Crystallographic Characterization of ATG Proteins and Their Interacting Partners.

Autophagosome formation and specific substrate recruitment during autophagy require ligation of the ubiquitin-like protein (UBL) Atg8 to the head group of the lipid phosphatidylethanolamine. Atg8 lipidation is mediated by distinctive UBL cascades involving autophagy-specific E1, E2, and E3 enzymes that differ substantially in sequence from components of other UBL conjugation cascades. Structural studies are important for elucidating the roles of Atg proteins that regulate multiple steps involved in autophagy. This chapter describes methods to prepare and crystallize selected proteins and complexes involved in autophagy UBL conjugation pathways, as a guide for strategies for structural and biochemical characterization of Atg proteins.

Production of Human ATG Proteins for Lipidation Assays.

Humans express several orthologs of yeast Atg8, in the LC3 and GABARAP families, which play crucial roles in autophagy through their covalent ligation to lipids, typically phosphatidylethanolamine (PE), in a process known as lipidation. Lipidation of LC3 and GABARAP regulates numerous facets of the autophagy process, including regulating expansion of the phagophore membrane, recruiting selected cargoes for degradation, and providing an autophagosome membrane-bound platform mediating dynamic interactions with other regulatory proteins. LC3 and GABARAP are families of related ubiquitin-like proteins (UBLs) (referred to here collectively as LC3/GABARAP), and their lipidation involves a divergent UBL conjugation cascade including ATG7, ATG3, and ATG12~ATG5-ATG16L1 acting as E1, E2, and E3 enzymes, respectively. ATG7 initiates LC3/GABARAP conjugation by catalyzing their C-terminal adenylation and conjugation to the catalytic cysteine of ATG3. Ultimately, the ATG12~ATG5-ATG16L1 complex catalyzes LC3/GABARAP ligation to a primary amino group on PE or other acceptor lipids. This chapter describes methods for expressing and purifying human LC3 or GABARAP, ATG7, ATG3, and the ATG12~ATG5-ATG16L1 complex for in vitro studies of LC3/GABARAP lipidation.

IDH1 and IDH2 mutations in pediatric acute leukemia.

To investigate the frequency of isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) mutations in pediatric acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL), we sequenced these genes in diagnostic samples from 515 patients (227 AMLs and 288 ALLs). Somatic IDH1/IDH2 mutations were rare in ALL (N=1), but were more common in AML, occurring in 3.5% (IDH1 N=3 and IDH2 N=5), with the frequency higher in AMLs with a normal karyotype (9.8%). The identified IDH1 mutations occurred in codon 132 resulting in replacement of arginine with either cysteine (N=3) or histidine (N=1). By contrast, mutations in IDH2 did not affect the homologous residue but instead altered codon 140, resulting in replacement of arginine with either glutamine (N=4) or tryptophan (N=1). Structural modeling of IDH2 suggested that codon 140 mutations disrupt the enzyme's ability to bind its substrate isocitrate. Accordingly, recombinant IDH2 R140Q/W were unable to carry out the decarboxylation of isocitrate to α-ketoglutarate (α-KG), but instead gained the neomorphic activity to reduce α-KG to R(-)-2-hydroxyglutarete (2-HG). Analysis of primary leukemic blasts confirmed high levels of 2-HG in AMLs with IDH1/IDH2 mutations. Interestingly, 3/5 AMLs with IDH2 mutations had FLT3-activating mutations, raising the possibility that these mutations cooperate in leukemogenesis.

Probing subtle differences in the hydrogen exchange behavior of variants of the human alpha-lactalbumin molten globule using mass spectrometry.

The hydrogen-exchange behavior of the low-pH molten globule of human alpha-lactalbumin, containing all four disulfides, has been examined and compared with that of a single disulfide variant, [28-111] alpha-lactalbumin, and of a series of proline variants of [28-111] alpha-lactalbumin. The small differences in hydrogen-exchange protection exhibited by these partially folded species were compared by mixing two or more proteins and monitoring their exchange simultaneously using mass spectrometry. The effect of single proline mutations within each alpha-domain helix on hydrogen-exchange protection has been investigated using six proline variants of [28-111] alpha-lactalbumin, L11P, L12P, M30P, I95P, K108P and Q117P. The results show that proline mutations in the A, B, C and D alpha-helices lead to a loss of hydrogen-exchange protection for residues in the local helix without perturbing hydrogen-exchange protection in other regions of the protein. Thus, local unfolding of the A, B, C and D helices does not significantly alter the packing and solvent accessibility of other regions of the molten globule. By contrast, introduction of a proline residue in the C-terminal 3(10) helix produces a larger and more widespread loss of hydrogen-exchange protection, demonstrating that longer-range perturbations of the molten globule have occurred. Thus, residues in this C-terminal region must be involved in contacts that are critical for the stabilisation of the compact molten globule structure.

Role of the F-box protein Skp2 in lymphomagenesis.

The F-box protein Skp2 (S-phase kinase-associated protein 2) positively regulates the G(1)-S transition by controlling the stability of several G(1) regulators, such as the cell cycle inhibitor p27. We show here that Skp2 expression correlates directly with grade of malignancy and inversely with p27 levels in human lymphomas. To directly evaluate the potential of Skp2 to deregulate growth in vivo, we generated transgenic mice expressing Skp2 targeted to the T-lymphoid lineage as well as double transgenic mice coexpressing Skp2 and activated N-Ras. A strong cooperative effect between these two transgenes induced T cell lymphomas with shorter latency and higher penetrance, leading to significantly decreased survival when compared with control and single transgenic animals. Furthermore, lymphomas of Nras single transgenic animals often expressed higher levels of endogenous Skp2 than tumors of double transgenic mice. This study provides evidence of a role for an F-box protein in oncogenesis and establishes SKP2 as a protooncogene causally involved in the pathogenesis of lymphomas.

Insights into SCF ubiquitin ligases from the structure of the Skp1-Skp2 complex.

F-box proteins are members of a large family that regulates the cell cycle, the immune response, signalling cascades and developmental programmes by targeting proteins, such as cyclins, cyclin-dependent kinase inhibitors, IkappaBalpha and beta-catenin, for ubiquitination (reviewed in refs 1-3). F-box proteins are the substrate-recognition components of SCF (Skp1-Cullin-F-box protein) ubiquitin-protein ligases. They bind the SCF constant catalytic core by means of the F-box motif interacting with Skp1, and they bind substrates through their variable protein-protein interaction domains. The large number of F-box proteins is thought to allow ubiquitination of numerous, diverse substrates. Most organisms have several Skp1 family members, but the function of these Skp1 homologues and the rules of recognition between different F-box and Skp1 proteins remain unknown. Here we describe the crystal structure of the human F-box protein Skp2 bound to Skp1. Skp1 recruits the F-box protein through a bipartite interface involving both the F-box and the substrate-recognition domain. The structure raises the possibility that different Skp1 family members evolved to function with different subsets of F-box proteins, and suggests that the F-box protein may not only recruit substrate, but may also position it optimally for the ubiquitination reaction.

Alpha-lactalbumin forms a compact molten globule in the absence of disulfide bonds.

Human alpha-lactalbumin (alpha-LA) is a four disulfide-bonded protein that adopts partially structured conformations under a variety of mildly denaturing conditions. At low pH, the protein is denatured but compact, with a high degree of secondary structure and a native-like fold. This is commonly referred to as a molten globule. A variant of alpha-LA, in which all eight cysteines have been mutated to alanine (all-Ala alpha-LA), has been studied using NMR spectroscopy. At low pH all-Ala alpha-LA is nearly as compact as wild type alpha-LA. Urea-induced unfolding experiments reveal that the residues that remain compact in the absence of disulfide bonds are those that are most resistant to unfolding in the wild-type alpha-LA molten globule. This is particularly remarkable because this stable core is formed by segments of the polypeptide chain from both the N- and C-termini. These results show that the overall architecture of the protein fold of alpha-LA is determined by the polypeptide sequence itself, and not as the result of cross-linking by disulfide bonds, and provide insight into the way in which the sequence codes for the fold.

Substrate recruitment to cyclin-dependent kinase 2 by a multipurpose docking site on cyclin A.

An important question in the cell cycle field is how cyclin-dependent kinases (cdks) target their substrates. We have studied the role of a conserved hydrophobic patch on the surface of cyclin A in substrate recognition by cyclin A-cdk2. This hydrophobic patch is approximately 35A away from the active site of cdk2 and contains the MRAIL sequence conserved among a number of mammalian cyclins. In the x-ray structure of cyclin A-cdk2-p27, this hydrophobic patch contacts the RNLFG sequence in p27 that is common to a number of substrates and inhibitors of mammalian cdks. We find that mutation of this hydrophobic patch on cyclin A eliminates binding to proteins containing RXL motifs without affecting binding to cdk2. This docking site is critical for cyclin A-cdk2 phosphorylation of substrates containing RXL motifs, but not for phosphorylation of histone H1. Impaired substrate binding by the cyclin is the cause of the defect in RXL substrate phosphorylation, because phosphorylation can be rescued by restoring a cyclin A-substrate interaction in a heterologous manner. In addition, the conserved hydrophobic patch is important for cyclin A function in cells, contributing to cyclin A's ability to drive cells out of the G1 phase of the cell cycle. Thus, we define a mechanism by which cyclins can recruit substrates to cdks, and our results support the notion that a high local concentration of substrate provided by a protein-protein interaction distant from the active site is critical for phosphorylation by cdks.

A residue-specific NMR view of the non-cooperative unfolding of a molten globule.

Molten globules are partially folded forms of proteins that are thought to be general intermediates in protein folding. Nonetheless, there is limited structural information about such species because they possess conformational heterogeneity and complex dynamical properties that lead to extreme line broadening in NMR spectra. Here we use a 2-D NMR approach that overcomes this difficulty by detecting the unfolding of individual residues in a molten globule in increasing concentrations of denaturant. The results show that the structure in the low pH form of alpha-lactalbumin (alpha-LA) is not formed cooperatively. Moreover, a core region remains collapsed under extremely denaturing conditions, even when the majority of the polypeptide chain is completely unfolded. Our results support a model for protein folding in which the core provides a template for correct assembly of the remainder of the structure.

Proline scanning mutagenesis of a molten globule reveals non-cooperative formation of a protein's overall topology.

Small proteins generally fold cooperatively: disruption of significant parts of the folded structure leads to unfolding of the rest of the protein. We show here, using proline scanning mutagenesis, that the native-like tertiary fold of the alpha-lactalbumin (alpha-LA) molten globule is formed by the non-cooperative assembly of its constituent helices. In contrast to the drastic destabilizing effects of proline substitutions in cooperatively folded proteins, proline mutations in the molten globule appear to cause only individual helices to unfold, without significantly influencing the other helices or the overall topology. Thus, the key determinants of a protein's overall fold may not be of the all-or-none type.

Disulfide determinants of calcium-induced packing in alpha-lactalbumin.

alpha-Lactalbumin (alpha-LA) is a two-domain calcium-binding protein that folds through a molten globule intermediate. Calcium binding to the wild-type alpha-LA molten globule induces a transition to the native state. Here we assess the calcium-binding properties of the alpha-LA molten globule by studying two variants of alpha-LA. alpha-LA(alpha) contains only the two disulfide bonds in the alpha-helical domain of alpha-LA, while alpha-LA(beta) contains only the beta-sheet domain and interdomain disulfide bonds. We found that only alpha-LA(beta) binds calcium, leading to the cooperative formation of substantial tertiary interactions. In addition, the beta-sheet domain acquires a native-like backbone topology. Thus, specific interactions within alpha-LA imposed by the beta-sheet domain and interdomain disulfide bonds, as opposed to the two alpha-helical domain disulfides, are necessary for the calcium-induced progression from the molten globule toward more native-like structure. Our results suggest that organization of the beta-sheet domain, coupled with calcium binding, comprises the locking step in the folding of alpha-LA from the molten globule to the native state.

Different subdomains are most protected from hydrogen exchange in the molten globule and native states of human alpha-lactalbumin.

alpha-Lactalbumin (alpha-LA) is a two-domain, calcium-binding protein that forms one of the best studied molten globules. We present here amide hydrogen exchange studies of the molten globule formed by human alpha-LA at pH 2 and compare these results with a similar study of the native state at pH 6.3. The most persistent structure in the molten globule is localized in the helical domain, consistent with previous results. However, the helices most protected from hydrogen exchange in the molten globule are, in the native state, less protected from exchange than other regions of the protein. The molten globule appears to contain major elements of the native fold, but formation of the fully native state requires stabilization of structure around the calcium-binding site and domain interface.

Does the molten globule have a native-like tertiary fold?

One of the mysteries in protein folding is how folding intermediates direct a protein to its unique final structure. To address this question, we have studied the molten globule formed by the alpha-helical domain of alpha-lactalbumin (alpha-LA) and demonstrated that it has a native-like tertiary fold, even in the absence of rigid, extensive side chain packing. These studies suggest that the role of molten globule intermediates in protein folding is to maintain an approximate native backbone topology while still allowing minor structural rearrangements to occur.

Hydrogen exchange in BPTI variants that do not share a common disulfide bond.

Bovine pancreatic trypsin inhibitor (BPTI) is stabilized by 3 disulfide bonds, between cysteines 30-51, 5-55, and 14-38. To better understand the influence of disulfide bonds on local protein structure and dynamics, we have measured amide proton exchange rates in 2 folded variants of BPTI, [5-55]Ala and [30-51; 14-38]V5A55, which share no common disulfide bonds. These proteins resemble disulfide-bonded intermediates that accumulate in the BPTI folding pathway. Essentially the same amide hydrogens are protected from exchange in both of the BPTI variants studied here as in native BPTI, demonstrating that the variants adopt fully folded, native-like structures in solution. However, the most highly protected amide protons in each variant differ, and are contained within the sequences of previously studied peptide models of related BPTI folding intermediates containing either the 5-55 or the 30-51 disulfide bond.

Hydrogen-oxidizing electron transport components in the hyperthermophilic archaebacterium Pyrodictium brockii.

The hyperthermophilic archaebacterium Pyrodictium brockii grows optimally at 105 degrees C by a form of metabolism known as hydrogen-sulfur autotrophy, which is characterized by the oxidation of H2 by S0 to produce ATP and H2S. UV-irradiated membranes were not able to carry out the hydrogen-dependent reduction of sulfur. However, the activity could be restored by the addition of ubiquinone Q10 or ubiquinone Q6 to the UV-damaged membranes. A quinone with thin-layer chromatography migration properties similar to those of Q6 was purified by thin-layer chromatography from membranes of P. brockii, but nuclear magnetic resonance analysis failed to confirm its identity as a ubiquinone. P. brockii quinone was capable of restoring hydrogen-dependent sulfur reduction to UV-irradiated membranes. Hydrogen-reduced-minus-air-oxidized absorption difference spectra on membranes revealed absorption peaks characteristic of c-type cytochromes. A c-type cytochrome with alpha, beta, and gamma peaks at 553, 522, and 421 nm, respectively, was solubilized from membranes with 0.5% Triton X-100. Pyridine ferrohemochrome spectra confirmed its identity as a c-type cytochrome, and heme staining of membranes loaded on sodium dodecyl sulfate gels revealed a single heme-containing component of 13 to 14 kDa. Studies with the ubiquinone analog 2-n-heptyl-4-hydroxyquinoline-N-oxide demonstrated that the P. brockii quinone is located on the substrate side of the electron transport chain with respect to the c-type cytochrome. These first characterizations of the strictly anaerobic, presumably primitive P. brockii electron transport chain suggest that the hydrogenase operates at a relatively high redox potential and that the H2-oxidizing chain more closely resembles those of aerobic eubacterial H2-oxidizing bacteria than those of the H2-metabolizing systems of anaerobes or the hyperthermophile Pyrococcus furiosus.

Active patient orientation.

The successful treatment of chronic ambulatory illness requires that patients be involved actively in the management of their own conditions. How health providers orient services toward patients is likely to affect the quality of patients' involvement. In the study reported here, three programs of care were evaluated in terms of the extent to which the care provided was oriented toward patients as active participants in the treatment process. Ninety-one hypertensive patients, randomly assigned among the three programs, rated their care in terms of active patient orientation (APO). On the basis of the structural characteristics of the three programs, it was hypothesized that the contingency-contracting approach would afford a stronger APO than either the educational or routine-care approach. The findings conform to expectation. Observed linkages among structure of care, level of APO reported, and patient participation are discussed in addition to the practice implications.

Active patient orientation and outcomes in hypertensive treatment: application of a socio-organizational perspective.

In the area of chronic ambulatory illness, it is well recognized that poor participation by patients in the treatment process greatly limits the potential benefits of effective medical technology. Patients' contributions to treatment outcomes might be enhanced if medical care was oriented to consider patients as active participants in the treatment process, rather than as passive-obedient recipients of care. A systematic attempt is needed to define and measure services along a specific dimension of "Active Patient Orientation" and to relate these measurements directly to treatment outcomes. Applying a socio-organizational perspective, the study reported here examined the link between an active patient orientation and treatment outcomes. Hypertensive patients were asked to characterize their care along the dimension of Active Patient Orientation (APO). Findings indicate that patients who are afforded a high degree of APO are significantly more likely to have their blood pressures under control and to exhibit more positive cognitive and behavioral responses to illness-management. Further, the data suggest that level of APO can be significantly increased through incremental changes in systems of routine clinic care.