Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. II. Interplay of local backbone conformational dynamics and long‐range hydrophobic interactions in hairpin formation

Two peptides, corresponding to the turn region of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus, consisting of residues 51–56 [IG(51–56)] and 50–57 [IG(50–57)], respectively, were studied by circular dichroism and NMR spectroscopy at various temperatures and by differential scanning calorimetry. Our results show that the part of the sequence corresponding to the β‐turn in the native structure (DDATKT) of the B3 domain forms bent conformations similar to those observed in the native protein. The formation of a turn is observed for both peptides in a broad range of temperatures (T = 283–323 K), which confirms the conclusion drawn from our previous studies of longer sequences from the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G (16, 14, and 12 residues), that the DDATKT sequence forms a nucleation site for formation of the β‐hairpin structure of peptides corresponding to the C‐terminal part of all the B domains of the immunoglobulin binding protein G. We also show and discuss the role of long‐range hydrophobic interactions as well as local conformational properties of polypeptide chains in the mechanism of formation of the β‐hairpin structure. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Hairpin ribozyme catalysis: A surface‐enhanced Raman spectroscopy study

The existence of an “RNA world” as an early step in the history of life increases the interest for the characterization of these biomolecules. The hairpin ribozyme studied here is a self‐cleaving/ligating motif found in the minus strand of the satellite RNA associated with Tobacco ringspot virus. Surface‐enhanced Raman spectroscopy (SERS) is a powerful tool to study trace amounts of RNA. In controlled conditions, a SERS signal is proportional to the amount of free residues adsorbed on the metal surface. On RNA cleavage, residues are unpaired and free to interact with metal. SERS procedures are used to monitor and quantify the catalysis of ribozyme cleavage at biological concentrations in real time; thus, they propose an interesting alternative to electrophoretic methods. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 384–390, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Conformational studies of the C-terminal 16-amino-acid-residue fragment of the B3 domain of the immunoglobulin binding protein G from Streptococcus

The structure and stability of the 16-amino-acid-residue fragment [IG(46-61)] corresponding to the C-terminal beta-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus was investigated by means of CD and NMR spectroscopy and by differential scanning calorimetry. The CD and 2D NMR experiments were carried out (i) in water at different temperatures and (ii) at one temperature (305 K), with only CD, at different TFE concentrations. Our results show that the IG(46-61) peptide possesses organized three-dimensional structure at all investigated temperatures. The three-dimensional structure of the IG(46-61) peptide resembles the general shape of a beta-hairpin that is also observed for this peptide in the experimental structure of the B3 domain in the whole G protein; the structure is stabilized by hydrophobic interactions between nonpolar side chains. Our study shows that the melting temperature of the IG(46-61) peptide is about 320 K which supports the hypothesis that the investigated peptide can serve as a folding initiation site of the B3 domain of the immunoglobulin binding protein G.     

Terminal sidechain packing of a designed β‐hairpin influences conformation and stability

While end capping in α‐helices is well understood, the concept of capping a β‐hairpin is a relatively recent development; to date, favorable Coulombic interactions are the only example of sidechains at the termini influencing the overall stability of a β‐hairpin. While cross‐strand hydrophobic residues generally provide hairpin stabilization, particular when flanking the turn region, those remote from this location appear to provide little stabilization. While probing for an optimal residue at a hydrogen bond position near the terminus of a designed β‐hairpin a conservative, hydrophobic, V → I mutation was observed to not only result in a significant change in fold population but also effected major changes in the structuring shifts at numerous sites in the peptide. Mutational studies reveal that there is an interaction between the sidechain at this H‐bonded site and the sidechain at the C‐terminal non‐H‐bonded site of the hairpin. This interaction, which appears to be hydrophobic in character, requires a highly twisted hairpin structure. Modifications at the C‐terminal site, for example an E → A mutation (ΔΔG_U = 6 kJ/mol), have profound affects on fold structure and stability. The data suggests that this may be a case of hairpin end capping by the formation of a hydrophobic cluster. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 557–564, 2009. This article was originally published online as an accepted preprint. The “Published Online”date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

A novel pocket in 14-3-3? is required to mediate specific complex formation with cdc25C and to inhibit cell cycle progression upon activation of checkpoint pathways

Mitotic progression requires the activity of the dual specificity phosphatase, cdc25C. Cdc25C function is inhibited by complex formation with two 14-3-3 isoforms, 14-3-3? and 14-3-3γ. To understand the molecular basis of specific complex formation between 14-3-3 proteins and their ligands, chimeric 14-3-3 proteins were tested for their ability to form a complex with cdc25C in vivo. Specific complex formation between cdc25C and 14-3-3? in vivo requires a phenylalanine residue at position 135 (F135) in 14-3-3?. Mutation of this residue to the corresponding residue present in other 14-3-3 isoforms (F135V) leads to reduced binding to cdc25C and a decrease in the ability to inhibit cdc25C function in vivo. Similarly, F135V failed to rescue the incomplete S phase and the G2 DNA damage checkpoint defects observed in cells lacking 14-3-3?. A comparative analysis of the 14-3-3 structures present in the database suggested that the F135 in 14-3-3? was required to maintain the integrity of a pocket that might be involved in secondary interactions with cdc25C. These results suggest that the specificity of the 14-3-3 ligand interaction may be dependent on structural motifs present in the individual 14-3-3 isoforms.     

BimL directly neutralizes Bcl-xL to promote Bax activation during UV-induced apoptosis

Bcl-2-interacting mediator of cell death (Bim) has been considered to initiate intrinsic apoptotic pathway through Bax activation. Previous studies indicated that BimL was involved in UV-induced apoptosis, but it remains unclear whether Bim activates Bax by directly engaging it or by releasing it from pro-survival relatives such as Bcl-xL. In this study, we attempt to determine the interactions between BimL and Bax/Bcl-xL during Ultraviolet (UV)-induced apoptosis. BimL activation appeared to be an important event in our experiments, as demonstrated by the significant inhibition of cell death, caspase-3 activity, and Bax translocation in cells with knockdown of endogenous BimL by RNAi approach. Both fluorescence resonance energy transfer (FRET) and Co-immunoprecipitation (CO-IP) assays indicated that Bcl-xL directly bound to Bax to inhibit its activation, while BimL directly neutralized Bcl-xL, followed by Bax release and activation upon UV irradiation. Not detected in our experiment was the interaction between BimL and Bax either using FRET approach in living cells or endogenous CO-IP assay. Thus, our findings provide strong evidence in living cells for the first time that BimL initiates apoptosis by abrogating Bcl-xL and promoting Bax activation under UV irradiation.

Structured summary

MINT-7034091: BIML (uniprotkb:O43521) physically interacts (MI:0218) with Bcl2-Xl (uniprotkb:Q92934) by anti bait coimmunoprecipitation (MI:0006)MINT-7034079: Bcl2-Xl (uniprotkb:Q92934) physically interacts (MI:0218) with BAX (uniprotkb:Q07812) and BIML (uniprotkb:O43521) by anti bait coimmunoprecipitation (MI:0006)MINT-7034069: BAX (uniprotkb:Q07812) physically interacts (MI:0218) with BIML (uniprotkb:O43521) by anti bait coimmunoprecipitation (MI:0006)MINT-7034114: BIML (uniprotkb:O43521) and BAX (uniprotkb:Q07812) physically interact (MI:0218) by fluorescent resonance energy transfer (MI:0055)     

FATP1 mediates fatty acid-induced activation of AMPK in 3T3-L1 adipocytes

Fatty acid transport proteins are integral membrane acyl-CoA synthetases implicated in adipocyte fatty acid influx and esterification. FATP-dependent production of AMP was evaluated using FATP4 proteoliposomes, and fatty acid-dependent activation of AMP-activated protein kinase (AMPK) was assessed in 3T3-L1 adipocytes. Insulin-stimulated fatty acid influx (palmitate or arachidonate) into cultured adipocytes resulted in an increase in the phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase. Consistent with the activation of AMPK, palmitate uptake into 3T3-L1 adipocytes resulted in an increase in intracellular [AMP]/[ATP]. The fatty acid-induced increase in AMPK activation was attenuated in a cell line expressing shRNA targeting FATP1. Taken together, these results demonstrate that, in adipocytes, insulin-stimulated fatty acid influx mediated by FATP1 regulates AMPK and provides a potential regulatory mechanism for balancing de novo production of fatty acids from glucose metabolism with influx of preformed fatty acids via phosphorylation of acetyl-CoA carboxylase.     

Sox9 Represses α-Sarcoglycan Gene Expression in Early Myogenic Differentiation

Alpha sarcoglycan (α-SG) is highly expressed in differentiated striated muscle, and its disruption causes limb-girdle muscular dystrophy. Accordingly, the myogenic master regulator MyoD finely modulates its expression. However, the mechanisms preventing α-SG gene expression at early stages of myogenic differentiation remain unknown. In this study, we uncovered Sox9, which was not previously known to directly bind muscle gene promoters, as a negative regulator of α-SG gene expression. Reporter gene and chromatin immunoprecipitation assays revealed three functional Sox-binding sites that mediate α-SG promoter activity repression during early myogenic differentiation. In addition, we show that Sox9-mediated inhibition of α-SG gene expression is independent of MyoD. Moreover, we provide evidence suggesting that Smad3 enhances the repressive activity of Sox9 over α-SG gene expression in a transforming growth factor-β-dependent manner. On the basis of these results, we propose that Sox9 and Smad3 are responsible for preventing precocious activation of α-SG gene expression during myogenic differentiation.