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Biomedical subjects

Min Lu

Publications and source records attributed to Min Lu.

At least 91 records · Page 5Linked to original sources
PubMed2003

Dietary glycotoxins promote diabetic atherosclerosis in apolipoprotein E-deficient mice.

Hyperglycemia derived advanced glycation endproducts (AGE) have been implicated in diabetic atherosclerosis (AS) but the role of exogenous (dietary) AGE in the development of this serious complication is not known. This study evaluates the influence of diet-related AGE on AS in genetically hypercholesterolemic apolipoprotein E-deficient (apoE(-/-)), streptozotocin-induced diabetic mice. Diabetic and non-diabetic apoE(-/-) mice (6-8 weeks old) were randomized into either a standard AIN-93G chow (AGE 12,500+/-700 U/mg, termed high-AGE diet, H-AGE), or the same chow having four to fivefold lower AGE level (L-AGE: 2,700+/-830 U/mg) based on ELISA. After 2 months of diabetes, compared to the diabetic mice fed standard (H-AGE) diet, the AS lesions at the aortic root of the L-AGE group were >50% smaller (0.17+/-0.03 vs. 0.31+/-0.05 mm(2), P<0.05). Serum AGE were lower in the diabetic L-AGE than in the H-AGE mice (by approximately 53%) (P<0.00001), as were in the non-diabetic L-AGE vs. H-AGE groups (P<0.05). No diet-related changes were noted in plasma glucose, triglycerides, or plasma cholesterol. Immunohistochemical comparisons showed markedly suppressed tissue AGE, AGE-Receptor-1, -2 and RAGE expression, reduced numbers of inflammatory cells, tissue factor, vascular cell adhesion molecule-1 and MCP-1 in the L-AGE diabetic group. The findings are supportive of an important link between dietary intake of pre-formed glycoxidation products, tissue-incorporated AGE, and diabetes-accelerated AS. The marked anti-atherogenic effects of an AGE-restricted diet in this model may provide the basis for relevant clinical studies.

Animals↗
PubMed2003

Dietary glycotoxins correlate with circulating advanced glycation end product levels in renal failure patients.

BACKGROUND: Levels of advanced glycation end products (AGEs), well-known proinflammatory compounds, are markedly elevated in patients with renal failure, raising the speculation that they have a role as cardiovascular risk factors in this population. Although elevated AGE levels in patients with renal failure have been attributed to impaired renal clearance and increased endogenous AGE formation, recent data suggest an important role for diet as a source of AGEs. METHODS: To determine the relationship between dietary AGE content and serum AGE levels, a cross-sectional study was performed in our long-term dialysis patients. Dietary AGE intake was estimated by means of dietary records and questionnaires, and sera were obtained for measurement of 2 well-characterized AGEs, carboxymethyl-lysine (CML) and methylglyoxal (MG) derivatives. RESULTS: The study population included 189 patients; 139 hemodialysis and 50 peritoneal dialysis patients. Serum CML level correlated significantly with dietary AGE intake, based on either 3-day food records (r = 0.5; P = 0.003) or dietary questionnaires (r = 0.22; P = 0.03). Although no correlation was observed with nutrient intake (protein, fat, saturated fat, or carbohydrate), both serum CML and MG levels correlated with blood urea nitrogen (r = 0.2; P = 0.03 and r = 0.2; P = 0.02, respectively) and serum albumin levels (r = 0.16; P = 0.04 and r = 0.18; P = 0.02, respectively). CONCLUSION: Data indicate that dietary AGE content, independently of other diet constituents, is an important contributor to excess serum AGE levels in patients with renal failure. Moreover, the lack of correlation between serum AGE levels and dietary protein, fat, and carbohydrate intake indicates that a reduction in dietary AGE content can be obtained safely without compromising the content of obligatory nutrients.

Cross-Sectional Studies↗
PubMed2003

Endoplasmic reticulum retention, degradation, and aggregation of olfactory G-protein coupled receptors.

The mammalian olfactory G-protein coupled receptor family is comprised of hundreds of proteins that mediate odorant binding and initiate signal transduction cascades leading to the sensation of smell. However, efforts to functionally express olfactory receptors and identify specific odorant ligand-olfactory receptor interactions have been severely impeded by poor olfactory receptor surface expression in heterologous systems. Therefore, experiments were performed to elucidate the cellular mechanism(s) responsible for inefficient olfactory receptor cell surface expression. We determined that the mouse odorant receptors mI7 and mOREG are not selected for export from the ER and therefore are not detectable at the Golgi apparatus or plasma membrane. Specifically, olfactory receptors interact with the ER chaperone calnexin, are excluded from ER export sites, do not accumulate in ER-Golgi transport intermediates at 15 degrees C, and contain endoglycosidase H-sensitive oligosaccharides, consistent with olfactory receptor exclusion from post-ER compartments. A labile pool of ER-retained olfactory receptors are post-translationally modified by polyubiquitination and targeted for degradation by the proteasome. In addition, olfactory receptors are sequestered into ER aggregates that are degraded by autophagy. Collectively, these data demonstrate that poor surface expression of olfactory receptors in heterologous cells is attributable to a combination of ER retention due to inefficient folding and poor coupling to ER export machinery, aggregation, and degradation via both proteasomal and autophagic pathways.

Animals↗
PubMed2003

Restriction of dietary glycotoxins reduces excessive advanced glycation end products in renal failure patients.

Advanced glycation endproduct (AGE) levels are elevated in renal failure patients and may contribute to the excessive cardiovascular disease in this population. Diet-derived AGE are major contributors to the total body AGE pool. It was postulated that a reduction in dietary AGE intake might impact on the high circulating AGE levels in renal failure patients. Twenty-six nondiabetic renal failure patients on maintenance peritoneal dialysis were randomized to either a high or a low AGE diet for 4 wk. Three-day dietary records, fasting blood, 24-h urine, and dialysis fluid collections were obtained at baseline and end of study. AGE levels were determined by ELISA for N(epsilon)-carboxymethyl-lysine (CML) and methylglyoxal-derivatives (MG). Eighteen patients completed the study. Low dietary AGE intake decreased serum CML (34%; P < 0.002), serum MG (35%; P < 0.008), CML-LDL (28%; P < 0.011), CML-apoB (25%; P < 0.028), dialysate CML (39%; P < 0.03), and dialysate MG output (40%; P < 0.04). High dietary AGE intake increased serum CML (29%; P < 0.028), serum MG (26%; P < 0.09), CML-LDL (50%; P < 0.011), CML-apoB (67%; P < 0.028), and dialysate CML output (27%; P < 0.01). Serum AGE correlated with BUN (r = 0.6, P < 0.002 for CML; r = 0.4, P < 0.05 for MG), serum creatinine (r = 0.76, P < 0.05 for CML; r = 0.55, P < 0.004 for MG), total protein (r = 0.4, P < 0.05 for CML; r = 0.4, P < 0.05 for MG), albumin (r = 0.4, P < 0.02 for CML; r = 0.4, P < 0.05 for MG), and phosphorus (r = 0.5, P < 0.006 for CML; r = 0.5, P < 0.01 for MG). It is concluded that dietary glycotoxins contribute significantly to the elevated AGE levels in renal failure patients. Moreover, dietary restriction of AGE is an effective and feasible method to reduce excess toxic AGE and possibly cardiovascular associated mortality.

Adult↗
PubMed2003

A constitutive decay element promotes tumor necrosis factor alpha mRNA degradation via an AU-rich element-independent pathway.

Tumor necrosis factor alpha (TNF-alpha) expression is regulated by transcriptional as well as posttranscriptional mechanisms, the latter including the control of mRNA decay through an AU-rich element (ARE) in the 3' untranslated region (UTR). Using two mutant cell lines deficient for ARE-mediated mRNA decay, we provide evidence for a second element, the constitutive decay element (CDE), which is also located in the 3' UTR of TNF-alpha. In stably transfected RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS), the CDE continues to target a reporter transcript for rapid decay, whereas ARE-mediated decay is blocked. Similarly, the activation of p38 kinase and phosphatidylinositol 3-kinase in NIH 3T3 cells inhibits ARE-mediated but not CDE-mediated mRNA decay. The CDE was mapped to an 80-nucleotide (nt) segment downstream of the ARE, and point mutation analysis identified within the CDE a conserved sequence of 15 nt that is required for decay activity. We propose that the CDE represses TNF-alpha expression by maintaining the mRNA short-lived, thereby preventing excessive induction of TNF-alpha after LPS stimulation. Thus, CDE-mediated mRNA decay is likely to be an important mechanism limiting LPS-induced pathologic processes.

3' Untranslated Regions↗
PubMed2003

Antibody-mediated blockade of the CXCR3 chemokine receptor results in diminished recruitment of T helper 1 cells into sites of inflammation.

Naïve T cells, when activated by specific antigen and cytokines, up-regulate adhesion molecules as well as chemokine receptors on their surface, which allows them to migrate to inflamed tissues. Human studies have shown that CXCR3 is one of the chemokine receptors that is induced during T cell activation. Moreover, CXCR3-positive T cells are enriched at inflammatory sites in patients with autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. In this study, we use a mouse model of inflammation to demonstrate that CXCR3 is required for activated T cell transmigration to inflamed tissue. Using an anti- mCXCR3 antibody, we have shown that in vitro-differentiated T helper (Th) 1 and Th2 cells up-regulated CXCR3 upon stimulation with specific antigen/major histocompatibility complex. However, only Th1 cells, when adoptively transferred to syngeneic recipients, are efficiently recruited to the peritoneum in an adjuvant-induced peritonitis model. Furthermore, the neutralizing anti-mCXCR3 antibody profoundly inhibits the recruitment of Th1 cells to the inflamed peritoneum. Real-time, quantitative reverse transcriptase-polymerase chain reaction analysis demonstrates that the CXCR3 ligands, interferon (IFN)-inducible protein 10 (CXCL10) and IFN-inducible T cell alpha chemoattractant (CXCL11), are among the many chemokines induced in the adjuvant-treated peritoneum. The anti-mCXCR3 antibody is also effective in inhibiting a delayed-type hypersensitivity response, which is largely mediated by enhanced trafficking of activated T cells to peripheral inflammatory sites. Collectively, our results suggest that CXCR3 has a critical role in T cell transmigration to sites of inflammation and thus, may serve as a molecular target for anti-inflammatory therapies.

Adoptive Transfer↗
PubMed2002

Integrin alpha8beta1 mediates adhesion to LAP-TGFbeta1.

The development of fibrosis is a common response to a variety of injuries and results in the net accumulation of matrix proteins and impairment of normal organ function. We previously reported that the integrin alpha8beta1 is expressed by alveolar interstitial cells in normal lung and is upregulated during the development of fibrosis. TGFbeta1 is an important mediator of the inflammatory response in pulmonary fibrosis. TGFbeta1 is secreted as a latent protein that is non-covalently associated with latency-associated peptide (LAP) and requires activation to exert its effects. LAP-TGFbeta1 and LAP-TGFbeta3 contain the tripeptide sequence, arginine-glycine-aspartic acid (RGD), a known integrin recognition motif. The integrin alpha8beta1 binds to several ligands such as fibronectin and vitronectin through the RGD sequence. Recent reports demonstrate that the integrins alphavbeta1, alphavbeta6 and alphavbeta8 adhere to LAP-TGFbeta1 through the RGD site. Therefore, we asked whether LAP-TGFbeta1 might be a ligand for alpha8beta1 and whether this may be important in the development of fibrosis. We found that cell lines transfected with alpha8 subunit were able to spread on and adhere to recombinant LAP-TGFbeta1 significantly better than mock transfected cell lines. alpha8-transfected cells were also able to adhere to LAP-TGFbeta3 significantly better than mock transfected cells. Adhesion to LAP-TGFbeta1 was enhanced by activation of alpha8beta1 by Mn(2+), or 8A2, an integrin beta1 activating antibody. Furthermore, cell adhesion was abolished when we used a recombinant LAP-TGFbeta1 protein in which the RGD site was mutated to RGE. alpha8beta1 binding to LAP-TGFbeta1 increased cell proliferation and phosphorylation of FAK and ERK, but did not activate of TGFbeta1. These data strongly suggest that LAP-TGFbeta1 is a ligand of alpha8beta1 and interaction of alpha8beta1 with LAP-TGFbeta1 may influence cell behavior.

Animals↗
PubMed2002

Molecular cloning, expression, and function of osteoclastic calcineurin Aalpha.

This study explores the role of the calmodulin- and Ca(2+)-sensitive phosphatase calcineurin A in the control of bone resorption by mature osteoclasts. We first cloned full-length calcineurin Aalpha and Abeta cDNA from a rabbit osteoclast library. Sequence analysis revealed an approximately 95 and 86% homology between the amino acid and the nucleotide sequences, respectively, of the two isoforms. The two rabbit isoforms also showed significant homology with the mouse, rat, and human homologs. In situ RT-PCR showed evidence of high levels of expression of calcineurin Aalpha mRNA in freshly isolated rat osteoclasts. Semiquantitative analysis of staining intensity revealed no significant difference in calcineurin Aalpha expression in cells treated with vehicle vs. those treated with the calcineurin (activity) inhibitors cyclosporin A (8 x 10(-7) M) and FK506 (5 x 10(-9) and 5 x 10(-7) M). We then constructed a fusion protein comprising calcineurin Aalpha and TAT, a 12-amino acid-long arginine-rich sequence of the human immunodeficiency virus protein. Others have previously shown that the fusion of proteins to this sequence results in their receptor-less transduction into cells, including osteoclasts. Similarly, unfolding of the TAT-calcineurin Aalpha fusion protein by shocking with 8 M urea resulted in its rapid influx, within minutes, into as many as 90% of all freshly isolated rat osteoclasts, as was evident on double immunostaining with anti-calcineurin Aalpha and anti-TAT antibodies. Pit assays performed with TAT-calcineurin Aalpha-positive osteoclasts revealed a concentration-dependent (10-200 nM) attenuation of bone resorption in the absence of cell cytotoxicity or changes in cell number. TAT-hemaglutinin did not produce significant effects on bone resorption or cell number. The study suggests the following: 1) the 61-kDa protein phosphatase calcineurin Aalpha can be effectively tranduced into osteoclasts by using the TAT-based approach, and 2) the transduced protein retains its capacity to inhibit osteoclastic bone resorption.

Amino Acid Sequence↗
PubMed2002

Prostaglandin E2 induces hypoxia-inducible factor-1alpha stabilization and nuclear localization in a human prostate cancer cell line.

Hypoxia-induced up-regulation of vascular endothelial growth factor (VEGF) expression is a critical event leading to tumor neovascularization. Hypoxia stimulates hypoxia-inducible factor-1alpha (HIF-1alpha), a transcriptional activator of VEGF. Cyclooxygenase (COX)-2, an inducible enzyme that catalyzes the formation of prostaglandins (PGs) from arachidonic acid, is also induced by hypoxia. We reported previously that COX-2 inhibition prevents hypoxic up-regulation of VEGF in human prostate cancer cells and that prostaglandin E(2) (PGE(2)) restores hypoxic effects on VEGF. We hypothesized that PGE(2) mediates hypoxic effects on VEGF by modulating HIF-1alpha expression. Addition of PGE(2) to PC-3ML human prostate cancer cells had no effect on HIF-1alpha mRNA levels. However, PGE(2) significantly increased HIF-1alpha protein levels, particularly in the nucleus. This effect of PGE(2) largely results from the promotion of HIF-1alpha translocation from the cytosol to the nucleus. PGE(2) addition to PC-3 ML cells transfected with a GFP-HIF-1alpha vector induced a time-dependent nuclear accumulation of the HIF-1alpha protein. Two selective COX-2 inhibitors, meloxicam and NS398, decreased HIF-1alpha levels and nuclear localization, under both normoxic and hypoxic conditions. Of several prostaglandins tested, only PGE(2) reversed the effects of a COX-2 inhibitor in hypoxic cells. Finally, PGE(2) effects on HIF-1alpha were specifically inhibited by PD98059 (a MAPK inhibitor). These data demonstrate that PGE(2) production via COX-2-catalyzed pathway plays a critical role in HIF-1alpha regulation by hypoxia and imply that COX-2 inhibitors can prevent hypoxic induction of HIF-mediated gene transcription in cancer cells.

Active Transport, Cell Nucleus↗
PubMed2002

The mechanism of inhibition of HIV-1 env-mediated cell-cell fusion by recombinant cores of gp41 ectodomain.

N36(L6)C34 is a recombinant protein that forms a six-helix bundle with high thermal stability. It consists of the N-terminal heptad-repeat region (N36 peptide) and the C-terminal heptad-repeat region (C34) of HIV-1 gp41, connected by six polar amino acids. The protein inhibits HIV-1 envelope-induced membrane fusion. Whether inhibition occurs while N36(L6)C34 is in its six-helix bundle configuration was investigated. Mutating a critical residue within the N36 region to promote dissociation of C34 from the grooves of the N36 coiled coil reduced bundle stability and increased the inhibition of fusion. In contrast, mutating a key residue within the C34 region to reduce bundle stability decreased inhibitory potency. The data provide strong evidence that the proteins inhibit fusion while they expose their C34 segments, rather than as six-helix bundles. Thus, despite high thermal stability of the bundle, the recombinants' less folded structures are present in sufficient concentration to inhibit fusion at physiological temperatures.

Amino Acid Sequence↗
PubMed2002

An alanine-zipper structure determined by long range intermolecular interactions.

A major challenge in protein folding is to identify and quantify specific structural determinants that allow native proteins to acquire their unique folded structures. Here we report the engineering of a 52-residue protein (Ala-14) that contains exclusively alanine residues at the hydrophobic a and d positions of a natural heptad-repeat sequence. Ala-14 is unfolded under normal solution conditions yet forms a parallel three-stranded alpha-helical coiled coil in crystals. Ala-14 trimers in the solid state associate with each other through the pairing of polar side chains and formation of an extended network of water-mediated hydrogen bonds. In contrast to the classical view that local intramolecular tertiary interactions dictate the three-dimensional structure of small single-domain proteins, Ala-14 shows that long range intermolecular interactions can be essential in determining the metastable alanine-zipper structure. A similar interplay between short range local and longer range global forces may underlie the conformational properties of the growing class of natively unstructured proteins in biological processes.

Alanine↗
PubMed2002

The common myelolymphoid progenitor: a key intermediate stage in hemopoiesis generating T and B cells.

We have previously shown that the common progenitors for myeloid, T, and B cell lineages are enriched in the earliest population of murine fetal liver. However, it remained unclear whether such multipotent progenitors represent the pluripotent progenitors capable of generating all hemopoietic cells or they also comprise progenitors restricted to myeloid, T, and B cell lineages. To address this issue, we have developed a new clonal assay covering myeloid, erythroid, T, and B cell lineages, and using this assay the developmental potential of individual cells in subpopulations of lineage marker-negative (Lin(-)) c-kit(+) murine fetal liver cells was investigated. We identified the progenitor generating myeloid, T, and B cells, but not erythroid cells in the Sca-1(high) subpopulation of Lin(-)c-kit(+) cells that can thus be designated as the common myelolymphoid progenitor (CMLP). Common myeloerythroid progenitors were also detected. These findings strongly suggest that the first branching point in fetal hemopoiesis is between the CMLP and common myeloerythroid progenitors. T and B cell progenitors may be derived from the CMLP through the previously identified myeloid/T and myeloid/B bipotent stages, respectively.

Animals↗
PubMed2002

Functional cloning of BRF1, a regulator of ARE-dependent mRNA turnover.

To identify regulators of AU-rich element (ARE)-dependent mRNA turnover we have followed a genetic approach using a mutagenized cell line (slowC) that fails to degrade cytokine mRNA. Accordingly, a GFP reporter construct whose mRNA is under control of the ARE from interleukin-3 gives an increased fluorescence signal in slowC. Here we describe rescue of slowC by a retroviral cDNA library. Flow cytometry allowed us to isolate revertants with reconstituted rapid mRNA decay. The cDNA was identified as butyrate response factor-1 (BRF1), encoding a zinc finger protein homologous to tristetraprolin. Mutant slowC carries frame-shift mutations in both BRF1 alleles, whereas slowB with intermediate decay kinetics is heterozygous. By use of small interfering (si)RNA, independent evidence for an active role of BRF1 in mRNA degradation was obtained. In transiently transfected NIH 3T3 cells, BRF1 accelerated mRNA decay and antagonized the stabilizing effect of PI3-kinase, while mutation of the zinc fingers abolished both function and ARE-binding activity. This approach, which identified BRF1 as an essential regulator of ARE-dependent mRNA decay, should also be applicable to other cis-elements of mRNA turnover.

3' Untranslated Regions↗
PubMed2002

Interhelical interactions in the gp41 core: implications for activation of HIV-1 membrane fusion.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein complex (gp120-gp41) promotes viral entry by mediating the fusion of viral and cellular membranes. Formation of a stable trimer-of-hairpins structure in the gp41 ectodomain brings the two membranes into proximity, leading to membrane fusion. The core of this hairpin structure is a six-helix bundle in which three carboxyl-terminal outer helices pack against an inner trimeric coiled coil. Here we investigate the role of these conserved interhelical interactions on the structure and function of both the envelope glycoprotein and the gp41 core. We have replaced each of the eight amino acids at the buried face of the carboxyl-terminal helix with a representative amino acid, alanine. Structural and physicochemical characterization of the alanine mutants shows that hydrophobic interactions are a dominant factor in the stabilization of the six-helix bundle. Alanine substitutions at the Trp628, Trp631, Ile635, and Ile642 residues also affected envelope processing and/or gp120-gp41 association and abrogated the ability of the envelope glycoprotein to mediate cell-cell fusion. These results suggest that the amino-terminal region of the gp41 outer-layer alpha-helix plays a key role in the sequence of events associated with HIV-1 entry and have implications for the development of antibodies and small-molecule inhibitors of this conserved element.

Alanine↗
PubMed2002

Core side-chain packing and backbone conformation in Lpp-56 coiled-coil mutants.

Native proteins exhibit precise geometric packing of atoms in their hydrophobic interiors. Nonetheless, controversy remains about the role of core side-chain packing in specifying and stabilizing the folded structures of proteins. Here we investigate the role of core packing in determining the conformation and stability of the Lpp-56 trimerization domain. The X-ray crystal structures of Lpp-56 mutants with alanine substitutions at two and four interior core positions reveal trimeric coiled coils in which the twist of individual helices and the helix-helix spacing vary significantly to achieve the most favored superhelical packing arrangement. Introduction of each alanine "layer" into the hydrophobic core destabilizes the superhelix by 1.4 kcal mol(-1). Although the methyl groups of the alanine residues pack at their optimum van der Waals contacts in the coiled-coil trimer, they provide a smaller component of hydrophobic interactions than bulky hydrophobic side-chains to the thermodynamic stability. Thus, specific side-chain packing in the hydrophobic core of coiled coils are important determinants of protein main-chain conformation and stability.

Alanine↗
PubMed2002

An effective method for the discrimination of motional anisotropy and chemical exchange.

Analysis of the ratio of transverse and longitudinal relaxation rates (R2/R1) is an approach commonly used for estimation of overall correlation time and identification of chemical exchange in biological macromolecules. However, this analysis fails to distinguish between chemical exchange and motional anisotropy. We describe a simple method for identifying chemical exchange and motional anisotropy using the product, R1R2. In the slow tumbling regime, the R1R2 product results in a constant value that is independent of overall correlation time and motional anisotropy. This analysis provides a simple method for rapidly estimating and dissociating the effects of motional anisotropy and chemical exchange in NMR heteronuclear spin relaxation data. We demonstrate the utility of the method with 15N relaxation data collected on the proteins E. coli ribonuclease H and the trimeric E. coli membrane associated lipoprotein lpp.

Anisotropy↗
PubMed2002

Mutations that destabilize the gp41 core are determinants for stabilizing the simian immunodeficiency virus-CPmac envelope glycoprotein complex.

The human and simian immunodeficiency viruses (HIV and SIV) envelope glycoprotein consists of a trimer of two noncovalently and weakly associated subunits, gp120 and gp41. Upon binding of gp120 to cellular receptors, this labile native envelope complex undergoes conformational changes, resulting in a stable trimer-of-hairpins structure in gp41. Formation of the hairpin structure is thought to mediate membrane fusion by placing the viral and cellular membranes in close proximity. An in vitro-derived variant of SIVmac251, denoted CPmac, has acquired an unusually stable virion-associated gp120-gp41 complex. This unique phenotype is conferred by five amino acid substitutions in the gp41 ectodomain. Here we characterize the structural and physicochemical properties of the N40(L6)C38 model of the CPmac gp41 core. The 1.7-A resolution crystal structure of N40(L6)C38 is very similar to the six-helix bundle structure present in the parent SIVmac251 gp41. In both structures, three N40 peptides form a central three-stranded coiled coil, and three C38 peptides pack in an antiparallel orientation into hydrophobic grooves on the coiled-coil surface. Thermal unfolding studies show that the CPmac mutations destabilize the SIVmac251 six-helix bundle by 15 kJ/mol. Our results suggest that the formation of the gp41 trimer-of-hairpins structure is thermodynamically coupled to the conformational stability of the native envelope glycoprotein and raise the intriguing possibility that introduction of mutations to destabilize the six-helix bundle may lead to the stabilization of the trimeric gp120-gp41 complex. This study suggests a potential strategy for the production of stably folded envelope protein immunogens for HIV vaccine development.

Circular Dichroism↗