High density lipoproteins (HDL) are considered athero-protective, primarily due to their role in reverse cholesterol transport, where they transport cholesterol from peripheral tissues to the liver for excretion. The current study was designed to determine the impact of HDL modification by acrolein, a highly reactive aldehyde found in high abundance in cigarette smoke, on the cholesterol transport functions of HDL. HDL was chemically-modified with acrolein and immunoblot and mass spectrometry analyses confirmed apolipoprotein crosslinking, as well as acrolein adducts on apolipoproteins A-I and A-II. The ability of acrolein-modified HDL (acro-HDL) to serve as an acceptor of free cholesterol (FC) from COS-7 cells transiently expressing SR-BI was significantly decreased. Further, in contrast to native HDL, acro-HDL promotes higher neutral lipid accumulation in murine macrophages as judged by Oil Red O staining. The ability of acro-HDL to mediate efficient selective uptake of HDL-cholesteryl esters (CE) into SR-BI-expressing cells was reduced compared to native HDL. Together, the findings from our studies suggest that acrolein modification of HDL produces a dysfunctional particle that may ultimately promote atherogenesis by impairing functions that are critical in the reverse cholesterol transport pathway.
High-density lipoproteins (HDLs) are athero-protective, primarily because of their ability to promote cholesterol flux from peripheral tissues to the liver by reverse cholesterol transport (RCT). The delivery of HDL-cholesteryl esters (CE) into cells is mediated by the HDL receptor, scavenger receptor class B type I (SR-BI), a promising target for enhancing whole body cholesterol disposal and preventing cardiovascular disease. A detailed understanding of the structural determinants underlying proper SR-BI/HDL alignment that supports the selective uptake of HDL-CE into cells remains lacking. To this end, we exploited CD36, a class B scavenger receptor with a predicted topology similar to that of SR-BI that binds HDL but is unable to mediate efficient selective uptake of HDL-CE. We generated a series of SR-BI/CD36 chimeric receptors that span the extracellular (EC) domain of SR-BI to delineate regions that are essential for SR-BI’s cholesterol transport functions. All 16 SR-BI/CD36 chimeras were transiently expressed in COS-7 cells, and their plasma membrane localization was confirmed. The majority of SR-BI/CD36 chimeric receptors displayed significant reductions in their ability to (i) bind HDL, (ii) deliver HDL-CE to cells, (iii) mediate efflux of free cholesterol (FC) to HDL, and (iv) redistribute plasma membrane domains of FC. We also demonstrated that changes in SR-BI function were independent of receptor oligomerization. Altogether, we have identified discrete subdomains, particularly in the N-terminal and C-terminal regions of the EC domain of SR-BI, that are critical for productive receptor–ligand interactions and the various cholesterol transport functions of SR-BI.
High density lipoproteins (HDL) are anti-atherogenic particles, primarily due to their role in the reverse cholesterol transport pathway whereby HDL delivers cholesteryl esters (CE) to the liver for excretion upon interaction with its receptor, scavenger receptor BI (SR-BI). We designed experiments to test the hypothesis that one or more of the eight highly conserved tryptophan (Trp; W) residues in SR-BI are critical for mediating function. We created a series of Trp-to-phenylalanine (Phe, F) mutant receptors, as well as Trp-less SR-BI (ΔW-SR-BI), and assessed their ability to mediate cholesterol transport. Wild-type (WT) or mutant SR-BI receptors were transiently expressed in COS-7 cells, and cell surface expression was confirmed. Next, we showed that Trp-less- and W415F-SR-BI had significantly decreased abilities to bind HDL and promote selective uptake of HDL-CE, albeit with higher selective uptake efficiency as compared to WT-SR-BI. Interestingly, only Trp-less-, but not W415F-SR-BI, showed an impaired ability to mediate efflux of free cholesterol (FC). Furthermore, both W415F- and Trp-less-SR-BI were unable to reorganize plasma membrane pools of FC based on lack of sensitivity to exogenous cholesterol oxidase. Restoration of Trp 415 into the Trp-less-SR-BI background was unable to rescue Trp-less-SR-BI’s impaired functions, suggesting that Trp 415 is critical, but not sufficient for full receptor function. Furthermore, with the exception of Trp 262, restoration of individual extracellular Trp residues, in combination with Trp 415, into the Trp-less-SR-BI background partially rescued SR-BI function, indicating that Trp 415 must be present in combination with other Trp residues for proper cholesterol transport functions.
Edited by Linda SpremulliStress-inducible heat shock protein 70 (hsp70) interacts with superoxide dismutase 2 (SOD2) in the cytosol after synthesis to transfer the enzyme to the mitochondria for subsequent activation. However, the structural basis for this interaction remains to be defined. To map the SOD2-binding site in hsp70, mutants of hsp70 were made and tested for their ability to bind SOD2. These studies showed that SOD2 binds in the amino acid 393-537 region of the chaperone. To map the hsp70-binding site in SOD2, we used a series of pulldown assays and showed that hsp70 binds to the amino-terminal domain of SOD2. To better define the binding site, we used a series of decoy peptides derived from the primary amino acid sequence in the SOD2-binding site in hsp70. This study shows that SOD2 specifically binds to hsp70 at 445 GERAMT 450 . Small peptides containing GERAMT inhibited the transfer of SOD2 to the mitochondria and decreased SOD2 activity in vitro and in vivo. To determine the amino acid residues in hsp70 that are critical for SOD2 interactions, we substituted each amino acid residue for alanine or more conservative residues, glutamine or asparagine, in the GERAMT-binding site. Substitutions of E446A/Q and R447A/Q inhibited the ability of the GERAMT peptide to bind SOD2 and preserved SOD2 function more than other substitutions. Together, these findings indicate that the GERAMT sequence is critical for hsp70-mediated regulation of SOD2 and that Glu 446 and Arg 447 cooperate with other amino acid residues in the GERAMT-binding site for proper chaperone-dependent regulation of SOD2 antioxidant function.
The interaction between scavenger receptor BI (SR-BI) and HDL reduces the risk for cardiovascular disease (CVD) by playing key roles in the reverse cholesterol transport pathway (RCT). In the beginning of RCT, SR-BI stimulates release of free cholesterol (FC) from peripheral macrophages to HDL, whereas in the final step of RCT, HDL binds to hepatic SR-BI for delivery of excess cholesteryl esters (CE) into the liver for excretion via bile. While “functioning” HDL is crucial in protection against CVD, environmental factors such as acrolein, a highly reactive aldehyde found in tobacco smoke, can induce oxidative stress that promotes HDL modifications and renders the particle “dysfunctional”. Although there is a strong correlation between cigarette smoking and CVD risk, much less is known about the effects of smoking on RCT. We hypothesized that acrolein modifications to HDL (acro-HDL) generate a dysfunctional particle by: (i) impairing SR-BI-mediated selective uptake of HDL-CE and efflux of FC and (ii) upregulating inflammatory pathways in macrophages that promote atherogenesis. To test this hypothesis, human HDL was modified with 250μM acrolein and acrolein adducts on HDL were confirmed by immunoblot analysis and mass spectrometry. To evaluate acro-HDL’s functionality at the beginning of RCT, SR-BI-mediated FC release to native- or acro-HDL was measured in SR-BI-transfected COS-7 cells. Our data revealed that there was 30% less efflux of FC in the presence of acro-HDL as compared to native HDL. To assess acro-HDL functionality at the end of RCT, HDL binding and HDL-CE uptake assays were performed and acro-HDL (0-50μg/mL) was 10-25% less efficient at delivering CE to cells via SR-BI. Finally, to determine whether acro-HDL was rendered atherogenic, we used qRT-PCR to demonstrate that acro-HDL, but not native HDL, paralleled the effects of oxidized LDL by upregulating mRNA scavenger receptor expression (CD36 and LOX-1) and inflammatory marker expression (MCP-1, IL-6, ICAM-1) in THP-1 macrophages. Altogether, our data reveal that acrolein modification produces a dysfunctional HDL particle that upregulates inflammatory pathways. More detailed investigations into the impact of acrolein adducts on HDL function may help prevent smoking-related CVD deaths.
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