Daniel Hirschberg

Structure and allosteric effects of low-molecular-weight activators on the protein kinase PDK1.

Protein phosphorylation transduces a large set of intracellular signals. One mechanism by which phosphorylation mediates signal transduction is by prompting conformational changes in the target protein or interacting proteins. Previous work described an allosteric site mediating phosphorylation-dependent activation of AGC kinases. The AGC kinase PDK1 is activated by the docking of a phosphorylated motif from substrates. Here we present the crystallography of PDK1 bound to a rationally developed low-molecular-weight activator and describe the conformational changes induced by small compounds in the crystal and in solution using a fluorescence-based assay and deuterium exchange experiments. Our results indicate that the binding of the compound produces local changes at the target site, the PIF binding pocket, and also allosteric changes at the ATP binding site and the activation loop. Altogether, we present molecular details of the allosteric changes induced by small compounds that trigger the activation of PDK1 through mimicry of phosphorylation-dependent conformational changes.

p53 targets identified by protein expression profiling

p53 triggers cell cycle arrest and apoptosis through transcriptional regulation of specific target genes. We have investigated the effect of p53 activation on the proteome using 2D gel electrophoresis analysis of mitomycin C-treated HCT116 colon carcinoma cells carrying wild-type p53. Approximately 5,800 protein spots were separated in overlapping narrow-pH-range gel strips, and 115 protein spots showed significant expression changes upon p53 activation. The identity of 55 protein spots was obtained by mass spectrometry. The majority of the identified proteins have no previous connection to p53. The proteins fall into different functional categories, such as mRNA processing, translation, redox regulation, and apoptosis, consistent with the idea that p53 regulates multiple cellular pathways. p53-dependent regulation of five of the up-regulated proteins, eIF5A, hnRNP C1/C2, hnRNP K, lamin A/C, and Nm23-H1, and two of the down-regulated proteins, Prx II and TrpRS, was examined in further detail. Analysis of mRNA expression levels demonstrated both transcription-dependent and transcription-independent regulation among the identified targets. Thus, this study reveals protein targets of p53 and highlights the role of transcription-independent effects for the p53-induced biological response.

Mechanism for activation of the growth factor-activated AGC kinases by turn motif phosphorylation

The growth factor/insulin‐stimulated AGC kinases share an activation mechanism based on three phosphorylation sites. Of these, only the role of the activation loop phosphate in the kinase domain and the hydrophobic motif (HM) phosphate in a C‐terminal tail region are well characterized. We investigated the role of the third, so‐called turn motif phosphate, also located in the tail, in the AGC kinases PKB, S6K, RSK, MSK, PRK and PKC. We report cooperative action of the HM phosphate and the turn motif phosphate, because it binds a phosphoSer/Thr‐binding site above the glycine‐rich loop within the kinase domain, promoting zipper‐like association of the tail with the kinase domain, serving to stabilize the HM in its kinase‐activating binding site. We present a molecular model for allosteric activation of AGC kinases by the turn motif phosphate via HM‐mediated stabilization of the αC helix. In S6K and MSK, the turn motif phosphate thereby also protects the HM from dephosphorylation. Our results suggest that the mechanism described is a key feature in activation of upto 26 human AGC kinases.

Sequential proteome alterations during genesis and progression of colon cancer

Changes in the proteome of colon mucosal cells accompany the transition from normal mucosa via adenoma and invasive cancer to metastatic disease. Samples from 15 patients with sporadic sigmoid cancers were analyzed. Proteins were separated by two-dimensional gel electrophoresis. Relative differences in expression levels between normal tissue, adenoma, carcinoma and metastasis were evaluated in both intra- and inter-patient comparisons. Up- and down-regulated proteins (<twofold) during development to cancer or metastasis were excised and submitted to peptide mass fingerprinting and MS/MS sequence analysis, facilitated by the use of a compact disc workstation. In total, 112 protein spots were found to be differentially regulated, of which 72 were determined as to protein identity, 46 being up-regulated toward the progression of cancer, and 26 down-regulated. Several of the identifications correlate with proteins of the cell cycle, cytoskeleton or metabolic pathways. The pattern changes now identified have the potential for design of marker panels for assistance in diagnostics and therapeutic strategies in colorectal cancer.

Ultraslow oligomerization equilibria of p53 and its implications.

The tumor suppressor p53 is in equilibrium at cellular concentrations between dimers and tetramers. Oncogenic mutant p53 (mut) exerts a dominant-negative effect on co-expression of p53 wild-type (wt) and mut alleles in cancer cells. It is believed that wt and mut form hetero-tetramers of attenuated activity, via their tetramerization domains. Using electrospray mass spectrometry on isotopically labeled samples, we measured directly the composition and rates of formation of p53 complexes in the presence and absence of response element DNA. The dissociation of tetramers was unexpectedly very slow (t1/2 = 40 min) at 37 °C, matched by slow association of dimers, which is approximately four times longer than the half-life of spontaneous denaturation of wt p53. On mixing wt tetramers with the oncogenic contact mutant R273H of low DNA affinity, we observed the same slow formation of only wt4, wt2mut2, and mut4, in the ratio 1:2:1, on a cellular time scale. On mixing wt and mut with response element DNAs P21 and BAX, we observed only the complexes wt4.DNA, wt2mut2.DNA, and mut4.DNA, with relative dissociation constants 1:4:71 and 1:13:85, respectively, accounting for the dominant-negative effect by weakened affinity. p53 dimers assemble rapidly to tetramers on binding to response element DNA, initiated by the p53 DNA binding domains. The slow oligomerization of free p53, competing with spontaneous denaturation, has implications for the possible regulation of p53 by binding proteins and DNA that affect tetramerization kinetics as well as equilibria.

Dynorphin A (1–17) induces apoptosis in striatal neurons in vitro through α-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor-mediated cytochrome C release and caspase-3 activation

Dynorphin A (1-17), an endogenous opioid neuropeptide, can have pathophysiological consequences at high concentrations through actions involving glutamate receptors. Despite evidence of excitotoxicity, the basic mechanisms underlying dynorphin-induced cell death have not been explored. To address this question, we examined the role of caspase-dependent apoptotic events in mediating dynorphin A (1-17) toxicity in embryonic mouse striatal neuron cultures. In addition, the role of opioid and/or glutamate receptors were assessed pharmacologically using dizocilpine maleate (MK(+)801), a non-equilibrium N-methyl-D-aspartate (NMDA) antagonist; 6-cyano-7-nitroquinoxaline-2,3-dione, a competitive alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate antagonist; or (-)-naloxone, a general opioid antagonist. The results show that dynorphin A (1-17) (>or=10 nM) caused concentration-dependent increases in caspase-3 activity that were accompanied by mitochondrial release of cytochrome c and the subsequent death of cultured mouse striatal neurons. Moreover, dynorphin A-induced neurotoxicity and caspase-3 activation were significantly attenuated by the cell permeable caspase inhibitor, caspase-3 inhibitor-II (z-DEVD-FMK), further suggesting an apoptotic cascade involving caspase-3. AMPA/kainate receptor blockade significantly attenuated dynorphin A-induced cytochrome c release and/or caspase-3 activity, while NMDA or opioid receptor blockade typically failed to prevent the apoptotic response. Last, dynorphin-induced caspase-3 activation was mimicked by the ampakine CX546 [1-(1,4-benzodioxan-6-ylcarbonyl)piperidine], which suggests that the activation of AMPA receptor subunits may be sufficient to mediate toxicity in striatal neurons. These findings provide novel evidence that dynorphin-induced striatal neurotoxicity is mediated by a caspase-dependent apoptotic mechanism that largely involves AMPA/kainate receptors.

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Specific Changes in the Local Flexibility of Plasminogen Activator Inhibitor 1 upon Binding to the Somatomedin B Domain of Vitronectin

The native fold of plasminogen activator inhibitor 1 (PAI-1) represents an active metastable conformation that spontaneously converts to an inactive latent form. Binding of the somatomedin B domain (SMB) of the endogenous cofactor vitronectin to PAI-1 delays the transition to the latent state and increases the thermal stability of the protein dramatically. We have used hydrogen/deuterium exchange mass spectrometry to assess the inherent structural flexibility of PAI-1 and to monitor the changes induced by SMB binding. Our data show that the PAI-1 core consisting of β-sheet B is rather protected against exchange with the solvent, while the remainder of the molecule is more dynamic. SMB binding causes a pronounced and widespread stabilization of PAI-1 that is not confined to the binding interface with SMB. We further explored the local structural flexibility in a mutationally stabilized PAI-1 variant (14-1B) as well as the effect of stabilizing antibody Mab-1 on wild-type PAI-1. The three modes of stabilizing PAI-1 (SMB, Mab-1, and the mutations in 14-1B) all cause a delayed latency transition, and this effect was accompanied by unique signatures on the flexibility of PAI-1. Reduced flexibility in the region around helices B, C, and I was seen in all three cases, which suggests an involvement of this region in mediating structural flexibility necessary for the latency transition. These data therefore add considerable depth to our current understanding of the local structural flexibility in PAI-1 and provide novel indications of regions that may affect the functional stability of PAI-1.

Solution structure of recombinant somatomedin B domain from vitronectin produced in Pichia pastoris

The cysteine‐rich somatomedin B domain (SMB) of the matrix protein vitronectin is involved in several important biological processes. First, it stabilizes the active conformation of the plasminogen activator inhibitor (PAI‐1); second, it provides the recognition motif for cell adhesion via the cognate integrins (αvβ3, αvβ5, and αIIbβ3); and third, it binds the complex between urokinase‐type plasminogen activator (uPA) and its glycolipid‐anchored receptor (uPAR). Previous structural studies on SMB have used recombinant protein expressed in Escherichia coli or SMB released from plasma‐derived vitronectin by CNBr cleavage. However, different disulfide patterns and three‐dimensional structures for SMB were reported. In the present study, we have expressed recombinant human SMB by two different eukaryotic expression systems, Pichia pastoris and Drosophila melanogaster S2‐cells, both yielding structurally and functionally homogeneous protein preparations. Importantly, the entire population of our purified, recombinant SMB has a solvent exposure, both as a free domain and in complex with PAI‐1, which is indistinguishable from that of plasma‐derived SMB as assessed by amide hydrogen (1H/2H) exchange. This solvent exposure was only reproduced by one of three synthetic SMB products with predefined disulfide connectivities corresponding to those published previously. Furthermore, this connectivity was also the only one to yield a folded and functional domain. The NMR structure was determined for free SMB produced by Pichia and is largely consistent with that solved by X‐ray crystallography for SMB in complex with PAI‐1.

Considerably Unfolded Transthyretin Monomers Preceed and Exchange with Dynamically Structured Amyloid Protofibrils

Despite numerous studies, a detailed description of the transthyretin (TTR) self-assembly mechanism and fibril structure in TTR amyloidoses remains unresolved. Here, using a combination of primarily small -angle X-ray scattering (SAXS) and hydrogen exchange mass spectrometry (HXMS) analysis, we describe an unexpectedly dynamic TTR protofibril structure which exchanges protomers with highly unfolded monomers in solution. The protofibrils only grow to an approximate final size of 2,900 kDa and a length of 70 nm and a comparative HXMS analysis of native and aggregated samples revealed a much higher average solvent exposure of TTR upon fibrillation. With SAXS, we reveal the continuous presence of a considerably unfolded TTR monomer throughout the fibrillation process, and show that a considerable fraction of the fibrillating protein remains in solution even at a late maturation state. Together, these data reveal that the fibrillar state interchanges with the solution state. Accordingly, we suggest that TTR fibrillation proceeds via addition of considerably unfolded monomers, and the continuous presence of amyloidogenic structures near the protofibril surface offers a plausible explanation for secondary nucleation. We argue that the presence of such dynamic structural equilibria must impact future therapeutic development strategies.

Thr94 in bovine myelin basic protein is a second phosphorylation site for 42-kDa mitogen-activated protein kinase (ERK2).

Treatment of bovine brain myelin basic protein with 42-kDa mitogen-activated protein kinase [p42 MAPK or extracellular signal-regulated kinase 2 (ERK2)] in the presence of ATP and Mg2+ results in phosphorylation of Thr94 and Thr97. Thr94 is not previously known to be an ERK2 phosphorylation site. Both residues are phosphorylated to about the same extent and are in the highly conserved segment Asn91-Ile-Val-Thr94-Pro-Arg-Thr97-Pro-Pro-Pro-Ser101. MALDI mass spectrometry before and after ERK2 treatment revealed the addition of two phosphate groups to the protein. Tryptic cleavage resulted in a single fragment (positions 91–104) carrying the observed mass increase. Tandem mass spectrometry applied to the tryptic peptide showed that both Thr94 and Thr97 are acceptors of phosphate. A singly phosphorylated species could not be detected. Identification of the ERK2 phosphorylation site Thr94 in bovine myelin basic protein reveals a nontraditional phosphate acceptor position, preceded by three noncharged residues (Asn-Ile-Val). Proline at position −2 or −3 from the phosphorylation site, typical for the recognition sequence of proline-directed kinases, is missing. The results provide information for delineation of a further substrate consensus motif for ERK2 phosphorylation.