Anna-Liisa Hänninen

Genome organization of membrane-containing bacteriophage PRD1

We have determined the nucleotide sequence of the late region (11 kbp) of the lipid-containing bacteriophage PRD1. Gene localization was carried out by complementing nonsense phage mutants with genomic clones containing specific reading frames. The localization was confirmed by sequencing the N-termini of isolated gene products as well as sequencing the N-termini of tryptic fragments of the phage membrane-associated proteins. This, with the previously obtained sequence of the early regions, allowed us to organize most of the phage genes in the phage genome.

Trehalose is required for conformational repair of heat-denatured proteins in the yeast endoplasmic reticulum but not for maintenance of membrane traffic functions after severe heat stress.

Saccharomyces cerevisiae cells grown at physiological temperature 24°C require preconditioning at 37°C to acquire tolerance towards brief exposure to 48–50°C. During preconditioning, the cytosolic trehalose content increases remarkably and in the absence of trehalose synthesis yeast cannot acquire thermotolerance. It has been speculated that trehalose protects proteins and membranes under environmental stress conditions, but recently it was shown to assist the Hsp104 chaperone in refolding of heat‐damaged proteins in the yeast cytosol. We have demonstrated that heat‐denatured proteins residing in the endoplasmic reticulum (ER) also can be refolded once the cells are returned to physiological temperature. Unexpectedly, not only ER chaperones but also the cytosolic Hsp104 chaperone is required for conformational repair events in the ER lumen. Here we show that trehalose facilitates refolding of glycoproteins in the ER after severe heat stress. In the absence of Tps1p, a subunit of trehalose synthase, refolding of heat‐damaged glycoproteins to bioactive and secretion‐competent forms failed or was retarded. In contrast, membrane traffic operated many hours after severe heat stress even in the absence of the TPS1 gene, demonstrating that trehalose had no role in thermoprotection of membranes engaged in vesicular traffic. However, cytosolic proteins were aggregated and protein synthesis abolished, resulting finally in cell death.

Vascular endothelial growth factor C acts as a neurotrophic factor for dopamine neurons in vitro and in vivo

Neurotrophic factors regulate the development and maintenance of the nervous system and protect and repair dopaminergic neurons in animal models of Parkinsons disease (PD). Vascular endothelial growth factors A (VEGF-A) and B have also neurotrophic effects on various types of neurons, including dopaminergic neurons. We examined the ability of the key lymphangiogenic factor VEGF-C to protect dopaminergic cells in vitro and in vivo. The study was initiated by a finding from microarray profiling of Neuro2A-20 cells which revealed up-regulation of VEGF-C by glial cell-line-derived neurotrophic factor (GDNF). Next, we observed that VEGF-C can rescue embryonic dopaminergic neurons and activate the mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) pathway in vivo. VEGF receptors 1-2 and co-receptors, neuropilins 1-2, were expressed both in mouse embryonic cultures and adult midbrains. In vivo, VEGF-C had a robust functional effect in the rat unilateral 6-hydroxydopamine (6-OHDA) model of PD and there was a small additive effect on the survival of tyrosine hydroxylase (TH)-positive cells with GDNF. The neuroprotective effect of VEGF-C is most likely due to a combination of direct and indirect neurotrophic effects because, VEGF-C, unlike GDNF, induced also angiogenesis in the striatum following 6-OHDA insult as it did in human umbilical vein endothelial cells (HUVEC). However, we detected activation of astroglia and microglia as well as blood-brain barrier disruption after intracerebral delivery of VEGF-C, raising a concern of its safe usage as a therapeutic molecule. Our results provide evidence of VEGF-C as a neurotrophic factor that influences the dopaminergic system through multiple mechanisms.

Validation of the Hsp150 Polypeptide Carrier and HSP150 Promoter in Expression of Rat α2,3-Sialyltransferase in Yeasts

Heterologous glycoproteins usually do not fold properly in yeast cells and fail to leave the endoplasmic reticulum. Here we show that the Hsp150Δ polypeptide carrier promoted proper folding and secretion of the catalytic ectodomain of rat α2,3‐sialyltransferase (ST3Ne) in Pichia pastoris.The efficiency of the Hsp150Δ carrier in P. pastoris and Saccharomyces cerevisiae was at least as high as that of the MFα carrier. Most of Hsp150Δ‐ST3Ne and MFα‐ST3Ne remained noncovalently attached to the cell wall via the ST3Ne portion. The strength of the HSP150promoter was found to be comparable to that of the GAL1 promoter.

Production of Heterologous Proteins in Yeast With the Aid of the Hsp150Δ Carrier

Proper folding, and consequently exit from the endoplasmic reticulum (ER) and secretion of heterologous exocytic proteins in yeast can be rescued by fusing the proteins to certain yeast-derived polypeptides. Biologically active mammalian glycoproteins can be produced in Saccharomyces cerevisiae and Pichia pastoris by joining them to a fragment of a natural secretory glycoprotein of S. cerevisiae, Hsp150delta. The performance of the Hsp150delta carrier in both yeasts appears to exceed that of the MFalpha leader, which is widely used in industrial protein production. Here we describe the use of the Hsp150delta carrier in P. pastoris in both shake flask and fermentor cultivations. As a reporter protein we use the periplasmic disulfide-bonded Escherichia coli enzyme beta-lactamase.