Inhibiting transthyretin amyloid fibril formation via protein stabilization

Transthyretin (TTR) amyloid fibril formation is observed systemically in familial amyloid polyneuropathy and senile systemic amyloidosis and appears to be the causative agent in these diseases. Herein, we demonstrate conclusively that thyroxine (10.8 μM) inhibits TTR fibril formation efficiently in vitro and does so by stabilizing the tetramer against dissociation and the subsequent conformational changes required for amyloid fibril formation. In addition, the nonnative ligand 2,4,6-triiodophenol, which binds to TTR with slightly increased affinity also inhibits TTR fibril formation by this mechanism. Sedimentation velocity experiments were employed to show that TTR undergoes dissociation (linked to a conformational change) to form the monomeric amyloidogenic intermediate, which self-assembles into amyloid in the absence, but not in the presence of thyroxine. These results demonstrate the feasibility of using small molecules to stabilize the native fold of a potentially amyloidogenic human protein, thus preventing the conformational changes, which appear to be the common link in several human amyloid diseases. This strategy and the compounds resulting from further development should prove useful for critically evaluating the amyloid hypothesis—i.e., the putative cause-and-effect relationship between TTR amyloid deposition and the onset of familial amyloid polyneuropathy and senile systemic amyloidosis.

Preferential exclusion of sucrose from recombinant interleukin-1 receptor antagonist: Role in restricted conformational mobility and compaction of native state

Understanding the mechanism for sucrose-induced protein stabilization is important in many diverse fields, ranging from biochemistry and environmental physiology to pharmaceutical science. Timasheff and Lee [Lee, J. C. & Timasheff, S. N. (1981) J. Biol. Chem. 256, 7193–7201] have established that thermodynamic stabilization of proteins by sucrose is due to preferential exclusion of the sugar from the protein’s surface, which increases protein chemical potential. The current study measures the preferential exclusion of 1 M sucrose from a protein drug, recombinant interleukin 1 receptor antagonist (rhIL-1ra). It is proposed that the degree of preferential exclusion and increase in chemical potential are directly proportional to the protein surface area and that, hence, the system will favor the protein state with the smallest surface area. This mechanism explains the observed sucrose-induced restriction of rhIL-1ra conformational fluctuations, which were studied by hydrogen–deuterium exchange and cysteine reactivity measurements. Furthermore, infrared spectroscopy of rhlL-1ra suggested that a more ordered native conformation is induced by sucrose. Electron paramagnetic resonance spectroscopy demonstrated that in the presence of sucrose, spin-labeled cysteine 116 becomes more buried in the protein’s interior and that the hydrodynamic diameter of the protein is reduced. The preferential exclusion of sucrose from the protein and the resulting shift in the equilibrium between protein states toward the most compact conformation account for sucrose-induced effects on rhIL-1ra.

Implications of a Developmental-Stage-Dependent Thylakoid-Bound Protease in the Stabilization of the Light-Harvesting Pigment-Protein Complex Serving Photosystem II during Thylakoid Biogenesis in Red Kidney Bean1

Intact etioplasts of bean (Phaseolus vulgaris) plants exhibit proteolytic activity against the exogenously added apoprotein of the light-harvesting pigment-protein complex serving photosystem II (LHCII) that increases as etiolation is prolonged. The activity increases in the membrane fraction but not in the stroma, where it remains low and constant and is mainly directed against LHCII and protochlorophyllide oxidoreductase. The thylakoid proteolytic activity, which is low in etioplasts of 6-d-old etiolated plants, increases in plants pretreated with a pulse of light or exposed to intermittent-light (ImL) cycles, but decreases during prolonged exposure to continuous light, coincident with chlorophyll (Chl) accumulation. To distinguish between the control of Chl and/or development on proteolytic activity, we used plants exposed to ImL cycles of varying dark-phase durations. In ImL plants exposed to an equal number of ImL cycles with short or long dark intervals (i.e. equal Chl accumulation but different developmental stage) proteolytic activity increased with the duration of the dark phase. In plants exposed to ImL for equal durations to such light-dark cycles (i.e. different Chl accumulation but same developmental stage) the proteolytic activity was similar. These results suggest that the protease, which is free to act under limited Chl accumulation, is dependent on the developmental stage of the chloroplast, and give a clue as to why plants in ImL with short dark intervals contain LHCII, whereas those with long dark intervals possess only photosystem-unit cores and lack LHCII.

Ubiquitin-dependent degradation of IκBα is mediated by a ubiquitin ligase Skp1/Cul 1/F-box protein FWD1

Activation of the transcription factor nuclear factor kappa B (NF-κB) is controlled by proteolysis of its inhibitory subunit (IκB) via the ubiquitin-proteasome pathway. Signal-induced phosphorylation of IκBα by a large multisubunit complex containing IκB kinases is a prerequisite for ubiquitination. Here, we show that FWD1 (a mouse homologue of Slimb/βTrCP), a member of the F-box/WD40-repeat proteins, is associated specifically with IκBα only when IκBα is phosphorylated. The introduction of FWD1 into cells significantly promotes ubiquitination and degradation of IκBα in concert with IκB kinases, resulting in nuclear translocation of NF-κB. In addition, FWD1 strikingly evoked the ubiquitination of IκBα in the in vitro system. In contrast, a dominant-negative form of FWD1 inhibits the ubiquitination, leading to stabilization of IκBα. These results suggest that the substrate-specific degradation of IκBα is mediated by a Skp1/Cull 1/F-box protein (SCF) FWD1 ubiquitin-ligase complex and that FWD1 serves as an intracellular receptor for phosphorylated IκBα. Skp1/Cullin/F-box protein FWD1 might play a critical role in transcriptional regulation of NF-κB through control of IκB protein stability.

Phosphorylation of Ser-20 mediates stabilization of human p53 in response to DNA damage

Stabilization of p53 in response to DNA damage is caused by its dissociation from Mdm2, a protein that targets p53 for degradation in the proteasome. Dissociation of p53 from Mdm2 could be caused by DNA damage-induced p53 posttranslational modifications. The ATM and ATR kinases, whose activation in response to ionizing radiation (IR) and UV light, respectively, is required for p53 stabilization, directly phosphorylate p53 on Ser-15. However, phosphorylation of Ser-15 is critical for the apoptotic activity of p53 and not for p53 stabilization. Thus, whether any p53 modifications, and which, underlie disruption of the p53–Mdm2 complex after DNA damage remains to be determined. We analyzed the IR- and UV light-induced stabilization of p53 proteins with substitutions of Ser known to be posttranslationally modified after DNA damage. Substitution of Ser-20 was sufficient to abrogate p53 stabilization in response to both IR and UV light. Furthermore, both IR and UV light induced phosphorylation of p53 on Ser-20, which involved the majority of nuclear p53 protein and weakened the interaction of p53 with Mdm2 in vitro. ATM and ATR cannot phosphorylate p53 on Ser-20. We therefore propose that ATM and ATR activate an, as yet unidentified, kinase that stabilizes p53 by phosphorylating it on Ser-20.

GCS1, an Arf Guanosine Triphosphatase-activating Protein in Saccharomyces cerevisiae, Is Required for Normal Actin Cytoskeletal Organization In Vivo and Stimulates Actin Polymerization In Vitro

Recent cloning of a rat brain phosphatidylinositol 3,4,5-trisphosphate binding protein, centaurin α, identified a novel gene family based on homology to an amino-terminal zinc-binding domain. In Saccharomyces cerevisiae, the protein with the highest homology to centaurin α is Gcs1p, the product of the GCS1 gene. GCS1 was originally identified as a gene conditionally required for the reentry of cells into the cell cycle after stationary phase growth. Gcs1p was previously characterized as a guanosine triphosphatase-activating protein for the small guanosine triphosphatase Arf1, and gcs1 mutants displayed vesicle-trafficking defects. Here, we have shown that similar to centaurin α, recombinant Gcs1p bound phosphoinositide-based affinity resins with high affinity and specificity. A novel GCS1 disruption strain (gcs1Δ) exhibited morphological defects, as well as mislocalization of cortical actin patches. gcs1Δ was hypersensitive to the actin monomer-sequestering drug, latrunculin-B. Synthetic lethality was observed between null alleles of GCS1 and SLA2, the gene encoding a protein involved in stabilization of the actin cytoskeleton. In addition, synthetic growth defects were observed between null alleles of GCS1 and SAC6, the gene encoding the yeast fimbrin homologue. Recombinant Gcs1p bound to actin filaments, stimulated actin polymerization, and inhibited actin depolymerization in vitro. These data provide in vivo and in vitro evidence that Gcs1p interacts directly with the actin cytoskeleton in S. cerevisiae.

Mapping of a Myosin-binding Domain and a Regulatory Phosphorylation Site in M-Protein, a Structural Protein of the Sarcomeric M Band

The myofibrils of cross-striated muscle fibers contain in their M bands cytoskeletal proteins whose main function seems to be the stabilization of the three-dimensional arrangement of thick filaments. We identified two immunoglobin domains (Mp2–Mp3) of M-protein as a site binding to the central region of light meromyosin. This binding is regulated in vitro by phosphorylation of a single serine residue (Ser76) in the immediately adjacent amino-terminal domain Mp1. M-protein phosphorylation by cAMP-dependent kinase A inhibits binding to myosin LMM. Transient transfection studies of cultured cells revealed that the myosin-binding site seems involved in the targeting of M-protein to its location in the myofibril. Using the same method, a second myofibril-binding site was uncovered in domains Mp9–Mp13. These results support the view that specific phosphorylation events could be also important for the control of sarcomeric M band formation and remodeling.

Activation of Androgen Receptor Function by a Novel Nuclear Protein Kinase

Androgen receptor (AR) belongs to the nuclear receptor superfamily and mediates the biological actions of male sex steroids. In this work, we have characterized a novel 130-kDa Ser/Thr protein kinase ANPK that interacts with the zinc finger region of AR in vivo and in vitro. The catalytic kinase domain of ANPK shares considerable sequence similarity with the minibrain gene product, a protein kinase suggested to contribute to learning defects associated with Down syndrome. However, the rest of ANPK sequence, including the AR-interacting interface, exhibits no apparent homology with other proteins. ANPK is a nuclear protein that is widely expressed in mammalian tissues. Its overexpression enhances AR-dependent transcription in various cell lines. In addition to the zinc finger region, ligand-binding domain and activation function AF1 of AR are needed, as the activity of AR mutants devoid of these domains was not influenced by ANPK. The receptor protein does not appear to be a substrate for ANPK in vitro, and overexpression of ANPK does not increase the extent of AR phosphorylation in vivo. In view of this, it is likely that ANPK-mediated activation of AR function is exerted through modification of AR-associated proteins, such as coregulatory factors, and/or through stabilization of the receptor protein against degradation.

Degradation of a Short-lived Glycoprotein from the Lumen of the Endoplasmic Reticulum: The Role of N-linked Glycans and the Unfolded Protein Response

We are studying endoplasmic reticulum–associated degradation (ERAD) with the use of a truncated variant of the type I ER transmembrane glycoprotein ribophorin I (RI). The mutant protein, RI332, containing only the N-terminal 332 amino acids of the luminal domain of RI, has been shown to interact with calnexin and to be a substrate for the ubiquitin-proteasome pathway. When RI332 was expressed in HeLa cells, it was degraded with biphasic kinetics; an initial, slow phase of ∼45 min was followed by a second phase of threefold accelerated degradation. On the other hand, the kinetics of degradation of a form of RI332 in which the single used N-glycosylation consensus site had been removed (RI332-Thr) was monophasic and rapid, implying a role of the N-linked glycan in the first proteolytic phase. RI332 degradation was enhanced when the binding of glycoproteins to calnexin was prevented. Moreover, the truncated glycoprotein interacted with calnexin preferentially during the first proteolytic phase, which strongly suggests that binding of RI332 to the lectin-like protein may result in the slow, initial phase of degradation. Additionally, mannose trimming appears to be required for efficient proteolysis of RI332. After treatment of cells with the inhibitor of N-glycosylation, tunicamycin, destruction of the truncated RI variants was severely inhibited; likewise, in cells preincubated with the calcium ionophore A23187, both RI332 and RI332-Thr were stabilized, despite the presence or absence of the N-linked glycan. On the other hand, both drugs are known to trigger the unfolded protein response (UPR), resulting in the induction of BiP and other ER-resident proteins. Indeed, only in drug-treated cells could an interaction between BiP and RI332 and RI332-Thr be detected. Induction of BiP was also evident after overexpression of murine Ire1, an ER transmembrane kinase known to play a central role in the UPR pathway; at the same time, stabilization of RI332 was observed. Together, these results suggest that binding of the substrate proteins to UPR-induced chaperones affects their half lives.

Nonneuronal isoforms of STOP protein are responsible for microtubule cold stability in mammalian fibroblasts

A number of cycling mammalian cells, such as NIH 3T3, contain abundant subsets of cold-stable microtubules. The origin of such microtubule stabilization in nonneuronal cells is unknown. We have previously described a neuronal protein, stable tubule-only polypeptide (STOP), that binds to microtubules and induces cold stability. We find that NIH 3T3 fibroblasts contain a major 42-kDa isoform of STOP (fibroblastic STOP, F-STOP). F-STOP contains the central repeats characteristic of brain STOP but shows extensive deletions of N- and C-terminal protein domains that are present in brain STOP. These deletions arise from differences in STOP RNA splicing. Despite such deletions, F-STOP has full microtubule stabilizing activity. F-STOP accumulates on cold-stable microtubules of interphase arrays and is present on stable microtubules within the mitotic spindle of NIH 3T3 cells. STOP inhibition by microinjection of affinity-purified STOP central repeat antibodies into NIH 3T3 cells abolishes both interphase and spindle microtubule cold stability. Similar results were obtained with Rat2 cells. These results show that STOP proteins have nonneuronal isoforms that are responsible for the microtubule cold stability observed in mammalian fibroblasts.

Free energy of burying hydrophobic residues in the interface between protein subunits

We have obtained an experimental estimate of the free energy change associated with variations at the interface between protein subunits, a subject that has raised considerable interest since the concept of accessible surface area was introduced by Lee and Richards [Lee, B. & Richards, F. M. (1971) J. Mol. Biol. 55, 379–400]. We determined by analytical ultracentrifugation the dimer–tetramer equilibrium constant of five single and three double mutants of human Hb. One mutation is at the stationary α1β1 interface, and all of the others are at the sliding α1β2 interface where cleavage of the tetramer into dimers and ligand-linked allosteric changes are known to occur. A surprisingly good linear correlation between the change in the free energy of association of the mutants and the change in buried hydrophobic surface area was obtained, after corrections for the energetic cost of losing steric complementarity at the αβ dimer interface. The slope yields an interface stabilization free energy of −15 ± 1.2 cal/mol upon burial of 1 Å2 of hydrophobic surface, in very good agreement with the theoretical estimate given by Eisenberg and McLachlan [Eisenberg, D. & McLachlan, A. D. (1986) Nature (London) 319, 199–203].

Herpes simplex virus 1 α regulatory protein ICP0 functionally interacts with cellular transcription factor BMAL1

The infected cell protein no. 0 (ICP0) of herpes simplex virus 1 (HSV-1) is a promiscuous transactivator shown to enhance the expression of gene introduced into cells by infection or transfection. At the molecular level, ICP0 is a 775-aa ring finger protein localized initially in the nucleus and late in infection in the cytoplasm and mediates the degradation of several proteins and stabilization of others. None of the known functions at the molecular level account for the apparent activity of ICP0 as a transactivator. Here we report that ICP0 functionally interacts with cellular transcription factor BMAL1, a member of the basic helix–loop–helix PER-ARNT-SIM (PAS) super family of transcriptional regulators. Specifically, sequences mapped to the exon II of ICP0 interacted with BMAL1 in the yeast two-hybrid system and in reciprocal pull-down experiments in vitro. Moreover, the enhancement of transcription of a luciferase reporter construct whose promoter contained multiple BMAL1-binding sites by ICP0 and BMAL1 was significantly greater than that observed by ICP0 or BMAL1 alone. Although the level of BMAL1 present in nuclei of infected cells remained unchanged between 3 and 8 h after infection, the level of cytoplasmic BMAL1 was reduced at 8 h after infection. The reduction of cytoplasmic BMAL1 was significantly greater in cells infected with the ICP0-null mutant than in the wild-type virus-infected cells, suggesting that ICP0 mediates partial stabilization of the protein. These results indicate that ICP0 interacts physically and functionally with at least one cellular transcription-regulatory factor.

X-ray structure of the human hyperplastic discs protein: An ortholog of the C-terminal domain of poly(A)-binding protein

The poly(A)-binding protein (PABP) recognizes the 3′ mRNA poly(A) tail and plays an essential role in eukaryotic translation initiation and mRNA stabilization/degradation. PABP is a modular protein, with four N-terminal RNA-binding domains and an extensive C terminus. The C-terminal region of PABP is essential for normal growth in yeast and has been implicated in mediating PABP homo-oligomerization and protein–protein interactions. A small, proteolytically stable, highly conserved domain has been identified within this C-terminal segment. Remarkably, this domain is also present in the hyperplastic discs protein (HYD) family of ubiquitin ligases. To better understand the function of this conserved region, an x-ray structure of the PABP-like segment of the human HYD protein has been determined at 1.04-Å resolution. The conserved domain adopts a novel fold resembling a right-handed supercoil of four α-helices. Sequence profile searches and comparative protein structure modeling identified a small ORF from the Arabidopsis thaliana genome that encodes a structurally similar but distantly related PABP/HYD domain. Phylogenetic analysis of the experimentally determined (HYD) and homology modeled (PABP) protein surfaces revealed a conserved feature that may be responsible for binding to a PABP interacting protein, Paip1, and other shared interaction partners.

The specificity of the secondary DNA binding site of RecA protein defines its role in DNA strand exchange.

The RecA protein-single-stranded DNA (ssDNA) filament can bind a second DNA molecule. Binding of ssDNA to this secondary site shows specificity, in that polypyrimidinic DNA binds to the RecA protein-ssDNA filament with higher affinity than polypurinic sequences. The affinity of ssDNA, which is identical in sequence to that bound in the primary site, is not always greater than that of nonhomologous DNA. Moreover, this specificity of DNA binding does not depend on the sequence of the DNA bound to the RecA protein primary site. We conclude that the specificity reflects an intrinsic property of the secondary site of RecA protein rather than an interaction between DNa molecules within nucleoprotein filament--i.e., self-recognition. The secondary DNA binding site displays a higher affinity for ssDNA than for double-stranded DNA, and the binding of ssDNA to the secondary site strongly inhibits DNA strand exchange. We suggest that the secondary binding site has a dual role in DNA strand exchange. During the homology search, it binds double-stranded DNA weakly; upon finding local homology, this site binds, with higher affinity, the ssDNA strand that is displaced during DNA strand exchange. These characteristics facilitate homologous pairing, promote stabilization of the newly formed heteroduplex DNA, and contribute to the directionality of DNA strand exchange.

Surface protein phosphorylation by ecto-protein kinase is required for the maintenance of hippocampal long-term potentiation.

During the induction of long-term potentiation (LTP) in hippocampal slices adenosine triphosphate (ATP) is secreted into the synaptic cleft, and a 48 kDa/50 kDa protein duplex becomes phosphorylated by extracellular ATP. All the criteria required as evidence that these two proteins serve as principal substrates of ecto-protein kinase activity on the surface of hippocampal pyramidal neurons have been fulfilled. This phosphorylation activity was detected on the surface of pyramidal neurons assayed after synaptogenesis, but not in immature neurons nor in glial cells. Addition to the extracellular medium of a monoclonal antibody termed mAb 1.9, directed to the catalytic domain of protein kinase C (PKC), inhibited selectively this surface protein phosphorylation activity and blocked the stabilization of LTP induced by high frequency stimulation (HFS) in hippocampal slices. This antibody did not interfere with routine synaptic transmission nor prevent the initial enhancement of synaptic responses observed during the 1-5 min period immediately after the application of HFS (the induction phase of LTP). However, the initial increase in the slope of excitatory postsynaptic potentials, as well as the elevated amplitude of the population spike induced by HFS, both declined gradually and returned to prestimulus values within 30-40 min after HFS was applied in the presence of mAb 1.9. A control antibody that binds to PKC but does not inhibit its activity had no effect on LTP. The selective inhibitory effects observed with mAb 1.9 provide the first direct evidence of a causal role for ecto-PK in the maintenance of stable LTP, an event implicated in the process of learning and the formation of memory in the brain.