Binding immunoglobulin protein

Binding immunoglobulin protein (BiP) also known as 78 kDa glucose-regulated protein (GRP-78) or heat shock 70 kDa protein 5 (HSPA5) is a protein that in humans is encoded by the HSPA5 gene.

BiP is a HSP70 molecular chaperone located in the lumen of the endoplasmic reticulum (ER) that binds newly synthesized proteins as they are translocated into the ER, and maintains them in a state competent for subsequent folding and oligomerization. BiP is also an essential component of the translocation machinery, as well as playing a role in retrograde transport across the ER membrane of aberrant proteins destined for degradation by the proteasome. BiP is an abundant protein under all growth conditions, but its synthesis is markedly induced under conditions that lead to the accumulation of unfolded polypeptides in the ER.

Structure

BiP contains two functional domains: a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). NBD binds and hydrolyzes ATP; the substrates for SBD are extended polypeptides.

NBD consists of two large globular subdomains (I and II), each further divided into two small subdomains (A and B). The subdomains are separated by a cleft where the nucleotide, one Mg²⁺ and two K⁺ ions bind and connect all four domains (IA, IB, IIA, IIB). SBD is divided into two subdomains: SBDβ and SBDα. SBDβ serves as a binding pocket for client proteins or peptide and SBDα serves as a helical lid to cover the binding pocket. An inter-domain linker connects NBD and SBD, favoring formation of an NBD–SBD interface.

Mechanism

The activity of BiP is regulated by its allosteric ATPase cycle: when ATP is bound to the NBD, the SBDα lid is open, which leads to the conformation of SBD with low affinity to substrate. Upon ATP hydrolysis, ADP is bound to the NBD and the lid closes on the bound substrate. This creates a low off rate for high-affinity substrate binding and protects the bound substrate from premature folding or aggregation. Exchange of ADP for ATP results in the opening of the SBDα lid and subsequent release of the substrate, which then is free to fold. The ATPase cycle can be synergistically enhanced by protein disulfide isomerase (PDI), and its cochaperones.

Function

When Chinese hamster K12 cells are starved of glucose, the synthesis of several proteins, called glucose-regulated proteins (GRPs), is markedly increased. GRP78 (HSPA5), also referred to as 'immunoglobulin heavy chain-binding protein' (BiP), a member of the heat-shock protein-70 (HSP70) family, is involved in the folding and assembly of proteins in the endoplasmic reticulum (ER). The level of GRP78 is strongly correlated with the amount of secretory proteins (e.g. IgG) within the ER.

Substrate release and binding by BiP facilitates diverse functions in the ER such as folding and assembly of newly synthesized proteins, binding to misfolded proteins to prevent protein aggregation, translocation of secretory proteins, and initiation of the UPR.

Protein foldase and holdase

BiPs can actively fold their substrates (acting as a foldase) or simply bind and restrict a substrate from folding or aggregating (acting as a holdase). Intact ATPase activity and peptide binding activity are required to act as a foldase: temperature-sensitivie mutants of BiP with defective ATPase activity (called class I mutations) and mutants of BiP with defective peptide binding activity (called class II mutations) both fail to fold carboxypeptidase Y (CPY) at non-permissive temperature.

ER translocation

As an ER molecular chaperone, BiP is also required to import polypeptide into the ER lumen or ER membrane in an ATP dependent manner. ATPase mutants of BiP were found to cause a block in translocation of a number of proteins (invertase, carboxypeptidase Y, a-factor) into the lumen of the ER.

ERAD degradation

BiP is also indicated to play a role in ER associated degradation (ERAD). The most studied ERAD substrate is CPY*, a constitutively misfolded CPY completely imported into the ER and modified by glycosylation. BiP is the first chaperone that contacts CPY* and is required for CPY* degradation. ATPase mutants (including allosteric mutants) of BiP have been shown to significantly slow down the degradation rate of CPY*.

UPR pathway

BiP is both a target of the ER stress response, or unfolded protein response (UPR) and an essential regulator of the UPR pathway. Two models of UPR pathway initiation by misfolded protein have been proposed: one model poses a competition between unfolded proteins and the sensor domain of IRE1 for binding to BiP. According to this model, unfolded proteins activate IRE1 by binding to BiP and causing its dissociation from the sensor domain. An alternative model poses direct binding of unfolded proteins to the sensor domain of IRE1.

Interactions

BiP’s ATPase cycle is facilitated by its co-chaperones, both nucleotide binding factors (NEFs), which facilitate ATP binding upon ADP release, and J proteins, which promote ATP hydrolysis. Specific binding to J proteins and NEFs directs BiP’s ability to achieve a wide array of functions. Two NEFs, Grp170 and Sil1, facilitate substrate release from BiP by stimulating the release of ADP and allowing ATP to rebind and open the lid on the SBD. Seven ERdj co-factors, facilitate ATP hydrolysis of BiP via their J-domains. They assist BiP in various of functions during protein translocation (ERdj2), protein folding (ERdj3 and ERdj6) and ERAD (ERdj4 and ERdj5). The functions of ERdj1 and ERdj7 are not well understood.

Conservation of BiP cysteines

BiP is highly conserved among eukaryotes including mammals (Table 1). it is also widely-expression among all tissue types in human. In the human BiP, there are two highly conserved cysteines. These cysteines have been shown to undergo post-translational modifications in both yeast and mammalian cells. In yeast cells, the N-terminus cysteine has been shown to be sulfenylated and glutathionylated upon oxidative stress. Both modifications enhance BiP's ability to prevent protein aggregation. In mice cells, the conserved cysteine pair forms disulfide bond upon activation of GPx7 (NPGPx). Disulfide bond enhances BiP's binding to denatured proteins.

Table 1. Conservation of BiP in mammalian cells
	Species common name	Species scientific name	Conservation of BiP	Conservation of BiP's cysteine	Cysteine number
Primates	Human	Homo sapiens	Yes	Yes	2
	Macaque	Macaca fuscata	Yes	Yes	2
	Vervet	Chlorocebus sabaeus	Predicted*	Yes	2
	Marmoset	Callithrix jacchus	Yes	Yes	2
Rodents	Mouse	Mus musculus	Yes	Yes	2
	Rat	Rattus norvegicus	Yes	Yes	3
	Guinea pig	Cavia porcellus	Predicted	Yes	3
	Naked mole rat	Heterocephalus glaber	Yes	Yes	3
	Rabbit	Oryctolagus cuniculus	Predicted	Yes	2
	Tree shrew	Tupaia chinensis	Yes	Yes	2
Ungulates	Cow	Bos taurus	Yes	Yes	2
	Minke whale	Balaenoptera acutorostrata scammoni	Yes	Yes	2
	Pig	Sus scrofa	Predicted	Yes	2
Carnivores	Dog	Canis familiaris	Predicted	Yes	2
	Cat	Hammondia hammondi	Yes	Yes	3
	Ferret	Mustela putorius furo	Predicted	Yes	2
Marsupials	Opossum	Monodelphis domestica	Predicted	Yes	2
	Tasmanian Devil	Sarcophilus harrisii	Predicted	Yes	2
*Predicted: Predicted sequence according to NCBI protein

Clinical significance

Immunological diseases

Like many stress and heat shock proteins, BiP/GRP78 has potent immunological activity when released from the internal environment of the cell into the extracelluar space. Specifically, it feeds anti-inflammatory and pro-resolutory signals into immune networks, thus helping to resolve inflammation.

The mechanisms underlying BiP's immunological activity are incompletely understood. However, it has been shown that it binds to a receptor on the surface of monocytes and induces anti-inflammatory cytokine secretion dominated by IL-10, IL-1Ra, and soluble TNFR. Furthermore, it downregulates critical molecules involved in T-lymphocyte activation such as HLA-DR and CD86. It also modulates the differentiation pathway of monocytes into dendritic cells, causing them to develop tolerogenic characteristics, which, in turn, can facilitate the development of regulatory T-lymphocytes.

The potent immunomodulatory activities of BiP/GRP78 have also been demonstrated in animal models of autoimmune disease including collagen-induced arthritis, a murine disease that resembles human rheumatoid arthritis. Prophylactic or therapeutic parenteral delivery of BiP has been shown to ameliorate clinical and histological signs of inflammatory arthritis.

Heart diseases

BiP is recognized by heart infiltrating and peripheral T cells from rheumatic heart disease (RHD) patients. RHD is the major manifestation of RF, is characterized by inflammation of heart valves and myocardium.Identification of BiP as the target antigen suggests BiP may be involved in the autoimmune reactions that leads to valve damage.

As a novel class of anticancer agents, Proteasome inhibitors trigger heart failure through elevation of ER stress. In rat neonatal cardiomyocytes, overexpression of BiP attenuates cardiomyocyte death induced by proteasome inhibition.

BiP inhibitors and enhancers

As a therapeutic target, several BiP inhibitors and enhancers have been developed. Inhibitors of BiP target the ATP-binding domain. Honokiol, a Magnolia grandiflora derivative, is a BiP inhibitor. OSU-03012 (AR-12), The BiP inhibitor OSU-03012 (AR-12), a derivative of the drug celecoxib (Celebrex), interacting with sildenafil (Viagra) or tadalafil (Cialis) can rapidly reduce BiP levels in eukaryotes. Inducers of BiP were also found including, BiP inducer X (BIX) was identified in a screen for compounds that induce BiP expression.

External links

• HSPA5 protein, human at the US National Library of Medicine Medical Subject Headings (MeSH)