Accumulation of properly folded human type III procollagen molecules in specific intracellular membranous compartments in the yeast Pichia pastoris

It was recently reported that co-expression of the proal(III) chain of human type III procollagen with the subunits of human prolyl 4-hydroxylase in Pichia pastoris produces fully hydroxylated and properly folded recombinant type III procollagen molecules (Vuorela, A., Myllyharju, J., Nissi, R., Pihlajaniemi, T., Kivirikko, K.I., 1997. Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast Pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase. EMBO J, 16, 6702-6712). These properly folded molecules accumulated inside the yeast cell, however, only similar to 10% were found in the culture medium. We report here that replacement of the authentic signal sequence of the human pro alpha 1(III) with the Saccharomyces cerevisiae alpha mating factor prepro sequence led only to a minor increase in the amount secreted. Immunoelectron microscopy studies indicated that the procollagen molecules accumulate in specific membranous vesicular compartments that are closely associated with the nuclear membrane. Prolyl 4-hydroxylase, an endoplasmic reticulum (ER) lumenal enzyme, was found to be located in the same compartments. Non-helical pro alpha 1(III) chains produced by expression without recombinant prolyl 4-hydroxylase likewise accumulated within these compartments, The data indicate that properly folded recombinant procollagen molecules accumulate within the ER and do not proceed further in the secretory pathway. This may be related to the large size of the procollagen molecule. (C) 2000 Elsevier Science B.V./International Society of Matrix Biology. All rights reserved

Accumulation of properly folded human type III procollagen molecules in specific intracellular membranous compartments in the yeast Pichia pastoris

It was recently reported that co-expression of the proal(III) chain of human type III procollagen with the subunits of human prolyl 4-hydroxylase in Pichia pastoris produces fully hydroxylated and properly folded recombinant type III procollagen molecules (Vuorela, A., Myllyharju, J., Nissi, R., Pihlajaniemi, T., Kivirikko, K.I., 1997. Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast Pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase. EMBO J, 16, 6702-6712). These properly folded molecules accumulated inside the yeast cell, however, only similar to 10% were found in the culture medium. We report here that replacement of the authentic signal sequence of the human pro alpha 1(III) with the Saccharomyces cerevisiae alpha mating factor prepro sequence led only to a minor increase in the amount secreted. Immunoelectron microscopy studies indicated that the procollagen molecules accumulate in specific membranous vesicular compartments that are closely associated with the nuclear membrane. Prolyl 4-hydroxylase, an endoplasmic reticulum (ER) lumenal enzyme, was found to be located in the same compartments. Non-helical pro alpha 1(III) chains produced by expression without recombinant prolyl 4-hydroxylase likewise accumulated within these compartments, The data indicate that properly folded recombinant procollagen molecules accumulate within the ER and do not proceed further in the secretory pathway. This may be related to the large size of the procollagen molecule. (C) 2000 Elsevier Science B.V./International Society of Matrix Biology. All rights reserved.</p

Adiponectin Upregulates Prolyl-4-Hydroxylase α1 Expression in Interleukin 6-Stimulated Human Aortic Smooth Muscle Cells by Regulating ERK 1/2 and Sp1

Adiponectin is an anti-atherogenic adipokine that inhibits the development of plaque by mechanisms that are not completely understood. Extracellular matrix (ECM) may have a role in the pathogenesis of atherosclerosis. We explored the effect and mechanisms of adiponectin on the synthesis of prolyl-4-hydroxylase (P4H) in interleukin 6 (IL-6)-stimulated human aortic smooth muscle cells (HASMCs). P4Hα1 mRNA level was quantified by RT-PCR, the protein levels of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and P4Hα1 were quantified by western blot analysis, and activation of specific protein 1 (Sp1) was determined by electrophoretic mobility shift assay and subcellular localization of Sp1 by immunofluorescence analysis. Adiponectin significantly increased P4Hα1 mRNA and protein levels in IL-6-stimulated HASMCs in a dose- and time-dependent manner. As well, ERK1/2 and Sp1 played a crucial role in the effect of adiponectin upregulating P4Hα1 expression in IL-6-stimulated HASMCs. Adiponectin abrogated the effects of IL-6 on collagen III level, which may indicate that P4Hα1 is essential for folding the procollagen polypeptide chains into stabilized collagen. Adiponectin attenuates IL-6–inhibited P4Hα1 synthesis and stabilizes collagen formation in HASMCs through a Sp1-ERK1/2-P4Hα1-dependent pathway

The Classic: A Morphogenetic Matrix for Differentiation of Cartilage in Tissue Culture

This Classic Article is a reprint of the original work by Hiroshi Nogami and Marshall R. Urist, A Morphogenetic Matrix for Differentiation of Cartilage in Tissue Culture. An accompanying biographical sketch of Marshall R. Urist, MD is available at DOI 10.1007/s11999-009-1067-4; a second Classic Article is available at DOI 10.1007/s11999-009-1068-3; and a third Classic Article is available at DOI 10.1007/s11999-009-1070-9. The Classic Article is © 1970 by the Society for Experimental Biology and Medicine and is reprinted with permission from Nogami H, Urist MR. A morphogenetic matrix for differentiation of cartilage in tissue culture. Proc Soc Exp Biol Med. 1970;134;530–535

Neuronal Nitric Oxide Synthase-Rescue of Dystrophin/Utrophin Double Knockout Mice does not Require nNOS Localization to the Cell Membrane

Survival of dystrophin/utrophin double-knockout (dko) mice was increased by muscle-specific expression of a neuronal nitric oxide synthase (nNOS) transgene. Dko mice expressing the transgene (nNOS TG+/dko) experienced delayed onset of mortality and increased life-span. The nNOS TG+/dko mice demonstrated a significant decrease in the concentration of CD163+, M2c macrophages that can express arginase and promote fibrosis. The decrease in M2c macrophages was associated with a significant reduction in fibrosis of heart, diaphragm and hindlimb muscles of nNOS TG+/dko mice. The nNOS transgene had no effect on the concentration of cytolytic, CD68+, M1 macrophages. Accordingly, we did not observe any change in the extent of muscle fiber lysis in the nNOS TG+/dko mice. These findings show that nNOS/NO (nitric oxide)-mediated decreases in M2c macrophages lead to a reduction in the muscle fibrosis that is associated with increased mortality in mice lacking dystrophin and utrophin. Interestingly, the dramatic and beneficial effects of the nNOS transgene were not attributable to localization of nNOS protein at the cell membrane. We did not detect any nNOS protein at the sarcolemma in nNOS TG+/dko muscles. This important observation shows that sarcolemmal localization is not necessary for nNOS to have beneficial effects in dystrophic tissue and the presence of nNOS in the cytosol of dystrophic muscle fibers can ameliorate the pathology and most importantly, significantly increase life-span