Celluar Folding Determinants and Conformational Plasticity of Native C-Reactive Protein.

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.

Conformational folding and disulfide bonding drive distinct stages of protein structure formation.

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.

Topological localization of monomeric C-reactive protein determines proinflammatory endothelial cell responses.

The activation of endothelial cells (ECs) by monomeric C-reactive protein (mCRP) has been implicated in contributing to atherogenesis. However, the potent proinflammatory actions of mCRP on ECs in vitro appear to be incompatible with the atheroprotective effects of mCRP in a mouse model. Because mCRP is primarily generated within inflamed tissues and is rapidly cleared from the circulation, we tested whether these discrepancies can be explained by topological differences in response to mCRP within blood vessels. In a Transwell culture model, the addition of mCRP to apical (luminal), but not basolateral (abluminal), surfaces of intact human coronary artery EC monolayers evoked a significant up-regulation of MCP-1, IL-8, and IL-6. Such polarized stimulation of mCRP was observed consistently regardless of EC type or experimental conditions (e.g. culture of ECs on filters or extracellular matrix-coated surfaces). Accordingly, we detected enriched lipid raft microdomains, the major surface sensors for mCRP on ECs, in apical membranes, leading to the preferential apical binding of mCRP and activation of ECs through the polarized induction of the phospholipase C, p38 MAPK, and NF-κB signaling pathways. Furthermore, LPS and IL-1ß induction of EC activation also exhibited topological dependence, whereas TNF-α did not. Together, these results indicate that tissue-associated mCRP likely contributes little to EC activation. Hence, topological localization is an important, but often overlooked, factor that determines the contribution of mCRP and other proinflammatory mediators to chronic vascular inflammation.

[Characteristics of biofilm phase during the long-term degradation of a toluene-contaminated gas stream using BTF].

This study analyzes the accumulation and distribution of biomass and changes in properties of biofilm in a long-term biotrickling filter (BTF) system in order to investigate the correlation between the biofilm phase properties and the performance. After a long-term operation of 130 days, the BTF showed a deterioration in degradation performance and an increase in pressure drop with a gradual increase of biofilm thickness and uneven distribution of biomass. Meanwhile, the porosity of the upper and lower layers decreased from 85% and 82% in the start-up period to 65% and 40%, respectively, as a result of the excessive accumulation of biomass and its non-uniform distribution. The AWCD values showed a decreasing trend indicating that the biological activity decreased with the aging of biofilm in the long-term BTF. The contents of total extracellular polymeric substances (EPS) and protein in the later period were twice as much as those in the start-up period. The value of protein to polysaccharide (PN/PS) ratio increased from 0.3 to 0.95, and showed positive correlation with the surface hydrophobicity of the biofilm, which increased from 33% to 73% accordingly. The mean molecular weight of EPS decreased during the operation of BTF. The result of FTIR further showed that the main chemical composition of EPS changed in the long-term BTF accordingly, possibly resulting in the deterioration of performance in the long-term BTF. The above experimental results could be very helpful for reducing the clogging and performance deterioration in long-term BTF.