The causal relationship between conformational folding and disulfide bonding in protein oxidative folding remains incompletely defined. Here we show a stage-dependent interplay between the two events in oxidative folding of C-reactive protein (CRP) in live cells. CRP is composed of five identical subunits, which first fold spontaneously to a near-native core with a correctly positioned C-terminal helix. This process drives the formation of the intra-subunit disulfide bond between Cys36 and Cys97. The second stage of subunit folding, however, is a non-spontaneous process with extensive restructuring driven instead by the intra-subunit disulfide bond and guided by calcium binding-mediated anchoring. With the folded subunits, pentamer assembly ensues. Our results argue that folding spontaneity is the major determinant that dictates which event acts as the driver. The stepwise folding pathway of CRP further suggests that one major route might be selected out of the many in theory for efficient folding in the cellular environment.

The conformational conversion of pentameric C-reactive protein (pCRP) to monomeric CRP (mCRP) has been shown to play important roles in the action of CRP in inflammation regulation. In vivo studies revealed the origin of mCRP and provided insights into how pCRP dissociation affected its functions. However, the interplay and exact bioactivities of CRP isoforms still remain uncertain due to the rapid conformational conversion and complex milieu in vivo. Herein, we have used surface-immobilization of pCRP to generate a preservable intermediate with dual antigenicity expression of both pCRP and mCRP. The intermediate has been further shown to exhibit modified bioactivities, such as a high affinity with solution-phase pCRP and an enhanced capacity of complement interaction. These results thus not only provide the conformational conversion details of CRP, but also propose a simple way in vitro to study how the functions of CRP are tuned by distinct isoforms.

C-reactive protein is a critical checkpoint that limits destructive activation of complement in acetaminopheninduced liver injury, and could be exploited as a promising therapeutic approach to treat hepatotoxicity caused by drug overdose.BACKGROUND AND AIMS: C-reactive protein (CRP) is a hepatocyte-produced marker of inflammation yet with undefined function in liver injury. We aimed to examine the role of CRP in acetaminophen-induced liver injury (AILI). METHODS:The effects of CRP in AILI were investigated using CRP knockout mice and rats combined with human CRP rescue. The mechanisms of CRP action were investigated in vitro and in mice with Fcg receptor 2B knockout, C3 knockout, or hepatic expression of CRP mutants defective in complement interaction. The therapeutic potential of CRP was investigated by intraperitoneal administration at 2 or 6 hours post-AILI induction in wild-type mice.RESULTS: CRP knockout exacerbated AILI in mice and rats, which could be rescued by genetic knock-in, adeno-associated virus-mediated hepatic expression or direct administration of human CRP. Mechanistically, CRP does not act via its cellular receptor Fcg receptor 2B to inhibit the early phase injury to hepatocytes induced by acetaminophen; instead, CRP acts via factor H to inhibit complement overactivation on injured hepatocytes, thereby suppressing the late phase amplification of inflammation likely mediated by C3a-dependent actions of neutrophils. Importantly, CRP treatment effectively alleviated AILI with a significantly extended therapeutic time window than that of N-acetyl cysteine.CONCLUSION: Our results thus identify CRP as a crucial checkpoint that limits destructive activation of complement in acute liver injury, and we argue that long-term suppression of CRP expression or function might increase the susceptibility to AILI.

C-reactive protein (CRP) is an acute phase reactant secreted by hepatocytes as a pentamer. The structure formation of pentameric CRP has been demonstrated to proceed in a stepwise manner in live cells. Here, we further dissect the sequence determinants that underlie the key steps in cellular folding and assembly of CRP. The initial folding of CRP subunits depends on a leading sequence with a conserved dipeptide that licenses the formation of the hydrophobic core. This drives the bonding of the intra-subunit disulfide requiring a favorable niche largely conferred by a single residue within the C-terminal helix. A conserved salt bridge then mediates the assembly of folded subunits into pentamer. The pentameric assembly harbors a pronounced plasticity in inter-subunit interactions, which may form the basis for a reversible activation of CRP in inflammation. These results provide insights into how sequence constraints are evolved to dictate structure and function of CRP.

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Biophysical models suggest a dominant role of structural over functional constraints in shaping protein evolution. Selection on structural constraints is linked closely to expression levels of proteins, which together with structure-associated activities determine in vivo functions of proteins. Here we show that despite the up to two orders of magnitude differences in levels of C-reactive protein (CRP) in distinct species, the in vivo functions of CRP are paradoxically conserved. Such a pronounced level-function mismatch cannot be explained by activities associated with the conserved native structure, but is coupled to hidden activities associated with the unfolded, activated conformation. This is not the result of selection on structural constraints like foldability and stability, but is achieved by folding determinants-mediated functional selection that keeps a confined carrier structure to pass the stringent eukaryotic quality control on secretion. Further analysis suggests a folding threshold model which may partly explain the mismatch between the vast sequence space and the limited structure space of proteins.

Amyloid-β peptides (Aβs) are generated in a membrane-embedded state by sequential processing of amyloid precursor protein (APP). Although shedding of membrane-embedded Aβ is essential for its secretion and neurotoxicity, the mechanism behind shedding regulation is not fully elucidated. Thus, we devised a Langmuir film balance-based assay to uncover this mechanism. We found that Aβ shedding was enhanced under acidic pH conditions and in lipid compositions resembling raft microdomains, which are directly related to the microenvironment of Aβ generation. Furthermore, Aβ shedding efficiency was determined by the length of the C-terminal membrane-spanning region, whereas pH responsiveness appears to depend on the N-terminal ectodomain. These findings indicate that Aβ shedding may be directly coupled to its generation and represents an unrecognized control mechanism regulating the fate of membrane-embedded products of APP processing.

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