Proteomics of Endoplasmic Reticulum

Koch G.L.E., Macer DRJ. & Wooding F.B.P. (1989) "The major 55kDa calcium-binding Stains-all positive protein in mammalian cells is in the reticuloplasm of the endoplasmic reticulum", pp. 159-167 in Biochemical Approaches to Cellular Calcium (Methodological Surveys in Biochemistry and Analysis), eds., R.E. Reid, G.M.W. Cook & J.P. Luzio, Vol. 19 (London: Royal Society Chemistry 1989).

Objectives: To investigate the neuroprotective efficacy of  Valeriana
wallichii  DC rhizome extract against 1-methyl-4-phenlypyridinium
(MPP
+
) and  tunicamycin induced cell death.  Methods:  The 50%
methanolic extract of  V. wallichii  rhizome (VWE) was evaluated for
free radical scavenging properties. Cell viability analysis and IC50
determination for VWE in SH-SY5Y neuroblastoma cell line was
performed by MTT assay. The neuroprotective efficacy of VWE in
SH-SY5Y cells against the damages induced by the neurotoxin,
MPP
+
and the endoplasmic reticulum stress inducer, tunicamycin was
assessed by MTT assay and by morphological analysis with phase
contrast microscope. MTT assay was performed using 3 different
concentrations (0.1, 0.5 and 1 mg/mL) of VWE that were added at 0,
8 and 16 h post MPP
+
and tunicamycin treatment.  Results:  VWE
showed good free radical scavenging properties. From the cell
viability analysis the IC50
value of VWE was calculated as 2.207
mg/mL in SH-SY5Y cells. In MPP
+
treated cells 0.5mg and 1mg/mL
concentrations of VWE brought significant improvement in cell
viability at 0h, while at 8h and 16h post MPP
+
treatment the effect
was significant at 1mg/ml concentration. VWE exhibited significant
protective effect in tunicamycin treated cells only at 0h but not at 8h
and 16h post treatment. MPP
+
and tunicamycin induced cell
shrinkage and condensation were inhibited by treatment with
1mg/mL of VWE.  Conclusions:  The present work is the first study
that sheds light on the cytoprotective effect of  Valeriana wallichii
DC rhizome and provides lead for identification and isolation of
novel drugs for various neurodegenerative diseases

The location of endoplasmin in the endoplasmic reticulum was investigated by biochemical and immunoelectron microscopic analyses. The protein could be obtained in a soluble form by procedures that do not involve the use of any detergents. The soluble protein has the amino-and carboxy-terminal sequences of the intact molecule, showing that it has not been proteo-lysed. Application of the Triton X-114 phase-separation test does not reveal significant hydrophobicity in the molecule. Immunogold labelling studies on cells with a dilated endoplas-mic reticulum (ER) lumen show that endoplas-min is uniformly distributed throughout the lumen, with no evidence of a preferential association with the membrane. These studies clearly demonstrate that endoplasmin is a luminal protein of the ER, i.e. a reticuloplasmin, and not an integral membrane protein.

Numerous studies have established the key role of the Golgi apparatus (GA) in post-translational processing, transport and targeting of proteins destined for secretion, lysosomes and plasma membranes. Moreover, several studies performed in our laboratories have shown that the size of the immunocytochemically detected neuronal GA is a reliable index of neuronal activity in aging, Alzheimer's disease (AD) and amyotrophic lateral sclerosis. It has been suggested that in AD there is decreased neuronal activity, e.g. in terms of glucose metabolism and protein synthetic capability. To further explore the hypothesis of decreased neuronal activity in AD, in this study the size of the GA was measured in pyramidal neurons of the CA1 area of the hippocampus of non-demented controls and AD patients. The size of the GA was measured separately in neurons with and without neurofibrillary tangles (NFT). Moreover, in order to establish a correlation between the density of NFT and the size of the GA, the density of extraneuronal NFT was determined around each neuron and related to the size of its GA. The results, quantified by image analysis, indicate that there is a significant reduction in GA size in the neurons of the CA1 area of the hippocampus of AD patients. However, there was no significant relationship between the size of the GA and the presence or absence of intracellular NFT. In addition, there was no correlation between the density of extracellular NFT and GA size of adjacent neurons

Numerous studies have established the key role of the Golgi apparatus (GA) in post-translational processing, transport and targeting of proteins destined for secretion, lysosomes and plasma membranes. Moreover, several studies performed in our laboratories have shown that the size of the immunocytochemically detected neuronal GA is a reliable index of neuronal activity in aging, Alzheimer's disease (AD) and amyotrophic lateral sclerosis. It has been suggested that in AD there is decreased neuronal activity, e.g. in terms of glucose metabolism and protein synthetic capability. To further explore the hypothesis of decreased neuronal activity in AD, in this study the size of the GA was measured in pyramidal neurons of the CA1 area of the hippocampus of non-demented controls and AD patients. The size of the GA was measured separately in neurons with and without neurofibrillary tangles (NFT). Moreover, in order to establish a correlation between the density of NFT and the size of the GA, the density of extraneuronal NFT was determined around each neuron and related to the size of its GA. The results, quantified by image analysis, indicate that there is a significant reduction in GA size in the neurons of the CA1 area of the hippocampus of AD patients. However, there was no significant relationship between the size of the GA and the presence or absence of intracellular NFT. In addition, there was no correlation between the density of extracellular NFT and GA size of adjacent neurons.

A recently conducted chemical genetic screen for pharmaceuticals that can extend longevity of the yeast Saccharomyces cerevisiae has identified lithocholic acid as a potent anti-aging molecule. It was found that this hydrophobic bile acid is also a selective anti-tumor chemical compound; it kills different types of cultured cancer cells if used at concentrations that do not compromise the viability of non-cancerous cells. These studies have revealed that yeast can be successfully used as a model organism for high-throughput screens aimed at the discovery of selectively acting anti-tumor small molecules. Two metabolic traits of rapidly proliferating fermenting yeast, namely aerobic glycolysis and lipogenesis, are known to be similar to those of cancer cells. The mechanisms underlying these key metabolic features of cancer cells and fermenting yeast have been established; such mechanisms are discussed in this review. We also suggest how a yeast-based chemical genetic screen can be use...