinant baculoviruses were used to express wild-type serum response factor (SRF) and a mutant, SRF.CKIIA, which lacks all four serine residues in the major casein kinase II (CKII) site at residues 77-90. Purified recombinant SRF binds DNA with an affinity and specificity indistinguishable from that of HeLa cell SRF, and activates transcription in vitro. Comparative phosphopeptide analysis of the wild-type and mutant proteins demonstrated that the wild-type protein is phosphorylated at the major CKII site in insect cells. Dephosphorylation of recombinant SRF does not affect its affinity for the c-fos SRE, and results in only a 3-fold reduction in binding to the synthetic site ACT.L. However, dephosphorylation does cause a large decrease in the rates of association with and dissociation from either site. These effects are due solely to phosphorylation at the major CKII site: the binding properties of the SRF.CKIIA mutant are identical to those of dephosphorylated wild-type SRF, and CKII phosphorylation in vitro converts dephosphorylated wild-type SRF from a slow-binding to a fast-binding form without significantly changing binding affinity. CKII phosphorylation thus acts to potentiate SRF-DNA exchange rates rather than alter equilibrium binding affinity.

Animals↗

PubMed1992

Accelerated high-sensitivity microsequencing of proteins and peptides using a miniature reaction cartridge.

Greatly reduced automated protein sequencing degradation times of less than 25 min with concurrent on-line phenylthiohydantoin (PTH) derivative analysis times of less than 16 min have been achieved by using a miniaturized reaction cartridge with optimized chemical and analytical cycles. Using this method a wide range of standard and novel peptides and proteins have been sequenced with reproducibly high initial and repetitive cycle yields. In these accelerated analyses the recovery of the more labile PTH derivatives was markedly improved by using elevated pressure during cleavage steps and temperature programming throughout the Edman cycle.

Amino Acid Sequence↗

PubMed1989

Identification of a novel autophosphorylation site (P4) on the epidermal growth factor receptor.

Three major autophosphorylation sites are located near the C-terminus of the epidermal growth factor receptor, but a fourth site is repeatedly detected. We report here the purification and sequencing of a tryptic peptide containing this site, Tyr-1086. Furthermore, we demonstrate that additional phosphopeptides are observed following both partial digestion and overdigestion. Finally, we show that Tyr-1086 can be phosphorylated in intact cells.

Amino Acid Sequence↗

PubMed1989

Proteolytic generation of constitutive tyrosine kinase activity of the human insulin receptor.

Structural modification induced by partial digestion with trypsin has been shown to stimulate the tyrosine kinase activity of the insulin receptor both in solution and in intact cells [Tamura, Fujita-Yamaguchi & Larner (1983) J. Biol. Chem. 258, 14749-14752; Goren, White & Kahn (1987) Biochemistry 26, 2374-2382; Leef & Larner (1987) J. Biol. Chem. 262, 14837-14842]. Furthermore, experiments involving deletion of sequences encoding the extracellular domain of the insulin receptor suggest that it may function as a protooncogene in fibroblasts [Wang et al., (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5725-5729]. To further understand the structural requirements that generate this activity, the major activated fragments generated in solution following trypsin digestion have been characterized here, one of which is shown to have a similar amino acid sequence to a transforming protein. Furthermore, treatment with trypsin of intact Chinese hamster ovary cells that overexpress the human insulin receptor stimulates both autophosphorylation of the receptor and 2-deoxyglucose uptake into the cells, but does not enhance receptor internalization. Unlike digestion in solution, no proteolysis or loss of activity of the activated insulin receptor beta-subunit could be detected using intact cells, even at high trypsin concentrations, despite the existence of extracellular sites that are readily cleaved by trypsin in the solubilized receptor. These studies provide further detail of a mechanism used during trypsinization of cells in culture which mimics activation of the insulin receptor and contributes to stimulation of growth.

Amino Acid Sequence↗

PubMed1989

Structural basis for epidermal growth factor receptor function.

The receptor for epidermal growth factor (EGF) has been the subject of intense study primarily as a consequence of the pioneering studies of Cohen on growth factors and also because of its homology to the transforming protein encoded by the avian oncogene v-erbB, which is a truncated receptor and its consequent role in cancer. Although similar structural mutation of the EGF receptor has not yet been found in human tumours, aberrant overexpression of both EGF receptors and c-erbB2, a closely related putative receptor, have been found to occur in squamous cell carcinomas and glial tumours, and mammary carcinomas respectively. In addition to EGF, the related polypeptides transforming growth factor alpha (TGF alpha) and vaccinia virus growth factor are also ligands for the EGF receptor. Expression of TGF alpha occurs during embryonal development and in specific adult tissues; it may also play a role in cellular transformation These important properties, as well as the potential roles of both TGF alpha and EGF in wound repair, have emphasized the need to understand EGF receptor structure, function and regulation. This review discusses the structural properties of the EGF receptor and how these can be related to receptor function and regulation.

Amino Acid Sequence↗

PubMed1989

Transforming growth factors beta.

Since its discovery as a factor able to induce phenotypic transformation with transforming growth factor alpha, transforming growth factor beta (TGF beta) has been found to have unexpectedly widespread and frequently, seemingly paradoxical effects on a variety of cell types. Thus, a significant property of TGF beta activity is the ability to inhibit cellular growth and transformation. More recently, further important potential roles have also been found for TGF beta including the control of embryonal development, cellular differentiation, hormone secretion and immune function. Multiple forms of both TGF beta and TGF beta receptors are now known to exist, which may generate a complex and diverse range of interacting signals. The multifunctional actions of the TGF beta family are frequently defined in concert with other extracellular factors and much still has to be understood concerning these synergistic processes.

Animals↗

PubMed1989

Analysis of mammalian fibroblast transformation by normal and mutated human EGF receptors.

Activation of the EGF receptor (c-erbB) tyrosine kinase has been implicated in tumorigenesis, either by overexpression of the normal receptor in the presence of EGF, or through expression of a truncated receptor lacking the EGF binding domain as in the viral oncogene v-erbB. Here, normal and truncated human EGF receptors expressed in Rat1 fibroblasts were analysed for receptor tyrosine kinase activity and several transformation parameters in comparison with polyoma middle T and EJ-ras. Expression of a truncated EGF receptor lacking the extracellular ligand binding domain induced transformation of immortalized rodent fibroblasts and appears to activate the intrinsic tyrosine kinase. The transformed phenotype becomes enhanced by further truncation of the C-terminal domain containing the tyrosine autophosphorylation sites P1 and P2. Over expression of EGF receptors with an intact extracellular region in transfected Rat1 cells shows EGF dependent transformation, which is reduced by C-terminal truncation. Transformation is dependent on the cellular receptor concentration and can be selected as a stable phenotype. We conclude that expression of receptors with a truncated EGF-binding domain alone is sufficient to transform mammalian fibroblasts, in contrast to chick fibroblasts transformed by v-erbB where additional deletion of C-terminal receptor sequences appears to be an absolute requirement.

Animals↗

PubMed1988

Loss of three major auto phosphorylation sites in the EGF receptor does not block the mitogenic action of EGF.

The EGF receptor cDNA has been transfected into receptor-negative Chinese hamster ovary (CHO) cells. A mutant cell line (CHO 11) was isolated that expresses a receptor of lower molecular weight than the EGF receptor from A431 cells (150,000 daltons compared to 170,000 daltons) and which appeared as a doublet on SDS-PAGE. By digestion of the receptor with endoglycosidase F it was shown that an altered pattern of glycosylation could not account for the smaller size of the protein, although it could explain the appearance of the CHO 11 receptor as a doublet protein. A deletion was located to the transfected cDNA and shown to involve the removal of coding sequences for the most C-terminal 20,000 daltons of the EGF receptor, which contains the three major autophosphorylation sites. Despite the loss of these sites the EGF receptor from CHO 11 cells binds EGF, demonstrates protein tyrosine kinase activity in response to EGF, and transduces a mitogenic signal. The CHO 11 receptor protein is still autophosphorylated on alternative tyrosine residues. We conclude that phosphorylation of the three tyrosines (P1, P2, and P3) in the C-terminal domain of the receptor is not required for signal transduction by the EGF receptor in these cells.

Animals↗

PubMed1987

The cross-linking of tyrosine residues in apo-ovotransferrin by treatment with periodate anions.

The iron-binding ability of apotransferrins is rapidly abolished in the reaction with periodate anions, which destroys 4 mol of tyrosine per mol of protein. Treatment of ovotransferrin with cyanogen bromide and tryptic digestion of the glycopeptide fragment demonstrated the existence of an intramolecular cross-link in the C-terminal domain of the oxidized protein. The cross-linked residues were identified as Tyr-421 and Tyr-524 and the product is similar in structure to 3,3'-dityrosine.

Amino Acids↗

PubMed1986

The ability of salts to inhibit the reaction between periodate anions and ovotransferrin.

The reaction between periodate anions and apo-ovotransferrin results in the rapid abolition of the iron-binding ability of the protein and the loss of approximately 4 mol of tyrosine/mol of protein. The degree of inhibition exerted by a variety of salts on the rate of this reaction is found to be inconsistent with the lyotropic series and suggests the existence of a complex anion-binding site in the apoprotein. The existence of this site may explain the action of periodate anions on ovotransferrin.

Apoproteins↗