The essential endoplasmic reticulum chaperone Rot1 is required for protein N- and O-glycosylation in yeast

Abstract Rot1 is an essential yeast protein originally shown to be implicated in such diverse processes such as β-1,6-glucan synthesis, actin cytoskeleton dynamics, or lysis of autophagic bodies. More recently also a role as a molecular chaperone has been discovered. Here we report that Rot1 interacts in a synthetic manner with Ost3, one of the nine subunits of the oligosaccharyltransferase complex, the key enzyme of N-glycosylation. Deletion of OST3 in the rot1-1 mutant causes a temperature sensitive phenotype as well as sensitivity towards compounds interfering with cell wall biogenesis such as Calcofluor White, caffeine, Congo Red and hygromycin B, whereas deletion of OST6, a functional homolog of OST3, has no effect. Oligosaccharyltransferase activity in vitro determined in membranes from rot1-1ost3∆ cells was found to be decreased to 45% compared to wild-type membranes, and model glycoproteins of N-glycosylation, like carboxypeptidase CPY, Gas1 or DPAP B, displayed an underglycosylation pattern. By affinity chromatography a physical interaction between Rot1 and Ost3 was demonstrated. Moreover, Rot1 was found to be involved also in the Omannosylation process, as glycosylation of distinct glycoproteins of this type were affected as well. Altogether the data extend the role of Rot1 as a chaperone required to ensure proper glycosylation

The essential endoplasmic reticulum chaperone Rot1 is required for protein N- and O-glycosylation in yeast

Rot1 is an essential yeast protein originally shown to be implicated in such diverse processes such as β-1,6-glucan synthesis, actin cytoskeleton dynamics, or lysis of autophagic bodies. More recently also a role as a molecular chaperone has been discovered. Here we report that Rot1 interacts in a synthetic manner with Ost3, one of the nine subunits of the oligosaccharyltransferase complex, the key enzyme of N-glycosylation. Deletion of OST3 in the rot1-1 mutant causes a temperature sensitive phenotype as well as sensitivity towards compounds interfering with cell wall biogenesis such as Calcofluor White, caffeine, Congo Red and hygromycin B, whereas deletion of OST6, a functional homolog of OST3, has no effect. Oligosaccharyltransferase activity in vitro determined in membranes from rot1-1ost3Δ cells was found to be decreased to 45% compared to wild-type membranes, and model glycoproteins of N-glycosylation, like carboxypeptidase CPY, Gas1 or DPAP B, displayed an underglycosylation pattern. By affinity chromatography a physical interaction between Rot1 and Ost3 was demonstrated. Moreover, Rot1 was found to be involved also in the Omannosylation process, as glycosylation of distinct glycoproteins of this type were affected as well. Altogether the data extend the role of Rot1 as a chaperone required to ensure proper glycosylation. Keywords: ROT1/ N‐glycosylation/ O‐glycosylation/ oligosaccharyltransferase/ dolichol/ Saccharomyces cerevisiae Downloaded from http://glycob.oxfordjournals.org/ at Instytut Biochemii i Biofizyki PAN on April 17, 201

Impact of Yeast Glycosylation Pathway on Cell Integrity and Morphology, Glycosylation, Stefana Petrescu (Ed.), ISBN: 978-953-51-0771-2, InTech, Available from: http://www.intechopen.com/books/glycosylation/impact-of-yeast-glycosylation-pathway-on-cell-integrity-and-morphology

Protein glycosylation is a multi step reaction, well conserved in the eukaryotic cells. In N-glycosylation reactions dolichyl phosphate (DolP) serves as a lipid acceptor of sugar residues forming DolPPGlcNAc2Man9Glc3. Dolichyl phosphate mannose (DolPMan) is also a substrate for protein O-glycosylation, where it serves as a donor of the first mannose to be attached to hydroxyl groups of serine or treonine. DolPMan is also involved in the synthesis of the sugar part of glycosylphosphatidyl inositol anchor in yeast and other eukaryotes. Its remnant structure is responsible for the attachment of a large group of glycoproteins to the glucan polymers of the cell wall . Thus, a functional link could be predicted between the dolichol biosynthetic (mevalonate) pathway and subsequent N-glycosylation and O-mannosylation, cell wall assembly and/or fungus–host interaction. Moreover, on the basis of the data presented in this chapter, it can be assumed that the glycosylation pathway in yeast and fungi offers many levels of regulation, which might influence the final quality and quantity of cell wall glycoproteins and consequently cell surface immunogenicity. In this work we concentrate on early glycosylation defects, resulting from the impaired synthesis of dolichol (Dol) and dolichyl phosphate (DolP) or DolPP oligosaccharide (DolPPGlcNAc2Man9Glc3) assembly, and their effect on the cell integrity and morphology

A study of the activity and effectiveness of recombinant fibroblast growth factor (Q40P/S47I/H93G rFGF-1) in anti-aging treatment

Introduction : Fibroblast growth factor 1 (FGF-1) is a powerful mitogen involved in the stimulation of DNA synthesis and the proliferation of a wide variety of cell types. Fibroblast growth factor 1 was genetically modified to improve its thermal stability and resistance to protease degradation without losing its biological activity. Aim : To study the impact of Q40P/S47I/H93G rFGF-1 on skin cells, its penetration through the skin and the evaluation of the rFGF-1-cosmetic product properties. Material and methods : In vitro studies included the examination of primary fibroblast and keratinocyte viability after the incubation with rFGF-1. The penetration abilities of rFGF-1 in various formulations and carrier systems were examined ex vivo by the Raman spectroscopy. In vivo studies – HF Ultrasound and 3D Imaging System – were used to evaluate the anti-aging properties of creams containing rFGF-1. Results : In vitro studies demonstrated that rFGF-1 strongly enhanced the viability of the treated cells. The Raman Spectroscopy analysis indicated that rFGF-1 encapsulated in lipid spheres penetrate through the stratum corneum to the depth of 60 μm, and added to the o/w formulation – could penetrate to a depth of 90 μm. The results obtained from Primos revealed the reduction of the volume and the depth of the wrinkles. Changes in the skin structure in the analyzed areas were evaluated by HF Ultrasonography. Conclusions : Recombinant FGF-1 strongly stimulated fibroblast and keratinocyte proliferation. However, the transition of this protein through the SC required an appropriate carrier system – lipid spheres. All tests – in vitro , ex vivo and in vivo – have proved that rFGF-1 is a substance with a potentially wide spectrum of use

The Protective Effect of Simvastatin on the Systolic Function of the Heart in the Model of Acute Ischemia and Reperfusion Is Due to Inhibition of the RhoA Pathway and Independent of Reduction of MMP-2 Activity

The present study investigated whether Rho-associated protein kinase (RhoA/ROCK) signaling pathway inhibitor simvastatin inhibits matrix metalloproteinase 2 (MMP-2) activity in a rat ischemia-reperfusion injury (I/Ri) model by inhibiting the RhoA/ROCK pathway and reducing MMP-2 mRNA levels. Isolated rat hearts were subjected to aerobic perfusion or I/Ri control. The effect of simvastatin was assessed in hearts subjected to I/Ri. We determined cardiac mechanical function, the content of RhoA, phosphorylated myosin light chain subunit 1 (phospho-MYL9), troponin I, MMP-2, and MMP-2 mRNA in the heart homogenates, as well as MMP-2 activity in heart tissue. We showed that treatment with simvastatin caused improvement in the contractile function of the heart subjected to I/Ri which was accompanied by a decrease of MMP-2 activity in heart tissue along with inhibition of RhoA pathway, expressed in a reduction in both RhoA and its downstream product—phosphorylated myosin light chain (phospho-MYL9) in hearts treated with simvastatin. MMP-2 inactivation is not due to inhibition of MMP-2 m-RNA synthesis caused by inhibition of RhoA/ROCK pathway and is due, at least in part, to the direct drug action. The protective effect of simvastatin on systolic function in the acute ischemia-reperfusion model does not appear to be related to reduced MMP-2 activation, but other mechanisms related with the inhibition RhoA/ROCK pathway

New lupeol esters as active substances in the treatment of skin damage.

The purpose of the research was to obtain new derivatives of natural triterpene lupeol and to evaluate their potential as active substances in the treatment of skin damage. Four new lupeol esters (propionate, succinate, isonicotinate and acetylsalicylate) and lupeol acetate were obtained using an eco-friendly synthesis method. In the esterification process, the commonly used hazardous reagents in this type of synthesis were replaced by safe ones. This unconventional, eco-friendly, method is particularly important because the compounds obtained are potentially active substances in skin care formulations. Even trace amounts of hazardous reagents can have a toxic effect on damaged or irritated tissues. The molecular structure of the esters were confirmed by 1H NMR, 13C NMR and IR spectroscopy methods. Their crystal structures were determined using XRD method. To complete the analysis of their characteristics, physicochemical properties (melting point, lipophilicity, water solubility) and biological activity of the lupeol derivatives were studied. Results of an irritant potential test, carried out on Reconstructed Human Epidermis (RHE), confirmed that the synthesized lupeol derivatives are not cytotoxic and they stimulate a process of human cell proliferation. The safety of use for tested compounds was determined in a cell viability test (cytotoxicity detection kit based on the measurement of lactate dehydrogenase activity) for keratinocytes and fibroblasts. The results obtained showed that the modification of lupeol structure improve its bioavailability and activity. All of the esters penetrate the stratum corneum and the upper layers of the dermis better than the maternal lupeol. Lupeol isonicotinate, acetate and propionate were the most effective compounds in a stimulation of the human skin cell proliferation process. This combination resulted in an increase in the concentration of cells of more than 30% in comparison to control samples. The results indicate that the chemical modification of lupeol allows to obtain promising active substances for treatment of skin damage, including thermal, chemical and radiation burns

RhoA/ROCK Pathway Is Upregulated in Experimental Autoimmune Myocarditis and Is Inhibited by Simvastatin at the Stage of Myosin Light Chain Phosphorylation

Many studies have proven the involvement of the RhoA/ROCK pathway in autoimmune and cardiovascular diseases and the beneficial effects of its downregulation. Here, we examined whether the effect of simvastatin on experimental autoimmune myocarditis (EAM) may be through targeting the Ras homolog family member A/Rho-associated coiled-coil containing kinases (RhoA/ROCK) pathway and whether previously shown downregulation of metalloproteinase 9 (MMP-9) could be associated with MLC phosphorylation. Two doses of simvastatin were administered to experimental rats with autoimmune myocarditis by gastric gavage for 3 weeks, at the stage of development of the inflammatory process. Immunohistochemical staining for RhoA and ROCK1 was evaluated semi-quantitatively with H-score. The RhoA staining showed no significant differences in expression between the groups, but the ROCK1 expression was significantly upregulated in the hearts of the EAM group and was not downregulated by simvastatin. The Western blotting analysis of the last downstream product of the RhoA/ROCK axis, phosphorylated myosin light chain (phospho-MYL9), revealed that protein content increased in EAM hearts and it was prevented by the highest dose of simvastatin. Our findings suggest that the RhoA/ROCK pathway is upregulated in EAM, and simvastatin in EAM settings inhibits the RhoA/ROCK pathway at the stage of phosphorylation of myosin light chains and provides a new insight into the molecular pathology of autoimmune myocarditis