Characterization of dehydroascorbate-mediated modification of glutaredoxin by mass spectrometry.

Ascorbate is as a potent antioxidant in vivo protecting the organism against oxidative stress. In this process, ascorbate is oxidized in two steps to dehydroascorbate (DHA), which if not efficiently reduced back to ascorbate decomposes irreversibly to a complex mixture of products. We demonstrate that a component of this mixture specifically reacts with the thiol group of cysteine residues at physiological pH to give a protein adduct involving the addition of a 5-carbon fragment of DHA (+112 Da). Incubations of glutaredoxin-1 expressed in Escherichia coli and dehydroascorbate revealed abundant adducts of +112, +224 and +336 Da due to the addition of one, two and three conjugation products of DHA, respectively. ESI-MS of carbamidomethylated glutaredoxin-1 before incubation with DHA, deuterium exchange together with tandem mass spectrometry analysis and LC-ESIMS/MS of modified peptides confirmed structure and sites of modification in the protein. Modification of protein thiols by a DHA-derived product can be involved in oxidative stress-mediated cellular toxicity.

Methods for the expression, purification, preparation, and biophysical characterization of constructs of the c-Myc and Max b-HLH-LZs.

Specific heterodimerization and DNA binding by the b-HLH-LZ transcription factors c-Myc and Max is central to the activation and repression activities of c-Myc that lead to cell growth, proliferation, and tumorigenesis (Adhikary and Eilers, Nat Rev Mol Cell Biol 6:635-645, 2005; Eilers and Eisenman, Genes Dev 22:2755-2766, 2008; Grandori et al., Annu Rev Cell Dev Biol 16:653-699, 2000; Whitfield and Soucek, Cell Mol Life Sci 69:931-934, 2011). Although many c-Myc-interacting partner proteins are known to interact through their HLH domain (Adhikary and Eilers, Nat Rev Mol Cell Biol 6:635-645, 2005), current knowledge regarding the structure and the determinants of molecular recognition of these complexes is still very limited. Moreover, recent advances in the development and use of b-HLH-LZ dominant negatives (Soucek et al., Nature 455:679-683, 2008) and inhibitors of c-Myc interaction with its protein partners (Bidwell et al., J Control Release 135:2-10, 2009; Mustata et al., J Med Chem 52:1247-1250, 2009; Prochownik and Vogt, Genes Cancer 1:650-659, 2010) or DNA highlight the importance of efficient protocols to prepare such constructs and variants. Here, we provide methods to produce and purify high quantities of pure and untagged b-HLH-LZ constructs of c-Myc and Max as well as specific c-Myc/Max heterodimers for their biophysical and structural characterization by CD, NMR, or crystallography. Moreover, biochemical methods to analyze the homodimers and heterodimers as well as DNA binding of these constructs by native electrophoresis are presented. In addition to enable the investigation of the c-Myc/Max b-HLH-LZ complexes, the protocols described herein can be applied to the biochemical characterization of various mutants of either partner, as well as to ternary complexes with other partner proteins.

New structural determinants for c-Myc specific heterodimerization with Max and development of a novel homodimeric c-Myc b-HLH-LZ.

c-Myc must heterodimerize with Max to accomplish its functions as a transcription factor. This specific heterodimerization occurs through the b-HLH-LZ (basic region, helix 1-loop-helix 2-leucine zipper) domains. In fact, many studies have shown that the c-Myc b-HLH-LZ (c-Myc'SH) preferentially forms a heterodimer with the Max b-HLH-LZ (Max'SH). The primary mechanism underlying the specific heterodimerization lies on the destabilization of both homodimers and the formation of a more stable heterodimer. In this regard, it has been widely reported that c-Myc'SH has low solubility and homodimerizes poorly and that repulsions within the LZ domain account for the homodimer instability. Here, we show that replacing one residue in the basic region and one residue in Helix 1 (H(1)) of c-Myc'SH with corresponding residues conserved in b-HLH proteins confers to c-Myc'SH a higher propensity to form a stable homodimer in solution. In stark contrast to the wild-type protein, this double mutant (L362R, R367L) of the c-Myc b-HLH-LZ (c-Myc'RL) shows limited heterodimerization with Max'SH in vitro. In addition, c-Myc'RL forms highly stable and soluble complexes with canonical as well as non-canonical E-box probes. Altogether, our results demonstrate for the first time that structural determinants driving the specific heterodimerization of c-Myc and Max are embedded in the basic region and H(1) of c-Myc and that these can be exploited to engineer a novel homodimeric c-Myc b-HLH-LZ with the ability of binding the E-box sequence autonomously and with high affinity.

Influence of Ca2+ and pH on the folding of the prourotensin II precursor.

Proper folding is a crucial step for the trafficking of proteins through the secretory pathway. We hypothesized that the secretory granules of endocrine cells provide optimal folding conditions of prohormone precursors for cleavage. Here, using circular dichroism and in vitro processing on purified prourotensin II (ProUII), we show that the precursor undergoes pH- and Ca(2+)-dependent conformational and stability changes. ProUII has a stable tertiary structure at pH 5.5 in presence of Ca(2+) and is correctly cleaved in these conditions by prohormone convertases. Taken together, our results support the notion that precursors may need to be optimally folded in the lumen of secretory granules for their processing.

The Max homodimeric b-HLH-LZ significantly interferes with the specific heterodimerization between the c-Myc and Max b-HLH-LZ in absence of DNA: a quantitative analysis.

Specific heterodimerization plays a crucial role in the regulation of the biology of the cell. For example, the specific heterodimerization between the b-HLH-LZ transcription factors c-Myc and Max is a prerequisite for c-Myc transcriptional activity that leads to cell growth, proliferation and tumorigenesis. On the other hand, the Mad proteins can compete with c-Myc for Max. The Mad/Max heterodimer antagonizes the effect of the c-Myc/Max heterodimer. In this contribution, we have focused on the specific heterodimerization between the b-HLH-LZ domains of c-Myc and Max using CD and NMR. While the c-Myc and Max b-HLH-LZ domains are found to preferentially form a heterodimer; we demonstrate for the first time that a significant population of the Max homodimeric b-HLH-LZ can also form and hence interferes significantly with the specific heterodimerization. This indicates that the Max/Max homodimer can also interfere with c-Myc/Max functions, therefore adding to the complexity of the regulation of transcription by the Myc/Max/Mad network. The demonstration of the existence of the homodimeric population was made possible by the application of numerical routines that enable the simulation of composite spectroscopic signal (e.g. CD) as a function of temperature and total concentration of proteins. From a systems biology perspective, our routines may be of general interest as they offer the opportunity to treat many competing equilibriums in order to predict the probability of existence of protein complexes.

The self-association of two N-terminal interaction domains plays an important role in the tetramerization of TRPC4.

Transient receptor potential canonical (TRPC) channels function as cation channels. In a previous study, we identified the molecular determinants involved in promoting TRPC subunit assembly. In the present study, we used size-exclusion chromatography assays to show that the N-terminus of TRPC4 can self-associate and form a tetramer in cellulo. We further showed that the N-terminus of TRPC4 self-associates via the ankyrin repeat domain and the region downstream from the coiled-coil domain. GST pull-down, yeast two-hybrid, and circular dichroism approaches demonstrated that both domains can self-associate. These findings indicated that the self-association of two distinct domains in the N-terminus of TRPC4 is involved in the assembly of the tetrameric channel.

Mutation G827R in matriptase causing autosomal recessive ichthyosis with hypotrichosis yields an inactive protease.

Matriptase is a member of the novel family of type II transmembrane serine proteases. It was recently shown that a rare genetic disorder, autosomal recessive ichthyosis with hypotrichosis, is caused by a mutation in the coding region of matriptase. However, the biochemical and functional consequences of the G827R mutation in the catalytic domain of the enzyme have not been reported. Here we expressed the G827R-matriptase mutant in bacterial cells and found that it did not undergo autocatalytic cleavage from its zymogen to its active form as did the wild-type matriptase. Enzymatic activity measurements showed that the G827R mutant was catalytically inactive. When expressed in HEK293 cells, G827R-matriptase remained inactive but was shed as a soluble form, suggesting that another protease cleaved the full-length mature form of matriptase. Molecular modeling based on the crystal structure of matriptase showed that replacing Gly(827) by Arg blocks access to the binding/catalytic cleft of the enzyme thereby preventing autocatalysis of the zymogen form. Our study, thus, provides direct evidence that the G827R mutation in patients with autosomal recessive ichthyosis with hypotrichosis leads to the expression of an inactive protease.

Direct visualization of the binding of c-Myc/Max heterodimeric b-HLH-LZ to E-box sequences on the hTERT promoter.

Myc and Max belong to the b-HLH-LZ family of transcription factors. Heterodimerization between Myc and Max or homodimerization of Max allows these proteins to bind their cognate DNA sequence known as the E-box (CACGTG). Recent evidence has suggested that the c-Myc/Max heterodimeric b-HLH-LZ could interact to form a head-to-tail dimer of dimers and induce complex topologies such as loops in promoters containing more than one E-box sequence. In an attempt to shed light on this hypothesis, the interaction between the heterodimeric b-HLH-LZ of c-Myc/Max and a fragment of the hTERT promoter containing two E-box sequences was studied by atomic force microscopy. Specific binding events were observed at both E-box sites with equal probabilities. In accordance with previous results obtained by EMSA, we observed that the specific binding of the c-Myc/Max b-HLH-LZ bends the promoter. However no looping could be observed in a wide range of concentration encompassing the Ka (association constant) of the putative tetramer and the Ka for the specific binding of the heterodimer. In contrast, experiments performed with a mandatory c-Myc/Max b-HLH-LZ tetramer incubated with the hTERT promoter fragment allowed for the visualization of loops and cross-linked DNA strands originating from specific binding. Altogether, our results indicate that the c-Myc/Max b-HLH-LZ dimer binds specifically and equally to both E-box sites of the hTERT promoter and induces a significant bending of the promoter and that the suggested oligomerization of the c-Myc/Max heterodimeric b-HLH-LZ, if existing, is most likely too weak to induce the formation of a loop in a promoter.

Structural and thermodynamical characterization of the complete p21 gene product of Max.

The b-HLH-LZ family of transcription factors contains numerous proteins including the Myc and Mad families of proteins. Max heterodimerizes with other members to bind the E-Box DNA sequence in target gene promoters. Max is the only protein in this network that recognizes and binds E-Box DNA sequences as a homodimer in vitro and represses transcription of Myc target genes in vivo. Key information such as the structure of p21 Max, the complete gene product, and its KD in the absence of DNA are still unknown. Here, we report the characterization of the secondary and quaternary structures, the dimerization and DNA binding of p21 Max and a thermodynamically stable mutant. The helical content of p21 Max indicates that its N-terminal and C-terminal regions are unstructured in the absence of DNA. NMR experiments further support the location of folded and unfolded domains. We also show that p21 Max has an apparent KD (37 degrees C) of 7 x 10(-6), a value 10-100 times smaller than the b-HLH-LZ itself. We demonstrate that electrostatic repulsions are responsible for the higher KD of the b-HLH-LZ. Finally, we show that a p21 Max double mutant forms a very stable dimer with a KD (37 degrees C) of 3 x 10(-10) and that the protein/DNA complex depicts a higher temperature of denaturation than p21 Max/DNA complex. Our results indicate that Max could homodimerize, bind DNA, and repress transcription in vivo and that its mutant could be more efficient at repressing the expression of c-Myc target genes.

Toward the elucidation of the structural determinants responsible for the molecular recognition between Mad1 and Max.

Mad1 is a member of the Mad family. This family is part of the larger Myc/Max/Mad b-HLH-LZ eukaryotic transcription-factor network. Mad1 forms a specific heterodimer with Max and acts as a transcriptional repressor when bound to an E-box sequence (CACGTG) found in the promoter of c-Myc target genes. Mad1 cannot form a complex with DNA by itself under physiological conditions. A global model for the molecular recognition has emerged in which the Mad1 b-HLH-LZ homodimer is destabilized and the Mad/Max b-HLH-LZ heterodimer is favored. The detailed structural determinants responsible for the molecular recognition remain largely unknown. In this study, we focus on the elucidation of the structural determinants responsible for the destabilization of the Mad1 b-HLH-LZ homodimer. Conserved acidic residues at the dimerization interface (position a) of the LZ of all Max-interacting proteins have been hypothesized to be involved in the destabilization of the homodimeric states. In Mad1, this position corresponds to residue Asp 112. As reported for the complete gene product of Mad1, we show that wild-type b-HLH-LZ does not homodimerize or bind DNA under physiological conditions. On the other hand, the single mutation of Asp 112 to an Asn enables the b-HLH-LZ to dimerize and bind DNA. Our results suggest that Asp 112 is implicated in the destabilization of Mad1 b-HLH-LZ homodimer. Interestingly, this side chain is observed to form a salt bridge at the interface of the LZ domain in the crystal structure of Mad1/Max heterodimeric b-HLH-LZ bound to DNA [Nair, S. K., and Burley, S. K. (2003) Cell 112, 193-205]. This clearly suggests that Asp 112 plays a crucial role in the molecular recognition between Max and Mad1.