[show abstract][hide abstract] ABSTRACT: Dear Editor, Genetic alteration and inflammation underlie the de-velopment of cancer . C-reactive protein (CRP) is the most widely used nonspecific marker of inflammation , whose serum level has been shown to be associated with the risk and prognosis of several types of cancer . Accumulating evidence supports a role of CRP as a pattern recognition receptor in the innate immunity and inflammation ; however, its exact function remains to be defined because mouse is not an appropriate animal model for CRP . Moreover, as a prototypical acute phase reactant, the 2-3-order fluctuation in the serum lev-el of CRP has raised the concern that this molecule is not likely a fine modulator of inflammation . These make it difficult to dissect the contribution of CRP in diseases featured by chronic inflammation, including atheroscle-rosis  and cancer . What makes things even more complicated is the fact that large-scale genetic epidemio-logic studies do not support a causal association of CRP with these diseases [6, 7]. Therefore, it remains elusive whether CRP is a passive marker or plays a direct role in tumorigenesis. Recent studies have demonstrated that in addition to hepatocytes, CRP can also be produced by extra-hepatic cells . In searching for mechanism(s) involved in the regulation of CRP expression in human cancer cells, we incidentally found that the T-cell leukemia cell line Jurkat and the hepatoma cell line Hep3B appeared to possess the CA genotype, instead of the predominant CC genotype, at the position of 286 bp upstream of the TSS of CRP (Figure 1A). Further screening identified only 6 cases of –286 CC genotype of additional 13 cancer cell lines. The –286 C>A/T substitution is a known SNP of CRP (rs3091244), which is associated with elevated plasma concentrations of CRP . However, it is less likely that the cancer cell lines with the –286 CA/CT genotype all happened to be derived from individuals carrying the minor alleles. Alternatively, the –286 C>A/T transition may arise as a mutation during cancer develop-ment in the original hosts or during prolonged passage of the established cell lines. With the above hint, we sequenced the promoter of CRP in 453 matched tumor/normal sample pairs. Over-all, 109 tumors (24%) harbor –286 somatic mutations, the majority of which are –286 C>A (Figure 1B and Supplementary information, Tables S1 and S2). The –286 mutations were detected in 10 of 12 cancer types with the strongest prevalence (53.3%) for colon cancer (Figure 1C). The fractions of the mutated –286 allele are high in tumors (the average allelic fraction is 0.497; 95% CI: 0.477 to 0.517) and these mutations exhibit comparable mutational rates in frozen and formalin-fixed sample pairs. No additional recurrent mutations were identified within the near 700 bp sequenced range of CRP promoter (–601 ~ +76), which includes 14 other known CRP SNP sites. To exclude the possible misidentification of the –286 germline SNP as a somatic mutation, regular PCR sequencing results of 73 sample pairs were indepen-dently validated by TA clone sequencing (Supplementary information, Tables S2 and S3). Moreover, we genotyped 20 randomly selected SNP sites of 141 tumor/normal sample pairs, in which 123 (87.23%) were completely matched (Supplementary information, Table S4). This further demonstrate that the identified CRP –286 muta-tions were not due to mismatch of sample pairs or tech-nical artifacts. Therefore, we conclude that the promoter of CRP is specifically mutated at position –286 during cancer development. The mRNA levels of CRP in tumors were much high-er than that in matched controls (Figure 1D). Interest-ingly, the relative induction of CRP expression tends to be more pronounced in tumors with –286 C>A somatic mutation (Figure 1E), suggesting that this mutation en-hances CRP transcription. Indeed, CRP promoter con-struct carrying the –286 A allele shows higher luciferase reporter activity than the wild-type construct . Promoter CpG methylation is an essential epigenetic mechanism in gene silencing. There are only 5 CpG di-nucleotides, including the one containing –286 cytosine, within the range of 700 bp upstream of the TSS of CRP gene. Rabbit exhibits similar CRP expression pattern
[show abstract][hide abstract] ABSTRACT: Soluble oligomers of amyloid-β peptide (Aβ) are emerging as the primary neurotoxic species in Alzheimer disease, however, whether the membrane is among their direct targets that mediate the downstream adverse effects remains elusive. Herein, we show that multiple soluble oligomeric Aβ preparations, including Aβ-derived diffusible ligand, protofibril, and zinc-induced Aβ oligomer, exhibit much weaker capability to insert into the membrane than Aβ monomer. Aβ monomers prefer incorporating into membrane rather than oligomerizing in solution, and such preference can be reversed by the aggregation-boosting factor, zinc ion. Further analyses indicate that the membrane-embedded oligomers of Aβ are derived from rapid assembly of inserted monomers but not due to the insertion of soluble Aβ oligomers. By comparing the behavior of a panel of Aβ truncation variants, we demonstrate that the intra- and extra-membrane oligomerization are mutually exclusive processes that proceed through distinct motif interplay, both of which require the action of amino acids 37-40/42 to overcome the auto-inhibitory interaction between amino acids 29-36 and the N-terminal portion albeit via different mechanisms. These results indicate that intra- and extra-membrane oligomerization of Aβ are competing processes and emphasize a critical regulation of membrane on the behavior of Aβ monomer and soluble oligomers, which may determine distinct neurotoxic mechanisms.
Journal of Biological Chemistry 11/2011; 287(1):748-56. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: C-reactive protein (CRP) has been implicated in the regulation of inflammation underlying coronary artery disease; however, little is known about the molecular mechanisms responsible for the expression of its pro- or anti-inflammatory activities. Here, we have identified the intrasubunit disulfide bond as a conserved switch that controls the structure and functions of CRP. Conformational rearrangement in human pentameric CRP to monomeric CRP (mCRP) is the prerequisite for this switch to be activated by reducing agents, including thioredoxin. Immunohistochemical analysis revealed 36-79% colocalization of thioredoxin and mCRP in human advanced coronary atherosclerotic lesions. Nonreduced mCRP was largely inert in activating human coronary artery endothelial cells (HCAECs), whereas reduced or cysteine-mutated mCRP evoked marked release of IL-8 and monocyte chemoattractant protein-1 from HCAECs, with ~50% increase at a concentration of 1 μg/ml. Reduced mCRP was ~4 to 40-fold more potent than mCRP in up-regulating adhesion molecule expression, promoting U937 monocyte adhesion to HCAECs, and inducing cytokine release from rabbit arteries ex vivo and in mice. These actions were primarily due to unlocking the lipid raft interaction motif. Therefore, expression of proinflammatory properties of CRP on endothelial cells requires sequential conformational changes, i.e., loss of pentameric symmetry followed by reduction of the intrasubunit disulfide bond.
The FASEB Journal 06/2011; 25(9):3186-96. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: AMP-activated protein kinase (AMPK) is a heterotrimeric complex composed of α catalytic subunit, β scaffolding subunit, and γ regulatory subunit with critical roles in maintaining cellular energy homeostasis. However, the molecular architecture of the intact complex and the allostery associated with the adenosine binding-induced regulation of kinase activity remain unclear. Here, we determine the three-dimensional reconstruction and subunit organization of the full-length rat AMPK (α1β1γ1) through single-particle electron-microscopy. By comparing the structures of AMPK in ATP- and AMP-bound states, we are able to visualize the sequential conformational changes underlying kinase activation that transmits from the adenosine binding sites in the γ subunit to the kinase domain of the α subunit. These results not only make substantial revision to the current model of AMPK assembly, but also highlight a central role of the linker sequence of the α subunit in mediating the allostery of AMPK.
[show abstract][hide abstract] ABSTRACT: β-Amyloid peptide (Aβ42) is the core protein of amyloid plaque in Alzheimer disease. The intracellular accumulation of Aβ42
in the endosomal/lysosomal system has been under investigation for many years, but the direct link between Aβ42 accumulation
and dysfunction of the endosomal/lysosomal system is still largely unknown. Here, we found that both in vitro and in vivo, a major portion of Aβ42 was tightly inserted into and a small portion peripherally associated with the lysosomal membrane,
whereas its soluble portion was minimal. We also found that the Aβ42 molecules inserted into the membrane tended to form multiple
oligomeric aggregates, whereas Aβ40 peptides formed only dimers. Neutralizing lysosomal pH in differentiated PC12 cells decreased
the lysosomal membrane insertion of Aβ42 and moderated Aβ42-induced lysosomal labilization and cytotoxicity. Our findings,
thus, suggest that the membrane-inserted portion of Aβ42 accumulated in lysosomes may destabilize the lysosomal membrane and
Journal of Biological Chemistry 06/2010; 285(26):19986-19996. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Emerging evidence indicates that in addition to native pentameric C-reactive protein (CRP), monomeric CRP (mCRP) also plays an active role in inflammation associated with cardiovascular diseases. mCRP activates endothelial cells, one of the critical events in cardiovascular diseases; however, the underlying molecular mechanisms are incompletely understood. Here we report that association of mCRP with human aortic and coronary artery endothelial cells is predominantly due to membrane insertion rather than binding to the surface proteins Fc gammaRs and proteoglycans. We identify lipid rafts as the preferential membrane microdomains for mCRP anchorage. mCRP binding depends on membrane cholesterol content and is synergistically mediated by the putative cholesterol binding consensus sequence of CRP (aa 35-47) and the C-terminal octapeptide (aa 199-206). Conversely, disrupting lipid rafts with methyl-beta cyclodextrin or nystatin abrogated mCRP-induced cytokine release, reactive oxygen species generation, and adhesion molecule expression in endothelial cells. Furthermore, ex vivo treatment of rabbit thoracic aorta and carotid artery segments with nystatin prevented mCRP-induced IL-8 release. Our data identify mCRP-lipid raft interaction as an important mechanism in mediating cellular responses to mCRP and lend further support to the notion of mCRP regulation of endothelial cell function during inflammation.
The FASEB Journal 02/2009; 23(6):1806-16. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: Emerging evidence indicates that C-reactive protein (CRP) has at least two conformationally distinct isoforms, i.e., pentameric CRP (pCRP) and monomeric CRP (mCRP or CRP subunit). Both CRP isoforms are proposed to play roles in inflammation and may participate in the pathogenesis of cardiovascular disease. However, the origin of mCRP in situ and the interplay between the two CRP isoforms under physiological/pathological circumstances remain elusive. Herein, by probing conformational alteration, neoepitope expression, and direct visualization using electron-microscopy, we have shown that calcium-dependent binding of pCRP to membranes, including liposomes and cell membranes, led to a rapid but partial structural change, producing molecules that express CRP subunit antigenicity but with retained native pentameric conformation. This hybrid molecule is herein termed mCRP(m). The formation of mCRP(m) was associated with significantly enhanced complement fixation. mCRP(m) can further detach from membrane to form the well-recognized mCRP isoform converted in solution (mCRP(s)) and exert potent stimulatory effects on endothelial cells. The membrane-induced pCRP dissociation not only provides a physiologically relevant scenario for mCRP formation but may represent an important mechanism for regulating CRP function.
The FASEB Journal 02/2007; 21(1):284-94. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: The capacity of human C-reactive protein (CRP) to activate/regulate complement may be an important characteristic that links CRP and inflammation with atherosclerosis. Recent advances suggest that in addition to classical pentameric CRP, a conformationally distinct isoform of CRP, termed modified or monomeric CRP (mCRP), may also play an active role in atherosclerosis. Although the complement activation behavior of CRP has been well established, the capacity of mCRP to interact with and activate the complement cascade is unknown.
mCRP bound avidly to purified C1q, and this binding occurred primarily through collagen-like region of C1q. Fluid phase mCRP inhibited the activation of complement cascade via engaging C1q from binding with other complement activators. In contrast, when immobilized or bound to oxidized or enzymatically modified low-density lipoprotein, mCRP could activate classical complement pathway. Low-level generation of sC5b-9 indicated that the activation largely bypassed the terminal sequence of complement, which appears to involve recruitment of Factor H.
These results indicate that mCRP can both inhibit and activate the classical complement pathway by binding C1q, depending on whether it is in fluid phase or surface-bound state.
[show abstract][hide abstract] ABSTRACT: The interaction of C-reactive protein with low-density lipoprotein is considered to be one of the key properties that link C-reactive protein with atherosclerosis. However the data obtained to date are controversial, and hence make it difficult to conclude actual physiological or pathological impact of such interaction. The incompatible findings could be ascribed to the different structural state of C-reactive protein and/or low-density lipoprotein. We investigated in detail the interaction of various C-reactive protein isoforms with native and modified low-density lipoprotein. Our data showed "C-reactive protein" could indeed interact with each of native low-density lipoprotein, oxidized or enzymatically modified low-density lipoprotein, but that interaction occurs primarily when C-reactive protein is conformed in a modified form and not pentameric structure. Low level of modified C-reactive protein "contaminant" could confer C-reactive protein obvious low-density lipoprotein binding capacity. Interaction of modified C-reactive protein and low-density lipoprotein was mediated synergistically by both electrostatic association with ApoB and hydrophobic insertion into lipid layer. When complexed with modified C-reactive protein, macrophage binding/uptake of native and oxidized low-density lipoprotein was either increased 150% or decreased 35%, respectively. Thus the interaction of modified C-reactive protein with low-density lipoprotein may contribute to the regulation of low-density lipoprotein metabolism and foam cell formation in arterial wall. These results highlight an active role of modified C-reactive protein in atherosclerotic process.
The International Journal of Biochemistry & Cell Biology 02/2006; 38(4):648-61. · 4.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: C-reactive protein (CRP) is one of the most characteristic acute-phase proteins. Modified CRP is the monomeric form of native CRP and has recently been suggested to exist under physiological conditions. In the current work, CRP subunits were separated from stock CRP solution by size-exclusion chromatography. Two forms of two-dimensional crystals composed of monomeric CRP were obtained on negatively charged lipid monolayers: a previously reported form, MI, and a new form, MII. A projection map at 2.0 nm resolution of the two-dimensional MII crystals was obtained. The formation of the two forms of two-dimensional crystal exhibited a dependence on pH. At pH values of less than 5.5 the subunits assembled in MI packing, while at pH values greater than 6.5 they assembled in MII packing. When using modified CRP prepared by acidic denaturation, only MI crystals could be formed. The fact that CRP subunits produced by dissociation or denaturation could form highly ordered two-dimensional crystals indicates that they have a certain homogeneous structure, which supports the previous suggestion of the existence of modified CRP in vivo.
[show abstract][hide abstract] ABSTRACT: Neuronal exocytosis is mediated by Ca(2+)-triggered rearrangements between proteins and lipids that result in the opening and dilation of fusion pores. Synaptotagmin I (syt I) is a Ca(2+)-sensing protein proposed to regulate fusion pore dynamics via Ca(2+)-promoted binding of its cytoplasmic domain (C2A-C2B) to effector molecules, including anionic phospholipids and other copies of syt. Functional studies indicate that Ca(2+)-triggered oligomerization of syt is a critical step in excitation-secretion coupling; however, this activity has recently been called into question. Here, we show that Ca(2+) does not drive the oligomerization of C2A-C2B in solution. However, analysis of Ca(2+).C2A-C2B bound to lipid monolayers, using electron microscopy, revealed the formation of ring-like heptameric oligomers that are approximately 11 nm long and approximately 11 nm in diameter. In some cases, C2A-C2B also assembled into long filaments. Oligomerization, but not membrane binding, was disrupted by neutralization of two lysine residues (K326,327) within the C2B domain of syt. These data indicate that Ca(2+) first drives C2A-C2B.membrane interactions, resulting in conformational changes that trigger a subsequent C2B-mediated oligomerization step. Ca(2+)-mediated rearrangements between syt subunits may regulate the opening or dilation kinetics of fusion pores or may play a role in endocytosis after fusion.
Proceedings of the National Academy of Sciences 03/2003; 100(4):2082-7. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: beta-Amyloid peptide (A beta), a normal constituent of neuronal and non-neuronal cells, has been proven to be the major component of extracellular plaque of Alzheimer's disease. Interactions between A beta and neuronal membranes have been postulated to play an important role in the neuropathology of Alzheimer's disease. Here we show that A beta is able to insert into lipid bilayer. The membrane insertion ability of A beta is critically controlled by the ratio of cholesterol to phospholipids. In a low concentration of cholesterol A beta prefers to stay in membrane surface region mainly in a beta-sheet structure. In contrast, as the ratio of cholesterol to phospholipids rises above 30 mol%, A beta can insert spontaneously into lipid bilayer by its C terminus. During membrane insertion A beta generates about 60% alpha-helix and removes almost all beta-sheet structure. Fibril formation experiments show that such membrane insertion can reduce fibril formation. Our findings reveal a possible pathway by which A beta prevents itself from aggregation and fibril formation by membrane insertion.
Journal of Biological Chemistry 03/2002; 277(8):6273-9. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: C-reactive protein (CRP) is a prototypic human acute phase reactant composed of five identical subunits. Emerging evidence indicates that CRP is not merely a predictor of cardiovascular disease, but may also be a direct mediator. However, the diverse and sometimes contradictory activities of CRP have considerably hampered the attempts to define the exact role of CRP in atherogenesis. Here, we review the multiple layers of regulation of CRP’s structure and function, highlighting how local inflammation conditions, such as the abundance of damaged cell membranes and redox homeostasis, can tip the balance of the pro- and anti-inflammatory activities of CRP. We propose that the highly controlled interplay between different structural conformations of CRP underlies its intrinsic property as a fine modulator of inflammation and atherogenesis.
Chinese Science Bulletin 58(14). · 1.32 Impact Factor