Dynamic Conformations of Nucleophosmin (NPM1) at a Key Monomer-Monomer Interface Affect Oligomer Stability and Interactions with Granzyme B

Nucleophosmin (NPM1) is an abundant, nucleolar tumor antigen with important roles in cell proliferation and putative contributions to oncogenesis. Wild-type NPM1 forms pentameric oligomers through interactions at the amino-terminal core domain. A truncated form of NPM1 found in some hepatocellular carcinoma tissue formed an unusually stable oligomer and showed increased susceptibility to cleavage by granzyme B. Initiation of translation at the seventh methionine generated a protein (M7-NPM) that shared all these properties. We used deuterium exchange mass spectrometry (DXMS) to perform a detailed structural analysis of wild-type NPM1 and M7-NPM, and found dynamic conformational shifts or local “unfolding” at a specific monomer-monomer interface which included the β-hairpin “latch.” We tested the importance of interactions at the β-hairpin “latch” by replacing a conserved tyrosine in the middle of the β-hairpin loop with glutamic acid, generating Y67E-NPM. Y67E-NPM did not form stable oligomers and further, prevented wild-type NPM1 oligomerization in a dominant-negative fashion, supporting the critical role of the β-hairpin “latch” in monomer-monomer interactions. Also, we show preferential cleavage by granzyme B at one of two available aspartates (either D161 or D122) in M7-NPM and Y67E-NPM, whereas wild-type NPM1 was cleaved at both sites. Thus, we observed a correlation between the propensity to form oligomers and granzyme B cleavage site selection in nucleophosmin proteins, suggesting that a small change at an important monomer-monomer interface can affect conformational shifts and impact protein-protein interactions.     

Localizing the Membrane Binding Region of Group VIA Ca2+-independent Phospholipase A2 Using Peptide Amide Hydrogen/Deuterium Exchange Mass Spectrometry

The Group VIA-2 Ca²⁺-independent phospholipase A₂ (GVIA-2 iPLA₂) is composed of seven consecutive N-terminal ankyrin repeats, a linker region, and a C-terminal phospholipase catalytic domain. No structural information exists for this enzyme, and no information is known about the membrane binding surface. We carried out deuterium exchange experiments with the GVIA-2 iPLA₂ in the presence of both phospholipid substrate and the covalent inhibitor methyl arachidonoyl fluorophosphonate and located regions in the protein that change upon lipid binding. No changes were seen in the presence of only methyl arachidonoyl fluorophosphonate. The region with the greatest change upon lipid binding was region 708–730, which showed a >70% decrease in deuteration levels at numerous time points. No decreases in exchange due to phospholipid binding were seen in the ankyrin repeat domain of the protein. To locate regions with changes in exchange on the enzyme, we constructed a computational homology model based on homologous structures. This model was validated by comparing the deuterium exchange results with the predicted structure. Our model combined with the deuterium exchange results in the presence of lipid substrate have allowed us to propose the first structural model of GVIA-2 iPLA₂ as well as the interfacial lipid binding region.The Group VIA phospholipase A₂ is a member of the phospholipase A₂ superfamily that cleaves fatty acids from the sn-2 position of phospholipids (1, 2). The human Group VIA PLA₂³ gene yields multiple splice variants, including GVIA-1, GVIA-2, GVIA-3 PLA₂, GVIA Ankyrin-1, and GVIA Ankyrin-2 (3, 4). At least two isoforms, GVIA-1 and GVIA-2 iPLA₂, are active. Our laboratory purified and characterized the first mammalian iPLA₂, the 85-kDa GVIA-2 iPLA₂ (5), which became the first cloned iPLA₂ (6). This enzyme can hydrolyze the sn-2 fatty acyl bond of phospholipids and also has potent lysophospholipase and transacylase activity (7). GVIA iPLA₂ is involved in cell proliferation (8), apoptosis (9–11), bone formation (12), sperm development (13), and glucose-induced insulin secretion (14, 15), so its function may vary by cell and tissue.The human GVIA-2 iPLA₂ (806 amino acids), the form of the enzyme studied here, contains seven ankyrin repeats (residues 152–382), a linker region (residues 383–474) with the eighth repeat disrupted by a 54-amino acid insert (16), and a catalytic domain (residues 475–806). The active site serine of the GVIA iPLA₂ lies within a lipase consensus sequence (Gly-X-Ser⁵¹⁹-X-Gly) (1). The activity of GVIA iPLA₂ has been reported to be regulated through several mechanisms. A caspase-3 cleavage site at the N terminus of the enzyme has been identified that is clipped in vitro (17). This truncated form of the enzyme was hyperactive and reduced cell viability when overexpressed in HEK293 cells (17). Another possible control mechanism is through ATP binding on the ⁴⁸⁵GXGXXG motif (18).The activity of phospholipases depends critically on the interaction of the protein with phospholipid membranes. In vitro, GVIA iPLA₂ does not have any specificity for the fatty acid in the sn-2 position of substrate phospholipids (5). GVIA-2 iPLA₂ was found to be membrane-associated when overexpressed in COS-7 cells, and this was further confirmed in rat vascular smooth muscle cells (4, 19). The other active splice variant, GVIA-1, is cytosolic and not specific in targeting membrane surfaces (4, 19), indicating two different regulatory mechanisms between these two splice variants. The 54-residue insertion in the eighth ankyrin repeat alters the property of GVIA-2 iPLA₂ for membrane association. The ankyrin repeats have been reported to be involved in protein-protein interactions, such as 53BP2-p53, GA-binding protein α-GA-binding protein β, p16^INK4a-CDK6, and IκBα-NFκB (16). The ankyrin repeats of GVIA iPLA₂ may directly or indirectly assist membrane association because the catalytic domain by itself does not have activity (3). Determining the regions of the protein that interact with the membrane surface will allow for a more in-depth analysis of the regulatory mechanisms of the enzyme.There is an increasing interest in GVIA iPLA₂ because of its various newly discovered functions in vivo and in vitro. However, there is no published crystal or NMR structure to facilitate analysis on the molecular level. Amide hydrogen/deuterium exchange coupled with mass spectrometry (DXMS) has been widely used to analyze the interface of protein-protein interactions (20), protein conformational changes (21, 22), and protein dynamics (23), and we have now introduced it to study protein-phospholipid interactions (24, 25). There are also reports of using DXMS with homology modeling to validate enzymes where structural information does not exist (26). We used deuterium exchange along with homology modeling to generate models of the ankyrin repeats based on the Ankyrin-R (Protein Data Bank code 1N11) and of the catalytic domain based on patatin (Protein Data Bank code 1OXW). To study the interfacial activation of GVIA iPLA₂, we generated 1-palmitoyl-2-arachidonoyl-sn-phosphatidylcholine (PAPC) vesicles containing the methyl arachidonyl fluorophosphonate (MAFP) inhibitor, which binds to the active site and irreversibly inhibits GVIA iPLA₂ (7). By applying DXMS to the iPLA₂ and using our structural model, we were now able to monitor how GVIA iPLA₂ associates with phospholipid membranes.     

Phosphorylation driven motions in the COOH-terminal Src kinase,CSK, revealed through enhanced hydrogen-deuterium exchange and mass spectrometry (DXMS)

Previous kinetic studies demonstrated that nucleotide-derived conformational changes regulate function in the COOH-terminal Src kinase. We have employed enhanced methods of hydrogen-deuterium exchange-mass spectrometry (DXMS) to probe conformational changes on CSK in the absence and presence of nucleotides and thereby provide a structural framework for understanding phosphorylation-driven conformational changes. High quality peptic fragments covering approximately 63% of the entire CSK polypeptide were isolated using DXMS. Time-dependent deuterium incorporation into these probes was monitored to identify short peptide segments that exchange differentially with solvent. Regions expected to lie in loops exchange rapidly, whereas other regions expected to lie in stable secondary structure exchange slowly with solvent implying that CSK adopts a modular structure. The ATP analog, AMPPNP, protects probes in the active site and distal regions in the large and small lobes of the kinase domain, the SH2 domain, and the linker connecting the SH2 and kinase domains. The product ADP protects similar regions of the protein but the extent of protection varies markedly in several crucial areas. These areas correspond to the activation loop and helix G in the kinase domain and several inter-domain regions. These results imply that delivery of the gamma phosphate group of ATP induces unique local and long-range conformational changes in CSK that may influence regulatory motions in the catalytic pathway.     

A two-gate model for the ryanodine receptor with allosteric modulation by caffeine and quercetin

We have developed a model of the tetrameric ryanodine receptor--the calcium channel of the sarcoplasmic reticulum. The model accurately describes published experimental data on channel activity at various concentrations of Ca2+, caffeine and quercetin. The proposed mechanisms involve allosteric regulation of Ca2+ affinity by both caffeine and quercetin, and the existence of two independent, A- and I-gates controlled by Ca2+ binding to an activating and an inhibitory module of the receptor. There are four different configurations of the receptor that affect ligand binding to the activation module, but not to the inhibition module. Consequently, there are four kinetic modes for the A-gate and one mode for the I-gate. At a certain moment, the receptor can be in any of the four possible conformations with equal probability. By fitting the data we are able to derive ligand affinities and Hill coefficients, to describe the observation that quercetin is an activating agent stronger than caffeine, and that caffeine and quercetin activate the channel at very low Ca2+ concentration (approximately 10(-11) M). We predict that the activation regime at saturating caffeine or quercetin should present four distinct regions at increasing Ca2+, corresponding to the four different gating modes. Another interesting prediction is the enlargement of the activity domain toward higher Ca2+ concentrations in the presence of caffeine or quercetin.     

Response of obligate heterozygotes for phytosterolemia to a low-fat diet and to a plant sterol ester dietary challenge

Twelve obligate heterozygotes from two kindreds were ascertained through phytosterolemic probands homozygous for molecular defects in the ATP binding cassette (ABC) half transporter, ABCG8. The response of these heterozygotes to a Step 1 diet low in fat, saturated fat, and cholesterol, and to 2.2 g daily of plant sterols (as esters) was determined in Protocol I (16 weeks) and Protocol II (28 weeks) during three consecutive feeding periods: Step 1/placebo spread; Step 1/plant sterol spread; and Step 1/placebo spread (washout). At baseline, half the heterozygotes had moderate dyslipidemia and one-third had mildly elevated campesterol and sitosterol levels. On the Step 1/placebo spread, mean LDL cholesterol decreased significantly, 11.2% in Protocol I (n = 12), and 16.0% in Protocol II (n = 7). Substitution with plant sterol spread produced a significant treatment effect on LDL levels in Protocols I and II. Conversely, the mean levels of campesterol and sitosterol increased 119% and 54%, respectively, during the use of plant sterol spread for 6 weeks in Protocol I, an effect mirrored for 12 weeks in Protocol II. During the placebo spread washouts, LDL levels increased, while those of plant sterols decreased to baseline levels in both protocols. In conclusion, phytosterolemic heterozygotes respond well to a Step 1 diet, and their response to a plant sterol ester challenge appears similar to that observed in normals.     

Molecular cloning and expression of rat interferon-γ

The nueclotide sequence data reported in this paper have been submitted to the EMBL nucleotide databse and have been assigned the accession number X86972     

Neuritic deposits of amyloid-beta peptide in a subpopulation of central nervous system-derived neuronal cells

Our goal is to understand the pathogenesis of amyloid-beta (Abeta) deposition in the Alzheimer's disease (AD) brain. We established a cell culture system where central nervous system-derived neuronal cells (CAD cells) produce and accumulate within their processes large amounts of Abeta peptide, similar to what is believed to occur in brain neurons, in the initial phases of AD. Using this system, we show that accumulation of Abeta begins within neurites, prior to any detectable signs of neurodegeneration or abnormal vesicular transport. Neuritic accumulation of Abeta is restricted to a small population of neighboring cells that express normal levels of amyloid-beta precursor protein (APP) but show redistribution of BACE1 to the processes, where it colocalizes with Abeta and markers of late endosomes. Consistently, cells that accumulate Abeta appear in isolated islets, suggesting their clonal origin from a few cells that show a propensity to accumulate Abeta. These results suggest that Abeta accumulation is initiated in a small number of neurons by intracellular determinants that alter APP metabolism and lead to Abeta deposition and neurodegeneration. CAD cells appear to recapitulate the biochemical processes leading to Abeta deposition, thus providing an experimental in vitro system for studying the molecular pathobiology of AD.     

Pro-interleukin (IL)-1�� Shares a Core Region of Stability as Compared with Mature IL-1�� While Maintaining a Distinctly Different Configurational Landscape: A COMPARATIVE HYDROGEN/DEUTERIUM EXCHANGE MASS SPECTROMETRY STUDY*

Interleukin-1β (IL-1β) is a master cytokine involved in initiating the innate immune response in vertebrates (Dinarello, C. A. (1994) FASEB J. 8, 1314–1325). It is first synthesized as an inactive 269-residue precursor (pro-interleukin-1β or pro-IL-1β). Pro-IL-1β requires processing by caspase-1 to generate the active, mature 153-residue cytokine. In this study, we combined hydrogen/deuterium exchange mass spectrometry, circular dichroism spectroscopy, and enzymatic digestion comparative studies to investigate the configurational landscape of pro-IL-1β and the role the N terminus plays in modulating the landscape. We find that the N terminus keeps pro-IL-1β in a protease-labile state while maintaining a core region of stability in the C-terminal region, the eventual mature protein. In mature IL-1β, this highly protected region maps back to the area protected earliest in the NMR studies characterizing an on-route kinetic refolding intermediate. This protected region also encompasses two important functional loops that participate in the IL-1β/receptor binding interface required for biological activity. We propose that the purpose of the N-terminal precursor region in pro-IL-1β is to suppress the function of the eventual mature region while keeping a structurally and also functionally important core region primed for the final folding into the native, active state of the mature protein. The presence of the self-inhibiting precursor region provides yet another layer of regulation in the life cycle of this important cytokine.Nearly all cell types respond to interleukin (IL)-1β,⁴ in a very sensitive manner, via binding to the interleukin-1 receptor type 1 (IL-1RI) (2). Although essential in the immune response, overproduction of IL-1β can lead to both acute (sepsis) as well as chronic (rheumatoid arthritis, atherosclerosis, obesity, and diabetes) disease states (3). Thus, the expression, activation, and secretion of this cytokine are tightly controlled (4). Although many cell types express IL-1β, it is predominately produced and secreted by monocytes and macrophages (1). The protein is synthesized as a biologically inactive 269-residue precursor molecule, pro-interleukin-1β (pro-IL-1β), and the 153-residue active mature IL-1β is generated from the C-terminal domain. Processing of the proprotein involves the recently discovered NALP-1 and NALP-3 inflammasomes, which are responsible for activating procaspase-1 (5). The inflammasome function is integral in wound repair as well as for combating infection (6–9).In vivo, the 31-kDa pro-IL-1β precursor is processed to the active C-terminal 17-kDa form by the interleukin-1 converting enzyme, caspase-1 (10, 11). Caspase-1 is a cysteine protease that recognizes two cleavage sites in pro-IL-1β, the Asp²⁷↓Gly²⁸ and Asp¹¹⁶↓Ala¹¹⁷ peptide bonds (Fig. 1A). These cleavage sites are conserved across mammals (12–14). The activation pathway is believed to proceed with cleavage first at Asp²⁷↓Gly²⁸ (site 1) followed by Asp¹¹⁶↓Ala¹¹⁷ (site 2). These processing events lead to the generation of the mature, active IL-1β from the C-terminal domain of pro-IL-1β (15). After cleavage, the mature protein is exported via a cell-specific non-classical pathway (16). The events leading from caspase-1 activation to active IL-1β secretion are poorly understood and constitute an area of active research (16–20).Open in a separate windowFIGURE 1.A, a schematic of pro-interleukin-1β processing by caspase-1. The two caspase-1 cleavage sites are labeled by residue/number. The products for the cleavage scenario are represented as smaller blocks, and the final mature protein as the actual three-dimensional structure shown in blue (Protein Data Bank code 6I1B (74)). B, panel i, important features are highlighted on the structure of mature IL-1β. Residues Tyr⁶⁸ (residue 184 in pro-IL-1β) and Trp¹²⁰ (236 in pro-IL-1β) are indicated by red side chain stick representation. The two loops important for binding at the third Ig domain of the receptor are indicated by blue spheres (the basic/hydrophobic 90s loop, which encompasses residues 85–99 in mature and 201–216 in pro-IL-1β) and yellow spheres (the β-bulge, residues 46–53 and 162–169). The numbering corresponds to mature and pro-IL-1β, respectively. Panel ii, after rotating the structure 90°, the individual trefoils are labeled by color (trefoil 1 in orange, trefoil 2 in yellow, and trefoil 3 in blue). The structural features described in panel i maintain the same coloring. Panel iii, the two-dimensional splay diagram of the trefoils labeled by color as in panel ii showing the 3-fold symmetry of the secondary structure elements.The native structure of IL-1β is classified as a β-trefoil. The global protein-fold contains three pseudo-symmetric βββloopβ motifs that coalesce to form a six-stranded barrel with three hairpins that form a six-stranded cap closing one end of the barrel (see Fig. 1B) (21). Mature IL-1β refolds relatively slowly (22), accessing multiple routes including a major route with a detectable intermediate population (23, 24). Recently, this slow folding has been attributed to repacking of a functionally important loop (the β-bulge) in the mature protein (see Fig. 1B, i) (25–27). Although much information is known about the structure, folding, and function of mature IL-1β, there is little information available on pro-IL-1β, despite the central importance of this molecule in mediating critical inflammatory processes (28–30). What is known is that the presence of the N-terminal 116 amino acids results in a highly protease-sensitive protein with no biological activity (31). Folding of mature IL-1β is believed to occur after cleavage of pro-IL-1β in vivo. Therefore, structural analysis of the precursor is essential for a better understanding of the role the precursor region plays in regulating folding events leading to the generation of the eventual mature protein.The crystal structure of pro-IL-1β has not been determined, despite approximately 25 years of intensive efforts directed toward this goal, as a result of the dynamic nature of this molecule (32–34). Therefore, we used structure-sensitive methods to compare pro-IL-1β in reference to the mature protein. Optical methods in combination with hydrogen/deuterium exchange mass spectrometric analysis (DXMS) and enzymatic digestion were used to investigate how the N-terminal precursor region modulates the properties of the C-terminal mature domain. DXMS is a well established technique for characterizing proteins refractory to standard crystallographic or NMR structure determination techniques (35–37). Taken together, our results indicate that the N terminus inhibits folding to the fully active trefoil structure in the C-terminal region, but maintains the protein in a conformation that is primed for efficient folding upon release after caspase-1 cleavage.