Areas Expuestas a Riesgo Medio por Inundación

The best characterised GRP is GRP78, also known as BiP, the immunoglobulin heavy chain-binding protein. GRP78 resides primarily in the ER, an essential perinuclear organelle for the synthesis and folding of secretory and membrane proteins, which accounts for about one third of the cell’s proteins. In addition to facilitating proper protein folding, preventing intermediates from aggregating, and targeting misfolded protein for proteosome degradation, GRP78 also binds Ca2+ and serves as an ER stress signalling regulator.(202) BiP was identified in 1983 when it was observed that immunoglobulin heavy chain synthesised by pre-B cell lymphocytes bound to a protein that inhibited their secretion in the absence of light chains, thus preventing secretion of incompletely folded immunoglobulins.(203) GRP78 and BiP were later found to be identical ER-resident proteins that were abundant in antibody-secreting cells.(204) GRP78 is very similar to hsp70, sharing about 60% of its amino acid sequence including the ATP-binding site required for

49 their role as a chaperone in protein folding. GRP78 differs from hsp70 in two important aspects. First, GRP78 is primarily an ER resident protein whereas hsp70 is cytosolic and, second, thermal stress does not significantly induce GRP78.(205)

1.3.1.1 Atypical GRP78

1.3.1.1.1 Cell surface GRP78

Although GRP78 is generally regarded as an ER resident chaperone protein, there is mounting evidence that GRP78 is also located on the cell surface of cancer cells but not normal cells, and that it can act as a receptor that regulates cellular activity.(206) In 1998, cell surface localized GRP78, induced by thapsigargin treatment in human rabdomyosarcoma cells, was confirmed by biotinylation and chromatography.(207) In an additional study, GRP78 was found to be expressed on the cell surface with MHC class1 in unstressed cells. GRP78 is known to associate with MHC class 1 heavy chain intracellularly during folding and assembly, but on the cell surface GRP78 does not appear reliant on MHC class 1 and, in fact, in the absence of MHC class 1, GRP78 is overexpressed.(208)

In prostate cancer cells, GRP78 acts as a high-affinity receptor for the protease inhibitor alpha-2-macroglobulin (α2M*) and binding of α2M* to cell surface GRP78 can promote cellular proliferation.(209-212) In a variety of cancer cell lines, global profiling of the cell surface proteome revealed that GRP78, among other chaperone proteins, was present in all cell types tested in relatively high abundance.(213) Circulating antibodies to cell surface GRP78 in the serum of prostate cancer patients correlates with more aggressive cancer phenotype and reduced survival. As proof of principle that cell surface GRP78 may have potential therapeutic value, a synthetic

50 chimeric peptide with GRP78 binding motifs fused to proapoptotic sequences could specifically target tumour cells in vivo with minimal effect on normal tissues and in human cancer cells ex vivo, confirming that cell surface GRP78 is a functional molecule leading to internalization of GRP78 binding ligands.(214, 215)

The question of how an ER resident protein such as GRP78 can localize to the cell surface is an interesting one. It has been shown that the murine tumour cell DnaJ-like protein-1 (MTJ-1), as well as being a transmembrane protein, also acts as a co- chaperone to GRP78 within the ER.(216) MTJ-1 co-immunoprecipitates with GRP78 in the plasma membranes of macrophages and silencing the MTJ-1 gene expression by RNA interference abolishes cell surface localization of GRP78; thus suggesting that MTJ-1 is essential for transport to the cell surface.(217) Another theory is that GRP78 is expressed to such high levels in tumours that it may oversaturate the ER protein retrieval mechanism, resulting in cotrafficking to the cell surface with its client proteins.(218, 219) In principle, drugs targeting cell surface GRP78 may have antitumour properties with minimal adverse effects on normal organs and tissues.

1.3.1.1.2 Cytoplasmic GRP78

GRP78va is a novel cytosolic isoform devoid of an ER signal sequence. It is generated through ER stress-induced alternative pre-mRNA splicing, a process through which a single gene may generate several distinct protein isoforms that may have diverse or even antagonistic functions.(220, 221) It is known to be elevated in leukaemic cell lines and in leukaemia patient samples and specifically enhances PERK signalling to promote cell survival under conditions of ER stress. It is not known which client proteins it interacts with in the cytosol.

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1.3.1.1.3 Mitochondrial GRP78

Cellular fractionation and protease digestion of isolated mitochondria suggested that a significant amount of GRP78 was localised to the mitochondria in ER stressed cells. This was confirmed by immunoelectron microscopy, demonstrating that during UPR activation, GRP78 localised to the mitochondrial membrane compartment.(222) Although its functions at this site are not yet understood, it is possible that it may modulate the mitochondrial component of the intrinsic apoptosis pathway.

1.3.2 ‘ER’ Stress

The induction of GRP78 is an important response to disturbance of ER homeostasis.(223) The ER is a unique oxidising compartment for the folding of membrane and secretory proteins, and has a role as a major signal-transducing organelle. It also provides a highly selective quality control system to ensure correct protein folding and assembly, and recognise unfolded proteins so that they can be repaired or targeted for proteosome degradation. A number of biochemical, physical and pathological stimuli, for example, ER calcium depletion, glucose deprivation, oxidative stress and DNA damage can disrupt ER homeostasis and lead to accumulation of unfolded or misfolded proteins in the ER – so called ‘ER stress’. Eukaryotic cells have evolved a coordinated cellular response to such stress termed the unfolded protein response (UPR).(198, 219, 224, 225)

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