JUSTIFICACIÓN DEL ANÁLISIS DE REQUERIMIENTOS

The ER is the site of synthesis and folding of proteins destined for entry into the secretory pathway. The folding of proteins in the ER is facilitated by a number of molecular chaperones whose chaperone activity require the hydrolysis of ATP, such as BiP (immunoglobulin heavy chain binding protein also known as Glucose-regulated protein Grp78, Haas and WabI, 1983) of the Hsp70 family of molecular chaperones and Grp94 (Sorger and Pelham, 1987) of the Hsp90 family of molecular chaperones. The members of the Hsp70 chaperones family are highly conserved ATPases with an ability to couple ATP hydrolysis to the binding and release of substrate proteins with regulatory imput from auxiliary proteins known as co-chaperones (Agashe and Marti 2000). The Hsp70s are thought to have a major role in facilitating protein folding and protect nascent and newly synthesised polypeptides against aggregation. Hsp70s have two functional domains, an N-terminal ATPase domain and a C-terminal peptide binding domain which contains a peptide binding cleft and an a-helical extension that acts as a lid covering the peptide binding site (Agashe and Marti 2000). Msp70s bind short hydrophobic peptides of approximately 7 amino acids in length. The binding of ATP by Msp70 opens the peptide binding cleft and hydrolysis of ATP by the N-terminal domain causes the cleft to close thereby retaining the bound peptide of a given polypeptide, which is then released on exchange of ADP for ATP at the N-terminal domain of Msp70. The net result of this reaction cycle is the binding and release of an unfolded polypeptide. Folding of the polypeptide occurs only after release from the chaperone, which interacts with hydrophobic regions, which are destined to be buried in the hydrophobic core of the folded protein. A number of proteins regulate Msp70 ATPase activity and nucleotide exchange including Bag-1, Msp40 and Mip, thereby regulating the binding and release of hydrophobic polypetides (Agashe and Marti 2000). Other chaperones which facilitate folding of proteins include the lectin chaperones, calnexin (CNX) (David et al., 1993) and calreticulin (CRT) (Peterson at a/., 1995) and proteins which catalyse disulfide bond formation (protein disulfide isomerase (PDI), Erp72, Erp57 etc).

Misfolded and incompletely assembled proteins are common side products of protein synthesis in the ER. An elaborate mechanism within the ER consisting of molecular chaperones stringently checks the fidelity of protein folding, retains misfolded or unassembled proteins and prevents their entry into the secretory pathway, eventually targeting these polypeptides for ER associated degradation (ERAD). This process of conformation dependent molecular sorting of newly synthesised proteins has been termed

quality control (Hurtley and Helenius, 1989). An important component of the quality control mechanism of the ER involves two homologous lectin chaperones; CNX, an integral membrane protein and CRT present in the lumen of the ER. These chaperones specifically associate with oligosaccharide moieties of glycoproteins after they have been trimmed by glucosidases I and II, generating the monoglucosylated form GIUilVlangGlcNAc2. The resulting monoglucosylated glycoproteins can now associate with

CNX and CRT. If there is a glycoslylation site within the first fifty amino acids of a nascent glycoprotein, interaction with CNX and CRT begins cotranslationally (Molinari and Helenius 2000). Also, Erp57, a thiol oxidoreductase homologue of protein disulfide isomerase (Oliver et a/., 1997) forms a complex with both of these lectin chaperones and short-lived disulphide bonds occur between Erp57 and cysteines in CNX and CRT bound glycoproteins (Molinari and Helenius 1999) which serve to facilitate correctly paired disulfide bonds within the bound glycoprotein (Huppa and Ploegh, 1998).

Association of glycoproteins with CNX and CRT involves a binding and release cycle driven by the opposite actions of two soluble ER enzymes, uridine diphosphate (UDP)- glucose:glycoprotein glucosyltransferase (UGGT) and glucosidase II (Hammond et al., 1994, Trombetta et a/., 1996, Parodi, 2000). In the first step of the cycle, the association of glycoprotein substrate with either CNX or CRT is terminated if the glucose residue of the oligosaccharide moiety is cleaved by glucosidase II. After release, if the glycoprotein remains unfolded/misfolded, it is recognised and reglucosylated by UGGT (Helenius and Aebi 2001). This allows the unfolded/misfolded glycoprotein to reassociate with CNX/CRT for another round of the refolding reaction (Fig. 1.9). Glycoproteins may go through this re-glucosylation cycle many times and in order to escape the cycle, a glycoprotein must refold so that it is no longer recognised by UGGT, and remain unglycosylated so as not to rebind CNX/CRT. The folded glycoprotein interacts with ER resident mannosidases I and II which trims mannose residues generating Man8-7GlcNAc2 glycoprotein which is free to

leave the ER because it has no glucose residue that could mediate its binding to CNX/CRT (Liu et a/., 1999). In a model proposed by Liu et a/., 1999, persistently unfolded/misfolded glycoproteins are marked for degradation by the action of mannosidase I, which removes the terminal mannose residue generating the GICiMan8GlcNAc2 moiety. After reglucosylation by UGGT the protein rebinds CNX/CRT

and because the GICiMan8GlcNAc2 form is a poor substrate for glucosidase II, as

compared to the GICiManglcNAc2 form (Grinna and Robbins, 1980), the glycoprotein

remains associated with CNX/CRT. The abolished dissociation of CNX and CRT with misfolded/unfolded glycoprotein initiates the entrance of the substrate into the ER

associated degradation pathway (ERAD). This involves the retrograde transport of the misfolded protein out of the ER followed by ubiquitin dependent degradation by the proteasome (section 1.8). The identity of the ER lectins involved in the ERAD pathway remain unknown but a recently discovered novel ER a-mannosidase-like protein (EDEM) which binds Man8GlcNAc2 glycoproteins is thought to be directly involved in the targeting

of misfolded glycoproteins for degradation (Hosokawa etaL, 2001).

Misfolded proteins within the ER which are destined for degradation are first exported from the ER lumen or ER membrane into the cytosol so as to gain access to the cytosolic proteasomal machinery (Plemper and Wolf, 1999). This mechanism is known as retrotranslocation and it is believed that proteins are exported from the ER via the Sec61 complex or translocon (Plemper and Wolf, 1999). The molecular chaperone Grp78 (BiP) in addition to facilitating protein folding in the ER is also thought to regulate the conformation of the translocon pore (Hamman etaL, 1998).

Ribosome

GD

GICgMang Glc^Mang Mang Man

( Man I )

9 c Man 1 )

Gl&i Mang Man, Man I

Cytosol

Golgi

Proteasome

Figure 1.9 Calnexin/Calreticulin mediated degradation of misfolded glycoproteins. Glucosidase I and II (GI/GII) trim the first two glucose residues from newly synthesised glycoproteins which then associate with calnexin (CNX) or calreticulin. The substrate glycoprotein is released from CNX when Gll removes the terminal glucose of the substrate. If misfolded the glycoprotein is reglucosylated by the folding sensor uridine diphosphate (UDP)-glucose:glycoprotein glucosyltransferase (UGGT, but GT in this diagram) and the unfolded glycoprotein rebinds CNX. Associated with CNX is ERp57 (not shown) which catalyses the formation of any disulphide bonds within the protein. If the protein is terminally misfolded, it is marked for degradation by the action of mannosidase I (Man I). Because Glc^MangGlcNAc^ is a poor substrate for Gll compared to the Glc^MangGlcNACg oligosaccharide the protein is not rapidly released from CNX and is thus targeted for ERAD (Figure from Ellgaard et al., 1999).