Background: The mechanism driving hepatic gene expression of Foxo1 in the fasted state remains unclear.
Results: Activation of cAMP-PKA pathway induces Foxo1 gene expression through CREB and co-activator P300.
Conclusion: P300 mediates Foxo1 gene expression by binding to Foxo1 proximal promoter.
Significance: Induction of the Foxo1 gene by cAMP-PKA via P300 fully activates the gluconeogenic program during fasting to maintain euglycemia.
FOXO1 is an important downstream mediator of the insulin signaling pathway. In the fed state, elevated insulin phosphorylates FOXO1 via AKT, leading to its nuclear exclusion and degradation. A reduction in nuclear FOXO1 levels then leads to suppression of hepatic glucose production. However, the mechanism leading to expression of Foxo1 gene in the fasted state is less clear. We found that Foxo1 mRNA and FOXO1 protein levels of Foxo1 were increased significantly in the liver of mice after 16 h of fasting. Furthermore, dibutyrl cAMP stimulated the expression of Foxo1 at both mRNA and protein level in hepatocytes. Because cAMP-PKA regulates hepatic glucose production through cAMP-response element-binding protein co-activators, we depleted these co-activators using adenoviral shRNAs. Interestingly, only depletion of co-activator P300 resulted in the decrease of Foxo1 mRNA and FOXO1 protein levels. In addition, inhibition of histone acetyltransferase activity of P300 significantly decreased hepatic Foxo1 mRNA and FOXO1 protein levels in fasted mice, as well as fasting blood glucose levels. By characterization of Foxo1 gene promoter, P300 regulates the Foxo1 gene expression through the binding to tandem cAMP-response element sites in the proximal promoter region of Foxo1 gene.
CREB; Foxo; Gluconeogenesis; Histone Acetylase; P300
Background: Following ligand-induced internalization, GPCRs are sorted by diverse receptor motifs and protein interactions.
Results: Distinct GPCRs are targeted to a pre-early endosome compartment for their sorting and MAPK signaling.
Conclusion: GPCR sorting motifs and their interacting proteins provide specificity in endosomal targeting and receptor signaling.
Significance: We describe a system to reprogram GPCR signaling at an unprecedented spatial level.
Postendocytic sorting of G protein-coupled receptors (GPCRs) is driven by their interactions between highly diverse receptor sequence motifs with their interacting proteins, such as postsynaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), zonula occludens-1 protein (zo-1) (PDZ) domain proteins. However, whether these diverse interactions provide an underlying functional specificity, in addition to driving sorting, is unknown. Here we identify GPCRs that recycle via distinct PDZ ligand/PDZ protein pairs that exploit their recycling machinery primarily for targeted endosomal localization and signaling specificity. The luteinizing hormone receptor (LHR) and β2-adrenergic receptor (B2AR), two GPCRs sorted to the regulated recycling pathway, underwent divergent trafficking to distinct endosomal compartments. Unlike B2AR, which traffics to early endosomes (EE), LHR internalizes to distinct pre-early endosomes (pre-EEs) for its recycling. Pre-EE localization required interactions of the LHR C-terminal tail with the PDZ protein GAIP-interacting protein C terminus, inhibiting its traffic to EEs. Rerouting the LHR to EEs, or EE-localized GPCRs to pre-EEs, spatially reprograms MAPK signaling. Furthermore, LHR-mediated activation of MAPK signaling requires internalization and is maintained upon loss of the EE compartment. We propose that combinatorial specificity between GPCR sorting sequences and interacting proteins dictates an unprecedented spatiotemporal control in GPCR signal activity.
Endocytosis; Endosomes; G Protein-coupled Receptor (GPCR); Receptor Recycling; Signaling; Sorting; GAIP-interacting Protein C Terminus (GIPC)
Plasmodium falciparum actin depolymerizing factor 1 (PfADF1) severs actin polymers without stable filament-binding, challenging current models for severing.
Results: Cross-linking mass spectrometry of PfADF1 with filamentous actin reveals a novel binding interface required for severing.
Conclusion: Filament severing by PfADF1 is via a previously unidentified binding interface.
Significance: We propose an alternative mechanism for actin filament severing potentially used across eukaryotic cells.
Actin depolymerizing factor (ADF)/cofilins are essential regulators of actin turnover in eukaryotic cells. These multifunctional proteins facilitate both stabilization and severing of filamentous (F)-actin in a concentration-dependent manner. At high concentrations ADF/cofilins bind stably to F-actin longitudinally between two adjacent actin protomers forming what is called a decorative interaction. Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament. To date, how these two contrasting modalities are achieved by the same protein remains uncertain. Here, we define the proximate amino acids between the actin filament and the malaria parasite ADF/cofilin, PfADF1 from Plasmodium falciparum. PfADF1 is unique among ADF/cofilins in being able to sever F-actin but do so without stable filament binding. Using chemical cross-linking and mass spectrometry (XL-MS) combined with structure reconstruction we describe a previously overlooked binding interface on the actin filament targeted by PfADF1. This site is distinct from the known binding site that defines decoration. Furthermore, total internal reflection fluorescence (TIRF) microscopy imaging of single actin filaments confirms that this novel low affinity site is required for F-actin severing. Exploring beyond malaria parasites, selective blocking of the decoration site with human cofilin (HsCOF1) using cytochalasin D increases its severing rate. HsCOF1 may therefore also use a decoration-independent site for filament severing. Thus our data suggest that a second, low affinity actin-binding site may be universally used by ADF/cofilins for actin filament severing.
Actin; Cofilin; Cytoskeleton; Electron Microscopy (EM); Malaria; Mass Spectrometry (MS); Plasmodium; Protein Cross-linking
Background: mTORC1 integrates cellular inputs and is often deregulated in cancer.
Results: In response to DNA damage, p53/TAp63 and AKT regulate mTORC1 through two independent parallel pathways.
Conclusion: DNA damage activates Akt, resulting in inhibition of S6K1, whereas Sestrin1/2 downstream of p53 and REDD1 downstream of p63 coordinate to suppress 4E-BP1.
Significance: mTORC1-dependent 4EBP1 inhibition by DNA damage is abrogated in most human cancers.
Under conditions of DNA damage, the mammalian target of rapamycin complex 1 (mTORC1) is inhibited, preventing cell cycle progression and conserving cellular energy by suppressing translation. We show that suppression of mTORC1 signaling to 4E-BP1 requires the coordinated activity of two tumor suppressors, p53 and p63. In contrast, suppression of S6K1 and ribosomal protein S6 phosphorylation by DNA damage is Akt-dependent. We find that loss of either p53, required for the induction of Sestrin 1/2, or p63, required for the induction of REDD1 and activation of the tuberous sclerosis complex, prevents the DNA damage-induced suppression of mTORC1 signaling. These data indicate that the negative regulation of cap-dependent translation by mTORC1 inhibition subsequent to DNA damage is abrogated in most human cancers.
Akt; DNA Damage Response; mTOR; mTOR Complex (mTORC); p53; p63; 4EBP1; REDD1; S6K1; Sestrin
Background: Prion disease causes chronic neurodegeneration via deposition of extracellular misfolded protein akin to Alzheimer disease.
Results: Proteomic analysis revealed a major nonproliferative astrogliosis encompassing GFAP, EAAT-2, Prdx6, and the acute phase protein Clusterin.
Conclusion: Brain changes are not reflective of measured circulating Clusterin.
Significance: Our data highlights caution in the use of circulating levels of biomarker as clinical measures of actual neurodegeneration.
Prion diseases are characterized by accumulation of misfolded protein, gliosis, synaptic dysfunction, and ultimately neuronal loss. This sequence, mirroring key features of Alzheimer disease, is modeled well in ME7 prion disease. We used iTRAQTM/mass spectrometry to compare the hippocampal proteome in control and late-stage ME7 animals. The observed changes associated with reactive glia highlighted some specific proteins that dominate the proteome in late-stage disease. Four of the up-regulated proteins (GFAP, high affinity glutamate transporter (EAAT-2), apo-J (Clusterin), and peroxiredoxin-6) are selectively expressed in astrocytes, but astrocyte proliferation does not contribute to their up-regulation. The known functional role of these proteins suggests this response acts against protein misfolding, excitotoxicity, and neurotoxic reactive oxygen species. A recent convergence of genome-wide association studies and the peripheral measurement of circulating levels of acute phase proteins have focused attention on Clusterin as a modifier of late-stage Alzheimer disease and a biomarker for advanced neurodegeneration. Since ME7 animals allow independent measurement of acute phase proteins in the brain and circulation, we extended our investigation to address whether changes in the brain proteome are detectable in blood. We found no difference in the circulating levels of Clusterin in late-stage prion disease when animals will show behavioral decline, accumulation of misfolded protein, and dramatic synaptic and neuronal loss. This does not preclude an important role of Clusterin in late-stage disease, but it cautions against the assumption that brain levels provide a surrogate peripheral measure for the progression of brain degeneration.
Astrocytes; Mass Spectrometry (MS); mRNA; Neurobiology; Prions; ME7; Neurodegeneration; Prion; iTRAQ
Background: A stability control region (SCR) (residues 97–118) is conserved in vertebrate tropomyosins and controls overall stability.
Results: An A109L mutation interrupts the transmission of stability information along the coiled-coil, inducing two domains.
Conclusion: The optimum stability provided by the SCR is critical for transmission of the stability signal.
Significance: Learning how stability information is transmitted along tropomyosin is crucial to understanding function and signaling.
Tropomyosin (Tm) is an actin-binding, thin filament, two-stranded α-helical coiled-coil critical for muscle contraction and cytoskeletal function. We made the first identification of a stability control region (SCR), residues 97–118, in the Tm sequence that controls overall protein stability but is not required for folding. We also showed that the individual α-helical strands of the coiled-coil are stabilized by Leu-110, whereas the hydrophobic core is destabilized in the SCR by Ala residues at three consecutive d positions. Our hypothesis is that the stabilization of the individual α-helices provides an optimum stability and allows functionally beneficial dynamic motion between the α-helices that is critical for the transmission of stabilizing information along the coiled-coil from the SCR. We prepared three recombinant (rat) Tm(1–131) proteins, including the wild type sequence, a destabilizing mutation L110A, and a stabilizing mutation A109L. These proteins were evaluated by circular dichroism (CD) and differential scanning calorimetry. The single mutation L110A destabilizes the entire Tm(1–131) molecule, showing that the effect of this mutation is transmitted 165 Å along the coiled-coil in the N-terminal direction. The single mutation A109L prevents the SCR from transmitting stabilizing information and separates the coiled-coil into two domains, one that is ∼9 °C more stable than wild type and one that is ∼16 °C less stable. We know of no other example of the substitution of a stabilizing Leu residue in a coiled-coil hydrophobic core position d that causes this dramatic effect. We demonstrate the importance of the SCR in controlling and transmitting the stability signal along this rodlike molecule.
Circular Dichroism (CD); Contractile Protein; Protein Stability; Recombinant Protein Expression; Tropomyosin; Stability Control Region; Tropomyosin N-domain 1–131; Differential Scanning Calorimetry (DSC); Stability Signal Transmission; Two-stranded Coiled-coils
Background: Induced pluripotent stem cells (iPSCs) constitute an attractive source of cells for regenerative medicine.
Results: MicroRNA-21 mediates endothelial differentiation derived from iPSCs in presence of VEGF.
Conclusion: MicroRNA-21 and TGF-β2 signaling pathways regulate iPSC differentiation to endothelial lineage.
Significance: Elucidation of the molecular mechanisms underlying microRNA-21-regulated iPSC differentiation might provide the basic information for stem cell therapy of vascular diseases.
Finding a suitable cell source for endothelial cells (ECs) for cardiovascular regeneration is a challenging issue for regenerative medicine. In this paper, we describe a novel mechanism regulating induced pluripotent stem cells (iPSC) differentiation into ECs, with a particular focus on miRNAs and their targets. We first established a protocol using collagen IV and VEGF to drive the functional differentiation of iPSCs into ECs and compared the miRNA signature of differentiated and undifferentiated cells. Among the miRNAs overrepresented in differentiated cells, we focused on microRNA-21 (miR-21) and studied its role in iPSC differentiation. Overexpression of miR-21 in predifferentiated iPSCs induced EC marker up-regulation and in vitro and in vivo capillary formation; accordingly, inhibition of miR-21 produced the opposite effects. Importantly, miR-21 overexpression increased TGF-β2 mRNA and secreted protein level, consistent with the strong up-regulation of TGF-β2 during iPSC differentiation. Indeed, treatment of iPSCs with TGFβ-2 induced EC marker expression and in vitro tube formation. Inhibition of SMAD3, a downstream effector of TGFβ-2, strongly decreased VE-cadherin expression. Furthermore, TGFβ-2 neutralization and knockdown inhibited miR-21-induced EC marker expression. Finally, we confirmed the PTEN/Akt pathway as a direct target of miR-21, and we showed that PTEN knockdown is required for miR-21-mediated endothelial differentiation. In conclusion, we elucidated a novel signaling pathway that promotes the differentiation of iPSC into functional ECs suitable for regenerative medicine applications.
Endothelial Cell; Induced Pluripotent Stem Cells; MicroRNA; Transforming Growth Factor Beta (TGFbeta); Vascular Endothelial Growth Factor (VEGF)
Background: Amyloidogenic D76N β2m variant escapes the intracellular quality control despite its instability.
Results: We show tridimensional structure and stability of D76N β2m assembled within MHCI compared with the wild type protein.
Conclusion: Assembly of D76N β2m within the MHCI totally masks its misfolding propensity.
Significance: The MHCI-mediated stabilization of amyloidogenic D76N β2m explains the failure of quality control in preventing its secretion.
To form extracellular aggregates, amyloidogenic proteins bypass the intracellular quality control, which normally targets unfolded/aggregated polypeptides. Human D76N β2-microglobulin (β2m) variant is the prototype of unstable and amyloidogenic protein that forms abundant extracellular fibrillar deposits. Here we focus on the role of the class I major histocompatibility complex (MHCI) in the intracellular stabilization of D76N β2m. Using biophysical and structural approaches, we show that the MHCI containing D76N β2m (MHCI76) displays stability, dissociation patterns, and crystal structure comparable with those of the MHCI with wild type β2m. Conversely, limited proteolysis experiments show a reduced protease susceptibility for D76N β2m within the MHCI76 as compared with the free variant, suggesting that the MHCI has a chaperone-like activity in preventing D76N β2m degradation within the cell. Accordingly, D76N β2m is normally assembled in the MHCI and circulates as free plasma species in a transgenic mouse model.
Amyloid; Major Histocompatibility Complex (MHC); Protein Aggregation; Protein Complexes; Protein Structure; Beta2 Microglobulin Amyloidosis; D76N Beta2-Microglobulin Variant
Background: Sar1 mediates the onward transport of ER cargo.
Results: Sar1B promotes VLDL secretion, whereas Sar1A antagonizes this activity, and a deficit of both reduces cholesterol biosynthesis.
Conclusion: Sar1B independently of and through its lipoprotein secretion function promotes the expression of genes regulating cholesterol biosynthesis.
Significance: Sar1B-mediated transport activities contribute to both the functional integrity of the ER membrane and blood cholesterol levels.
Triglycerides and cholesterol are essential for life in most organisms. Triglycerides serve as the principal energy storage depot and, where vascular systems exist, as a means of energy transport. Cholesterol is essential for the functional integrity of all cellular membrane systems. The endoplasmic reticulum is the site of secretory lipoprotein production and de novo cholesterol synthesis, yet little is known about how these activities are coordinated with each other or with the activity of the COPII machinery, which transports endoplasmic reticulum cargo to the Golgi. The Sar1B component of this machinery is mutated in chylomicron retention disorder, indicating that this Sar1 isoform secures delivery of dietary lipids into the circulation. However, it is not known why some patients with chylomicron retention disorder develop hepatic steatosis, despite impaired intestinal fat malabsorption, and why very severe hypocholesterolemia develops in this condition. Here, we show that Sar1B also promotes hepatic apolipoprotein (apo) B lipoprotein secretion and that this promoting activity is coordinated with the processes regulating apoB expression and the transfer of triglycerides/cholesterol moieties onto this large lipid transport protein. We also show that although Sar1A antagonizes the lipoprotein secretion-promoting activity of Sar1B, both isoforms modulate the expression of genes encoding cholesterol biosynthetic enzymes and the synthesis of cholesterol de novo. These results not only establish that Sar1B promotes the secretion of hepatic lipids but also adds regulation of cholesterol synthesis to Sar1B's repertoire of transport functions.
Apolipoproteins; Cholesterol Regulation; Endoplasmic Reticulum (ER); Lipoprotein Secretion; Transcriptomics
Background: Signaling by mTOR complex 1 requires its amino acid-stimulated binding to Rag GTPase heterodimers.
Results: mTOR complex 1-Rag binding in vitro is independent of Rag guanyl nucleotide charging, and withdrawal of amino acids from cells does not alter Rag GTP charging.
Conclusion: Amino acids promote Rag binding to mTOR complex 1 without changing Rag GTP charging.
Significance: Amino acids promote Rag-mTORC1 binding by undefined mechanisms.
Activation of mammalian target of rapamycin complex 1 (mTORC1) by amino acids is mediated in part by the Rag GTPases, which bind the raptor subunit of mTORC1 in an amino acid-stimulated manner and promote mTORC1 interaction with Rheb-GTP, the immediate activator. Here we examine whether the ability of amino acids to regulate mTORC1 binding to Rag and mTORC1 activation is due to the regulation of Rag guanyl nucleotide charging. Rag heterodimers in vitro exhibit a very rapid, spontaneous exchange of guanyl nucleotides and an inability to hydrolyze GTP. Mutation of the Rag P-loop corresponding to RasSer-17 abolishes guanyl nucleotide binding. Such a mutation in RagA or RagB inhibits, whereas in RagC or RagD it enhances, Rag heterodimer binding to mTORC1. The binding of wild-type and mutant Rag heterodimers to mTORC1 in vitro parallels that seen with transient expression, but binding to mTORC1 in vitro is entirely independent of Rag guanyl nucleotide charging. HeLa cells stably overexpressing wild-type or P-loop mutant RagC exhibit unaltered amino acid regulation of mTORC1. Despite amino acid-independent raptor binding to Rag, mTORC1 is inhibited by amino acid withdrawal as in parental cells. Rag heterodimers extracted from 32P-labeled whole cells, or just from the pool associated with the lysosomal membrane, exhibit constitutive [32P]GTP charging that is unaltered by amino acid withdrawal. Thus, amino acids promote mTORC1 activation without altering Rag GTP charging. Raptor binding to Rag, although necessary, is not sufficient for mTORC1 activation. Additional amino acid-dependent steps couple Rag-mTORC1 to Rheb-GTP.
Cell Signaling; GTPase; Insulin; Lysosomes; mTOR Complex (mTORC); Rag GTPase; Amino Acids; Raptor
Background: The angiotensin-1-converting enzyme (ACE) is a zinc protease unique in its chloride ion dependence.
Results: Key substrate interactions with the catalytic site affect the structure and thermodynamics of ACE, which modulate chloride ion-dependent hydrolysis.
Conclusion: Substrate composition influences chloride affinity in ACE.
Significance: The interdependence of ACE substrate composition, structure, and thermodynamics influence its chloride ion activation in biology.
Somatic angiotensin-converting enzyme (sACE), a key regulator of blood pressure and electrolyte fluid homeostasis, cleaves the vasoactive angiotensin-I, bradykinin, and a number of other physiologically relevant peptides. sACE consists of two homologous and catalytically active N- and C-domains, which display marked differences in substrate specificities and chloride activation. A series of single substitution mutants were generated and evaluated under varying chloride concentrations using isothermal titration calorimetry. The x-ray crystal structures of the mutants provided details on the chloride-dependent interactions with ACE. Chloride binding in the chloride 1 pocket of C-domain ACE was found to affect positioning of residues from the active site. Analysis of the chloride 2 pocket R522Q and R522K mutations revealed the key interactions with the catalytic site that are stabilized via chloride coordination of Arg522. Substrate interactions in the S2 subsite were shown to affect chloride affinity in the chloride 2 pocket. The Glu403-Lys118 salt bridge in C-domain ACE was shown to stabilize the hinge-bending region and reduce chloride affinity by constraining the chloride 2 pocket. This work demonstrated that substrate composition to the C-terminal side of the scissile bond as well as interactions of larger substrates in the S2 subsite moderate chloride affinity in the chloride 2 pocket of the ACE C-domain, providing a rationale for the substrate-selective nature of chloride dependence in ACE and how this varies between the N- and C-domains.
Biophysics; Crystallography; Hypertension; Protein Structure; Thermodynamics; Angiotensin-1-converting Enzyme; Chloride Ion Activation; Enzymology
Background: FIBCD1 is a tetrameric plasma membrane protein that uses a fibrinogen-like recognition domain (FReD) for pattern recognition of acetyl groups on chitin.
Results: The x-ray structure of the FIBCD1 FReD reveals how FIBCD1 binds acetylated and sulfated molecules.
Conclusion: FReD domains combine versatility with conservation to recognize their targets.
Significance: The structure suggests how FIBCD1 binds acetylated pathogen-associated molecular patterns (PAMPS) and endogenous glycosaminoglycans.
The high resolution crystal structures of a recombinant fragment of the C-terminal fibrinogen-like recognition domain of FIBCD1, a vertebrate receptor that binds chitin, have been determined. The overall tetrameric structure shows similarity in structure and aggregation to the horseshoe crab innate immune protein tachylectin 5A. The high affinity ligand N-acetylmannosamine (ManNAc) binds in the S1 site, predominantly via the acetyl group with the oxygen and acetamide nitrogen hydrogen-bonded to the protein and the methyl group inserted into a hydrophobic pocket. The binding of the ManNAc pyranose ring differs markedly between the two independent subunits, but in all structures the binding of the N-acetyl group is conserved. In the native structure, a crystal contact results in one of the independent protomers binding the first GlcNAc of the Asn340
N-linked glycan on the other independent protomer. In the ligand-bound structure this GlcNAc is replaced by the higher affinity ligand ManNAc. In addition, a sulfate ion has been modeled into the electron density at a location similar to the S3 binding site in L-ficolin, whereas in the native structure an acetate ion has been placed in the S1 N-acetyl binding site, and a sulfate ion has been placed adjacent to this site. These ion binding sites are ideally placed to receive the N-acetyl and sulfate groups of sulfated GalNAc residues of glycosaminoglycans such as chondroitin and dermatan sulfate. Together, these structures give insight into important determinants of ligand selectivity, demonstrating versatility in recognition and binding while maintaining conservation in N-acetyl and calcium binding.
Crystal Structure; Ligand-binding Protein; Pattern Recognition Receptor; Receptor Structure-Function; Structural Biology; FIBCD1; Acetyl Binding; Chitin Receptor; Fibrinogen-like Domain
Background: Cholesterol plays an important role in the pathogenesis of neurodegenerative diseases.
Results: Prion infection increased abundance of cholesterol transporter ABCA1 but reduced its functionality. Stimulation of ABCA1 reduced the conversion of prion into the pathological form.
Conclusion: ABCA1 plays a key role in pathogenesis of prion disease.
Significance: There is a reciprocal connection between prion infection and cholesterol metabolism.
Conversion of prion protein (PrPC) into a pathological isoform (PrPSc) during prion infection occurs in lipid rafts and is dependent on cholesterol. Here, we show that prion infection increases the abundance of cholesterol transporter, ATP-binding cassette transporter type A1 (ATP-binding cassette transporter type A1), but reduces cholesterol efflux from neuronal cells leading to the accumulation of cellular cholesterol. Increased abundance of ABCA1 in prion disease was confirmed in prion-infected mice. Mechanistically, conversion of PrPC to the pathological isoform led to PrPSc accumulation in rafts, displacement of ABCA1 from rafts and the cell surface, and enhanced internalization of ABCA1. These effects were abolished with reversal of prion infection or by loading cells with cholesterol. Stimulation of ABCA1 expression with liver X receptor agonist or overexpression of heterologous ABCA1 reduced the conversion of prion protein into the pathological form upon infection. These findings demonstrate a reciprocal connection between prion infection and cellular cholesterol metabolism, which plays an important role in the pathogenesis of prion infection in neuronal cells.
ABC Transporter; Cholesterol; Lipid Raft; Neurodegenerative Diseases; Prions
Background: How Hsp104 and ClpB coordinate polypeptide handover with Hsp70 to dissolve disordered protein aggregates is unknown.
Results: Conserved distal loop residues in the Hsp104 and ClpB middle domain contact NBD2 and enable Hsp70-dependent protein disaggregation.
Conclusion: Distal loop does not project out into solution and Hsp104 and ClpB are tuned differently for Hsp70 collaboration.
Significance: Understanding how protein disaggregases operate may empower strategies to counter protein-misfolding disorders.
The homologous hexameric AAA+ proteins, Hsp104 from yeast and ClpB from bacteria, collaborate with Hsp70 to dissolve disordered protein aggregates but employ distinct mechanisms of intersubunit collaboration. How Hsp104 and ClpB coordinate polypeptide handover with Hsp70 is not understood. Here, we define conserved distal loop residues between middle domain (MD) helix 1 and 2 that are unexpectedly critical for Hsp104 and ClpB collaboration with Hsp70. Surprisingly, the Hsp104 and ClpB MD distal loop does not contact Hsp70 but makes intrasubunit contacts with nucleotide-binding domain 2 (NBD2). Thus, the MD does not invariably project out into solution as in one structural model of Hsp104 and ClpB hexamers. These intrasubunit contacts as well as those between MD helix 2 and NBD1 are different in Hsp104 and ClpB. NBD2-MD contacts dampen disaggregase activity and must separate for protein disaggregation. We demonstrate that ClpB requires DnaK more stringently than Hsp104 requires Hsp70 for protein disaggregation. Thus, we reveal key differences in how Hsp104 and ClpB coordinate polypeptide handover with Hsp70, which likely reflects differential tuning for yeast and bacterial proteostasis.
Molecular Chaperone; Protein Aggregation; Protein Folding; Stress; Stress Response; ClpB; DNA; Hsp104; Hsp70; Disaggreg
Background: Telethonin mutations are associated with cardiomyopathy through unknown mechanisms.
Results: Telethonin is a substrate for CaMK family kinases and exists in a bis-phosphorylated state in cardiomyocytes, in which non-phosphorylated telethonin disrupts transverse tubule organization and intracellular calcium transients.
Conclusion: Telethonin phosphorylation is critical for the maintenance of normal cardiomyocyte morphology and calcium handling.
Significance: Disruption of phospho-telethonin functions may contribute to pathogenesis in cardiomyopathy.
Telethonin (also known as titin-cap or t-cap) is a muscle-specific protein whose mutation is associated with cardiac and skeletal myopathies through unknown mechanisms. Our previous work identified cardiac telethonin as an interaction partner for the protein kinase D catalytic domain. In this study, kinase assays used in conjunction with MS and site-directed mutagenesis confirmed telethonin as a substrate for protein kinase D and Ca2+/calmodulin-dependent kinase II in vitro and identified Ser-157 and Ser-161 as the phosphorylation sites. Phosphate affinity electrophoresis and MS revealed endogenous telethonin to exist in a constitutively bis-phosphorylated form in isolated adult rat ventricular myocytes and in mouse and rat ventricular myocardium. Following heterologous expression in myocytes by adenoviral gene transfer, wild-type telethonin became bis-phosphorylated, whereas S157A/S161A telethonin remained non-phosphorylated. Nevertheless, both proteins localized predominantly to the sarcomeric Z-disc, where they partially replaced endogenous telethonin. Such partial replacement with S157A/S161A telethonin disrupted transverse tubule organization and prolonged the time to peak of the intracellular Ca2+ transient and increased its variance. These data reveal, for the first time, that cardiac telethonin is constitutively bis-phosphorylated and suggest that such phosphorylation is critical for normal telethonin function, which may include maintenance of transverse tubule organization and intracellular Ca2+ transients.
CaMKII; Cardiac Muscle; Cardiomyopathy; Excitation-Contraction Coupling; Protein Kinase D (PKD); Protein Phosphorylation
Background: Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) cause Parkinson disease.
Results: LRRK2 binds directly to three β-tubulin isoforms at the luminal face of microtubules and suppresses α-tubulin acetylation. Interaction is weakened by the R1441G LRRK2 GTPase domain mutant.
Conclusion: LRRK2 modulates microtubule stability.
Significance: Deregulation of microtubule-dependent processes likely contribute to neurodegeneration in Parkinson disease.
Mutations in LRRK2, encoding the multifunctional protein leucine-rich repeat kinase 2 (LRRK2), are a common cause of Parkinson disease. LRRK2 has been suggested to influence the cytoskeleton as LRRK2 mutants reduce neurite outgrowth and cause an accumulation of hyperphosphorylated Tau. This might cause alterations in the dynamic instability of microtubules suggested to contribute to the pathogenesis of Parkinson disease. Here, we describe a direct interaction between LRRK2 and β-tubulin. This interaction is conferred by the LRRK2 Roc domain and is disrupted by the familial R1441G mutation and artificial Roc domain mutations that mimic autophosphorylation. LRRK2 selectively interacts with three β-tubulin isoforms: TUBB, TUBB4, and TUBB6, one of which (TUBB4) is mutated in the movement disorder dystonia type 4 (DYT4). Binding specificity is determined by lysine 362 and alanine 364 of β-tubulin. Molecular modeling was used to map the interaction surface to the luminal face of microtubule protofibrils in close proximity to the lysine 40 acetylation site in α-tubulin. This location is predicted to be poorly accessible within mature stabilized microtubules, but exposed in dynamic microtubule populations. Consistent with this finding, endogenous LRRK2 displays a preferential localization to dynamic microtubules within growth cones, rather than adjacent axonal microtubule bundles. This interaction is functionally relevant to microtubule dynamics, as mouse embryonic fibroblasts derived from LRRK2 knock-out mice display increased microtubule acetylation. Taken together, our data shed light on the nature of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 might contribute to the pathogenesis of Parkinson disease.
Lrrk2; Microtubules; Molecular Genetics; Parkinson Disease; Tubulin; GTPase Mutation; RocCOR; Cytoskeletal Dynamics; Growth Cone; Tubulin Acetylation
Background: ATP-gated (P2X) receptors form pores with second transmembrane helices from each of three subunits.
Results: Addition of an unbranched lipophilic group at I328C in P2X2 receptors opened the channel as effectively as ATP.
Conclusion: Destabilizing the Ile328/Val48 interaction allows the Ile328 side chain to move laterally and open the pore.
Significance: Transmembrane domains can open and close P2X receptors without ATP binding.
The ionic pore of the P2X receptor passes through the central axis of six transmembrane (TM) helices, two from each of three subunits. Val48 and Ile328 are at the outer end of TM1 and TM2, respectively. Homology models of the open and closed states of P2X2 indicate that pore opening is associated with a large lateral displacement of Ile328. In addition, molecular dynamics simulations suggest that lipids enter the interstices between the outer ends of the TM domains. The P2X2(I328C) receptor was activated by propyl-methanethiosulfonate (MTS) as effectively as by ATP, but cysteine substitutions elsewhere in TM2 had no such effect. Other lipophilic MTS compounds (methyl, ethyl, and tert-butylethyl) had a similar effect but not polar MTS. The properties of the conducting pathway opened by covalent attachment of propyl-MTS were the same as those opened by ATP, with respect to unitary conductance, rectification, and permeability of N-methyl-d-glucamine. The ATP-binding residue Lys69 was not required for the action of propyl-MTS, although propyl-MTS did not open P2X2(K308A/I328C) receptors. The propyl-MTS did not open P2X2 receptors in which the Val48 side chain was removed (P2X2(V48G/I328C)). The results suggest that an interaction between Val48 and Ile328 stabilizes the closed channel and that this is broken by covalent attachment of a larger lipophilic moiety at the I328C receptors. Lipid intercalation between the separating TM domains during channel opening would be facilitated in P2X2(I328C) receptors with attached propyl-MTS. The results are consistent with the channel opening mechanism proposed on the basis of closed and open crystal structures and permit the refinement of the position of the TMs within the bilayer.
ATP; Gating; Ion Channels; Molecular Dynamics; Purinergic Receptor; P2X2 Receptor; Permeation
Background: β-Arrestins are required for Wnt/β-catenin signaling, but the mechanism of their action is unclear.
Results: β-Arrestins bind PtdInsP2-interacting protein Amer1, bind PtdInsP2-producing kinases, and control Wnt3a-induced PtdInsP2 production and Amer1 membrane dynamics.
Conclusion: β-Arrestins bridge Wnt3a-induced Dvl-associated PtdInsP2 production to the phosphorylation of Lrp6 via control of Amer1 dynamics.
Significance: The first mechanistic explanation how β-arrestin regulates Wnt/β-catenin signaling is provided.
β-Arrestin is a scaffold protein that regulates signal transduction by seven transmembrane-spanning receptors. Among other functions it is also critically required for Wnt/β-catenin signal transduction. In the present study we provide for the first time a mechanistic basis for the β-arrestin function in Wnt/β-catenin signaling. We demonstrate that β-arrestin is required for efficient Wnt3a-induced Lrp6 phosphorylation, a key event in downstream signaling. β-Arrestin regulates Lrp6 phosphorylation via a novel interaction with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding protein Amer1/WTX/Fam123b. Amer1 has been shown very recently to bridge Wnt-induced and Dishevelled-associated PtdIns(4,5)P2 production to the phosphorylation of Lrp6. Using fluorescence recovery after photobleaching we show here that β-arrestin is required for the Wnt3a-induced Amer1 membrane dynamics and downstream signaling. Finally, we show that β-arrestin interacts with PtdIns kinases PI4KIIα and PIP5KIβ. Importantly, cells lacking β-arrestin showed higher steady-state levels of the relevant PtdInsP and were unable to increase levels of these PtdInsP in response to Wnt3a. In summary, our data show that β-arrestins regulate Wnt3a-induced Lrp6 phosphorylation by the regulation of the membrane dynamics of Amer1. We propose that β-arrestins via their scaffolding function facilitate Amer1 interaction with PtdIns(4,5)P2, which is produced locally upon Wnt3a stimulation by β-arrestin- and Dishevelled-associated kinases.
β-Catenin; Membrane Lipids; Phosphatidylinositol Kinase; Phosphatidylinositol Signaling; Wnt Signaling; Amer1/WTX/FAM123B; β-Arrestin; Dvl; Lrp6 Phosphorylation; Phosphatidylinositol Phosphate Kinase
Background: KirBac3.1 is a prokaryotic homolog of eukaryotic Kir channels.
Results: A high-resolution crystal structure of a mutant channel reveals a novel open conformation.
Conclusion: The intersubunit interface between the cytoplasmic domains controls channel gating.
Significance: These findings help define the structures important for gating in prokaryotic and eukaryotic Kir channels.
KirBac channels are prokaryotic homologs of mammalian inwardly rectifying potassium (Kir) channels, and recent structures of KirBac3.1 have provided important insights into the structural basis of gating in Kir channels. In this study, we demonstrate that KirBac3.1 channel activity is strongly pH-dependent, and we used x-ray crystallography to determine the structural changes that arise from an activatory mutation (S205L) located in the cytoplasmic domain (CTD). This mutation stabilizes a novel energetically favorable open conformation in which changes at the intersubunit interface in the CTD also alter the electrostatic potential of the inner cytoplasmic cavity. These results provide a structural explanation for the activatory effect of this mutation and provide a greater insight into the role of the CTD in Kir channel gating.
Crystal Structure; Ion Channels; Membrane Proteins; Molecular Dynamics; Potassium Channels; Channel Gating; Kir Channel; KirBac
Background: RP2 function in ciliogenesis is enigmatic.
Results: Basal body tethering of TbRP2 depends only on N-terminal TOF-LisH motifs, TbRP2 depletion affects recruitment of transition zone proteins, and TbRP2 encodes the epitope recognized by YL1/2, a monoclonal antibody classically used to detect tyrosinated α-tubulin.
Conclusion: The previous model for RP2 function in trypanosomatids is questioned.
Significance: We give new insight into the assembly of the ciliary transition zone.
The tubulin cofactor C domain-containing protein TbRP2 is a basal body (centriolar) protein essential for axoneme formation in the flagellate protist Trypanosoma brucei, the causal agent of African sleeping sickness. Here, we show how TbRP2 is targeted and tethered at mature basal bodies and provide novel insight into TbRP2 function. Regarding targeting, understanding how several hundred proteins combine to build a microtubule axoneme is a fundamental challenge in eukaryotic cell biology. We show that basal body localization of TbRP2 is mediated by twinned, N-terminal TOF (TON1, OFD1, and FOP) and LisH motifs, motifs that otherwise facilitate localization of only a few conserved proteins at microtubule-organizing centers in animals, plants, and flagellate protists. Regarding TbRP2 function, there is a debate as to whether the flagellar assembly function of specialized, centriolar tubulin cofactor C domain-containing proteins is processing tubulin, the major component of axonemes, or general vesicular trafficking in a flagellum assembly context. Here we report that TbRP2 is required for the recruitment of T. brucei orthologs of MKS1 and MKS6, proteins that, in animal cells, are part of a complex that assembles at the base of the flagellum to regulate protein composition and cilium function. We also identify that TbRP2 is detected by YL1/2, an antibody classically used to detect α-tubulin. Together, these data suggest a general processing role for TbRP2 in trypanosome flagellum assembly and challenge the notion that TbRP2 functions solely in assessing tubulin “quality” prior to tubulin incorporation into the elongating axoneme.
Centriole; Cilia; Protein Targeting; Trypanosoma Brucei; Tubulin; GTPase-activating Protein; Axoneme
Background: Resistance of ovarian cancer cells to chemotherapy is a major therapeutic problem.
Results: Hirsutenone induces cisplatin sensitivity via p53, X-linked inhibitor of apoptosis protein, and apoptosis-inducing factor.
Conclusion: Hirsutenone sensitizes resistant ovarian cancer cells to cisplatin.
Significance: Co-treatment with hirsutenone may have the potential to overcome chemoresistance.
Cisplatin (CDDP) and its derivatives are considered first-line treatments for ovarian cancer (OVCA). However, despite initial results that often appear promising, in most cases patients will return with recurrent disease that fails to respond to further chemotherapy. We assayed a number of food phytochemicals with reported PI3K inhibitory ability to identify candidates that can influence CDDP treatment outcomes in chemoresistant OVCA cell lines. A direct comparison revealed that the diarylheptanoid hirsutenone from the tree bark of Alnus hirsuta var. sibirica was superior at inducing CDDP sensitivity in a number of chemoresistant cancer cell lines. Whereas hirsutenone treatment activated p53, its modest efficacy in p53-mutant and -null cell lines suggested the existence of a p53-independent mode of action. Further investigation revealed that hirsutenone causes CDDP-dependent apoptosis in chemoresistant cells by ubiquitin-proteasome-dependent X-linked inhibitor of apoptosis degradation and by enhancing the translocation of apoptosis-inducing factor from the mitochondria to the nucleus. This was found to be, at least in part, under the influence of upstream Akt activity, linking hirsutenone-dependent PI3K inhibition with downstream effects on apoptosis-inducing factor, X-linked inhibitor of apoptosis, and apoptosis. Our findings provide rationale for further investigation of the effects of hirsutenone on chemoresistant OVCA in clinical studies.
Akt; Chemoresistance; Ovarian Cancer; p53; XIAP; AIF; Hirsutenone
Background: The MLL1 core complex mono- and dimethylates histone H3 lysine 4 (H3K4).
Results: MLL1 automethylates a conserved cysteine residue in its active site cleft.
Conclusion: MLL1 automethylation is inhibited by unmodified histone H3 but not by histones previously mono-, di-, or trimethylated at H3K4.
Significance: The pattern of automethylation inhibition is consistent with distinct active sites for mono- and dimethylation of H3K4.
The mixed lineage leukemia-1 (MLL1) core complex predominantly catalyzes mono- and dimethylation of histone H3 at lysine 4 (H3K4) and is frequently altered in aggressive acute leukemias. The molecular mechanisms that account for conversion of mono- to dimethyl H3K4 (H3K4me1,2) are not well understood. In this investigation, we report that the suppressor of variegation, enhancer of zeste, trithorax (SET) domains from human MLL1 and Drosophila Trithorax undergo robust intramolecular automethylation reactions at an evolutionarily conserved cysteine residue in the active site, which is inhibited by unmodified histone H3. The location of the automethylation in the SET-I subdomain indicates that the MLL1 SET domain possesses significantly more conformational plasticity in solution than suggested by its crystal structure. We also report that MLL1 methylates Ash2L in the absence of histone H3, but only when assembled within a complex with WDR5 and RbBP5, suggesting a restraint for the architectural arrangement of subunits within the complex. Using MLL1 and Ash2L automethylation reactions as probes for histone binding, we observed that both automethylation reactions are significantly inhibited by stoichiometric amounts of unmethylated histone H3, but not by histones previously mono-, di-, or trimethylated at H3K4. These results suggest that the H3K4me1 intermediate does not significantly bind to the MLL1 SET domain during the dimethylation reaction. Consistent with this hypothesis, we demonstrate that the MLL1 core complex assembled with a catalytically inactive SET domain variant preferentially catalyzes H3K4 dimethylation using the H3K4me1 substrate. Taken together, these results are consistent with a “two-active site” model for multiple H3K4 methylation by the MLL1 core complex.
Chromatin Histone Modification; Enzyme Mechanisms; Epigenetics; Histone Methylation; Leukemia; Automethylation; Self-methylation
Background: The aggregation and stereotypic spreading of Tau protein is associated with Alzheimer disease.
Results: Monomeric Tau enters neurons and nucleates and engages endogenous Tau to aggregate.
Conclusion: Endocytosis of soluble Tau triggers aggregation in vesicles and is sufficient to initiate the spreading of pathological species.
Significance: Increased levels of extracellular monomeric Tau may increase the risk of developing tauopathies.
Understanding the formation and propagation of aggregates of the Alzheimer disease-associated Tau protein in vivo is vital for the development of therapeutics for this devastating disorder. Using our recently developed live-cell aggregation sensor in neuron-like cells, we demonstrate that different variants of exogenous monomeric Tau, namely full-length Tau (hTau40) and the Tau-derived construct K18 comprising the repeat domain, initially accumulate in endosomal compartments, where they form fibrillar seeds that subsequently induce the aggregation of endogenous Tau. Using superresolution imaging, we confirm that fibrils consisting of endogenous and exogenous Tau are released from cells and demonstrate their potential to spread Tau pathology. Our data indicate a greater pathological risk and potential toxicity than hitherto suspected for extracellular soluble Tau.
Alzheimer Disease; Amyloid; Endocytosis; Protein Aggregation; Tau; Fluorescence Lifetime Imaging Microscopy; Propagation; Superresolution Microscopy
Background: p53 mutations give rise to a mutant p53 protein that acquires novel pro-oncogenic functions.
Results: Mutant p53 inhibits Dicer to promote receptor recycling, which is required for invasion and scattering.
Conclusion: Dicer is inhibited by mutant p53 via two mechanisms: TAp63-dependent and TAp63-independent.
Significance: Our findings contribute to a better understanding of the proinvasive functions of mutant p53.
The control and processing of microRNAs (miRs) is critical in the regulation of all cellular responses. Previous studies have suggested that a reduction in the expression of certain miRs, or an overall decrease in miR processing through the partial depletion of Dicer, can promote enhanced metastatic potential. We show here that Dicer depletion can promote the invasive behavior of cells that is reflected in enhanced recycling and activation of the growth factor receptors Met and EGF receptor. These responses are also seen in response to the expression of tumor-derived mutant p53s, and we show that mutant p53 can down-regulate Dicer expression through both direct inhibition of the TAp63-mediated transcriptional activation of Dicer and a TAp63-independent control of Dicer protein expression. Our results delineate a clear relationship between mutant p53, TAp63, and Dicer that might contribute to the metastatic function of mutant p53 but, interestingly, also reveal TAp63-independent functions of mutant p53 in controlling Dicer activity.
Cell Invasion; Cell Motility; Dicer; MicroRNA; p63; Cell Scattering; Mutant p53
Background: Langerin is a receptor in the immune system that binds sugars on pathogens.
Results: The effects of genetic variations that cause changes in the sugar-binding site of human langerin have been investigated.
Conclusion: Two common, linked, variations in the gene for human langerin change sugar specificity of langerin.
Significance: Individuals with variant forms of langerin may have altered susceptibility to pathogenic microorganisms.
Langerin, a C-type lectin on Langerhans cells, mediates carbohydrate-dependent uptake of pathogens in the first step of antigen presentation to the adaptive immune system. Langerin binds a diverse range of carbohydrates including high mannose structures, fucosylated blood group antigens, and glycans with terminal 6-sulfated galactose. Mutagenesis and quantitative binding assays indicate that salt bridges between the sulfate group and two lysine residues compensate for the nonoptimal binding of galactose at the primary Ca2+ site. A commonly occurring single nucleotide polymorphism (SNP) in human langerin results in change of one of these lysine residues, Lys-313, to isoleucine. Glycan array screening reveals that this amino acid change abolishes binding to oligosaccharides with terminal 6SO4-Gal and enhances binding to oligosaccharides with terminal GlcNAc residues. Structural analysis shows that enhanced binding to GlcNAc may result from Ile-313 packing against the N-acetyl group. The K313I polymorphism is tightly linked to another SNP that results in the change N288D, which reduces affinity for glycan ligands by destabilizing the Ca2+-binding site. Langerin with Asp-288 and Ile-313 shows no binding to 6SO4-Gal-terminated glycans and increased binding to GlcNAc-terminated structures, but overall decreased binding to glycans. Altered langerin function in individuals with the linked N288D and K313I polymorphisms may affect susceptibility to infection by microorganisms.
Carbohydrate-binding Protein; Crystal Structure; Genetic Polymorphism; Glycobiology; Lectin; CD207; Sulfated Glycans