Michael A. Massiah

Potency of Michael reaction acceptors as inducers of enzymes that protect against carcinogenesis depends on their reactivity with sulfhydryl groups

Induction of phase 2 enzymes and elevations of glutathione are major and sufficient strategies for protecting mammals and their cells against the toxic and carcinogenic effects of electrophiles and reactive forms of oxygen. Inducers belong to nine chemical classes and have few common properties except for their ability to modify sulfhydryl groups by oxidation, reduction, or alkylation. Much evidence suggests that the cellular “sensor” molecule that recognizes the inducers and signals the enhanced transcription of phase 2 genes does so by virtue of unique and highly reactive sulfhydryl functions that recognize and covalently react with the inducers. Benzylidene-alkanones and -cycloalkanones are Michael reaction acceptors whose inducer potency is profoundly increased by the presence of ortho- (but not other) hydroxyl substituent(s) on the aromatic ring(s). This enhancement correlates with more rapid reactivity of the ortho-hydroxylated derivatives with model sulfhydryl compounds. Proton NMR spectroscopy provides no evidence for increased electrophilicity of the β-vinyl carbons (the presumed site of nucleophilic attack) on the hydroxylated inducers. Surprisingly, these ortho-hydroxyl groups display a propensity for extensive intermolecular hydrogen bond formation, which may raise the reactivity and facilitate addition of mercaptans, thereby raising inducer potencies.

Purifying natively folded proteins from inclusion bodies using sarkosyl, Triton X-100, and CHAPS.

We describe a rapid, simple, and efficient method for recovering glutathione S-transferase (GST)- and His6-tagged maltose binding protein (MBP) fusion proteins from inclusion bodies. Incubation of inclusion bodies with 10% sarkosyl effectively solubilized >95% of proteins, while high-yield recovery of sarkosyl-solubilized fusion proteins was obtained with a specific ratio of Triton X-100 and CHAPS. We demonstrate for the first time that this combination of three detergents significantly improves binding efficiency of GST and GST fusion proteins to gluthathione (GSH) Sepharose.

Structure of the MID1 Tandem B-Boxes Reveals an Interaction Reminiscent of Intermolecular Ring Heterodimers†,‡

The tripartite motif (TRIM) protein family, defined by N-terminal RING, B-box, and coiled-coil (RBCC) domains, consists of either a single type 2 B-box domain or tandem B-box domains of type 1 and type 2 (B1B2). Here, we report the first structure of the B-box domains in their native tandem orientation. The B-boxes are from Midline-1, a putative ubiquitin E3 ligase that is required for the proteosomal degradation of the catalytic subunit of protein phosphatase 2A (PP2Ac). This function of MID1 is facilitated by the direct binding of Alpha4, a regulatory subunit of PP2Ac, to B-box1, while B-box2 appears to influence this interaction. Both B-box1 and B-box2 bind two zinc atoms in a cross-brace motif and adopt a similar betabetaalpha structure reminiscent of the RING, PHD, ZZ, and U-box domains, although they differ from each other and with RING domains in the spacing of their zinc-binding residues. The two B-box domains pack against each other with the interface formed by residues located on the structured loop consisting of the two antiparallel beta-strands. The surface area of the interface is 188 A2 (17% of the total surface). Consistent with the globular structure, the Tm of the tandem B-box domain (59 degrees C) is higher than the individual domains, supporting a stable interaction between the B-box 1 and 2 domains. Notably, the interaction is reminiscent of the interaction of recently determined RING dimers, suggesting the possibility of an evolutionarily conserved role for B-box2 domains in regulating functional RING-type folds.

Detection and characterization of the in vitro e3 ligase activity of the human MID1 protein.

Human MID1 (midline-1) is a microtubule-associated protein that is postulated to target the catalytic subunit of protein phosphatase 2A for degradation. It binds alpha4 that then recruits the catalytic subunit of protein phosphatase 2A. As a member of the TRIM (tripartite motif) family, MID1 has three consecutive zinc-binding domains-RING (really interesting new gene), Bbox1, and Bbox2-that have similar ββα-folds. Here, we describe the in vitro characterization of these domains individually and in tandem. We observed that the RING domain exhibited greater ubiquitin (Ub) E3 ligase activity compared to the Bbox domains. The amount of autopolyubiquitinated products with RING-Bbox1 and RING-Bbox1-Bbox2 domains in tandem was significantly greater than those of the individual domains. However, no polyubiquitinated products were observed for the Bbox1-Bbox domains in tandem. Using mutants of Ub, we observed that these MID1 domain constructs facilitate Ub chain elongation via Lys63 of Ub. In addition, we observed that the high-molecular-weight protein products were primarily due to polyubiquitination at one site (Lys154) on the Bbox1 domain of the RING-Bbox1 and RING-Bbox1-Bbox2 constructs. We observed that MID1 E3 domains could interact with multiple E2-conjugating enzymes. Lastly, a 45-amino-acid peptide derived from the C-terminus of alpha4 that binds tightly to Bbox1 was observed to be monoubiquitinated in the assay and appears to down-regulate the amount of polyubiquitinated products formed. These studies shed light on MID1 E3 ligase activity and show how its three zinc-binding domains can contribute to MID1s overall function.

The MID1 E3 ligase catalyzes the polyubiquitination of Alpha4 (α4), a regulatory subunit of protein phosphatase 2A (PP2A): novel insights into MID1-mediated regulation of PP2A.

Background: IGBP1/α4 interacts with microtubule-associated MID1 to regulate PP2A within the mTOR pathway. Results: MID1 catalyzes the polyubiquitination and degradation of α4, demonstrating for the first time a mechanism for α4 regulation. Conclusion: The tandem RING-Bbox domains are required for α4 polyubiquitination and degradation. Significance: Ectopic overexpression of IGBP1/α4 transforms cells. Ubiquitination of α4 impacts the stability and activity level of PP2A. Alpha4 (α4) is a key regulator of protein phosphatase 2A (PP2A) and mTOR in steps essential for cell-cycle progression. α4 forms a complex with PP2A and MID1, a microtubule-associated ubiquitin E3 ligase that facilitates MID1-dependent regulation of PP2A and the dephosphorylation of MID1 by PP2A. Ectopic overexpression of α4 is associated with hepatocellular carcinomas, breast cancer, and invasive adenocarcinomas. Here, we provide data suggesting that α4 is regulated by ubiquitin-dependent degradation mediated by MID1. In cells stably expressing a dominant-negative form of MID1, significantly elevated levels of α4 were observed. Treatment of cells with the specific proteasome inhibitor, lactacystin, resulted in a 3-fold increase in α4 in control cells and a similar level in mutant cells. Using in vitro assays, individual MID1 E3 domains facilitated monoubiquitination of α4, whereas full-length MID1 as well as RING-Bbox1 and RING-Bbox1-Bbox2 constructs catalyzed its polyubiquitination. In a novel non-biased functional screen, we identified a leucine to glutamine substitution at position 146 within Bbox1 that abolished MID1-α4 interaction and the subsequent polyubiquitination of α4, indicating that direct binding to Bbox1 was necessary for the polyubiquitination of α4. The mutant had little impact on the RING E3 ligase functionality of MID1. Mass spectrometry data confirmed Western blot analysis that ubiquitination of α4 occurs only within the last 105 amino acids. These novel findings identify a new role for MID1 and a mechanism of regulation of α4 that is likely to impact the stability and activity level of PP2Ac.

MID1 catalyzes the ubiquitination of protein phosphatase 2A and mutations within its Bbox1 domain disrupt polyubiquitination of alpha4 but not of PP2Ac.

MID1 is a microtubule-associated protein that belongs to the TRIM family. MID1 functions as an ubiquitin E3 ligase, and recently was shown to catalyze the polyubiquitination of, alpha4, a protein regulator of protein phosphatase 2A (PP2A). It has been hypothesized that MID1 regulates PP2A, requiring the intermediary interaction with alpha4. Here we report that MID1 catalyzes the in vitro ubiquitination of the catalytic subunit of PP2A (PP2Ac) in the absence of alpha4. In the presence of alpha4, the level of PP2Ac ubiquitination is reduced. Using the MID1 RING-Bbox1-Bbox2 (RB1B2) construct containing the E3 ligase domains, we investigate the functional effects of mutations within the Bbox domains that are identified in patients with X-linked Opitz G syndrome (XLOS). The RB1B2 proteins harboring the C142S, C145T, A130V/T mutations within the Bbox1 domain and C195F mutation within the Bbox2 domain maintain auto-polyubiquitination activity. Qualitatively, the RB1B2 proteins containing these mutations are able to catalyze the ubiquitination of PP2Ac. In contrast, the RB1B2 proteins with mutations within the Bbox1 domain are unable to catalyze the polyubiquitination of alpha4. These results suggest that unregulated alpha4 may be the direct consequence of these natural mutations in the Bbox1 domain of MID1, and hence alpha4 could play a greater role to account for the increased amount of PP2A observed in XLOS-derived fibroblasts.

Obtaining Soluble Folded Proteins from Inclusion Bodies Using Sarkosyl, Triton X‐100, and CHAPS: Application to LB and M9 Minimal Media

This unit describes a straightforward and efficient method of using sarkosyl to solubilize and recover difficult recombinant proteins, such as GST‐ and His6‐tagged fusion proteins, that are overexpressed in E. coli. This protocol is especially useful for rescuing recombinant proteins overexpressed in M9 minimal medium. Sarkosyl added to lysis buffers helps with both protein solubility and cell lysis. Higher percentage sarkosyl (up to 10%) can extract >95% of soluble protein from inclusion bodies. In the case of sarkosyl‐solubilized GST‐fusion proteins, batch‐mode affinity purification requires addition of a specific ratio of Triton X‐100 and CHAPS, while sarkosyl‐solubilized His6‐tagged fusion proteins can be directly purified on Ni2+ resin columns. Proteins purified by this method could be widely used in biological assays, structure analysis and mass spectrum assay.

Molecular dynamics simulation reveals insights into the mechanism of unfolding by the A130T/V mutations within the MID1 zinc-binding Bbox1 domain.

The zinc-binding Bbox1 domain in protein MID1, a member of the TRIM family of proteins, facilitates the ubiquitination of the catalytic subunit of protein phosphatase 2A and alpha4, a protein regulator of PP2A. The natural mutation of residue A130 to a valine or threonine disrupts substrate recognition and catalysis. While NMR data revealed the A130T mutant Bbox1 domain failed to coordinate both structurally essential zinc ions and resulted in an unfolded structure, the unfolding mechanism is unknown. Principle component analysis revealed that residue A130 served as a hinge point between the structured β-strand-turn-β-strand (β-turn-β) and the lasso-like loop sub-structures that constitute loop1 of the ββα-RING fold that the Bbox1 domain adopts. Backbone RMSD data indicate significant flexibility and departure from the native structure within the first 5 ns of the molecular dynamics (MD) simulation for the A130V mutant (>6 Å) and after 30 ns for A130T mutant (>6 Å). Overall RMSF values were higher for the mutant structures and showed increased flexibility around residues 125 and 155, regions with zinc-coordinating residues. Simulated pKa values of the sulfhydryl group of C142 located near A130 suggested an increased in value to ~9.0, paralleling the increase in the apparent dielectric constants for the small cavity near residue A130. Protonation of the sulfhydryl group would disrupt zinc-coordination, directly contributing to unfolding of the Bbox1. Together, the increased motion of residues of loop 1, which contains four of the six zinc-binding cysteine residues, and the increased pKa of C142 could destabilize the structure of the zinc-coordinating residues and contribute to the unfolding.

The structure and behavior of the NA-CATH antimicrobial peptide with liposomes.

Naja atra cathelicidin (NA-CATH) is a 34-amino acid highly cationic peptide identified in Chinese cobras to possess potent toxicity against gram-negative and gram-positive bacteria and low toxicity against host cells. Here, we report the NMR solution structure of the full-length NA-CATH peptide and its interaction with liposomes. The structure shows a well-defined α-helix between residues Phe3 to Lys23, on which one surface is lined by the side-chains of one arginine and 11 lysine residues, while the other side is populated by hydrophobic residues. The last eleven amino acids, which are predominately aromatic and hydrophobic in nature, have no defined structure. NMR data reveal that these residues do not interact with the hydrophobic residues of the helix, indicating that the C-terminal residues have random conformations. Fluorescence requenching experiments, in which liposomes serve as a mimic of the bacterial membranes, result in fluorophore leakage that is consistent with a membrane thinning or transient pore formation mechanism. NMR titration studies of the peptide-liposome interaction reveal that the peptide is in fast exchange with the liposome, consistent with the fluorescent studies. These data indicate that full length NA-CATH possesses a helical segment and unstructured C-terminal tail that disrupts the bilayer to induce leakage and lysing.

NMR Studies of the C-Terminus of alpha4 Reveal Possible Mechanism of Its Interaction with MID1 and Protein Phosphatase 2A

Alpha4 is a regulatory subunit of the protein phosphatase family of enzymes and plays an essential role in regulating the catalytic subunit of PP2A (PP2Ac) within the rapamycin-sensitive signaling pathway. Alpha4 also interacts with MID1, a microtubule-associated ubiquitin E3 ligase that appears to regulate the function of PP2A. The C-terminal region of alpha4 plays a key role in the binding interaction of PP2Ac and MID1. Here we report on the solution structure of a 45-amino acid region derived from the C-terminus of alpha4 (alpha45) that binds tightly to MID1. In aqueous solution, alpha45 has properties of an intrinsically unstructured peptide although chemical shift index and dihedral angle estimation based on chemical shifts of backbone atoms indicate the presence of a transient α-helix. Alpha45 adopts a helix-turn-helix HEAT-like structure in 1% SDS micelles, which may mimic a negatively charged surface for which alpha45 could bind. Alpha45 binds tightly to the Bbox1 domain of MID1 in aqueous solution and adopts a structure consistent with the helix-turn-helix structure observed in 1% SDS. The structure of alpha45 reveals two distinct surfaces, one that can interact with a negatively charged surface, which is present on PP2A, and one that interacts with the Bbox1 domain of MID1.