Aggregation of α-synuclein is a pathological hallmark of sporadic or familial PD. However, the detailed molecular mechanism responsible for the aggregation of α-synuclein has not been properly explored. In the present study, we have identified a novel role of an anti-tumorigenic BTB/POZ domain containing protein-2 (BPOZ-2) in the regulation of α-synuclein accumulation in dopaminergic (DA) neurons. MPP+, an etiological factor for PD, significantly downregulated the expression of BPOZ-2 ahead of α-synuclein upregulation. Moreover, siRNA knockdown of BPOZ-2 alone stimulated the aggregation of α-synuclein protein; the effect was further induced in presence of MPP+ in mouse primary DA neurons. Finally, the absence of BPOZ-2 in α-synuclein expressing neuronal populations of MPTP-intoxicated mouse and primate nigra indicates that the suppression of BPOZ-2 could be involved in the accumulation of α-synuclein protein.
ABTB2; BPOZ-2; Neuron; α-Synuclein
Polarized cell morphogenesis requires actin cytoskeleton rearrangement for polarized transport of proteins, organelles and secretory vesicles, which fundamentally underlies cell differentiation and behavior. During yeast mating, S. cerevisiae responds to extracellular pheromone gradients by extending polarized projections, which are likely maintained through vesicle transport to (exocytosis) and from (endocytosis) the membrane. We experimentally demonstrate that the projection morphology is pheromone concentration-dependent, and propose the underlying mechanism through mathematical modeling. The inclusion of membrane flux and dynamically evolving cell boundary into our yeast mating signaling model shows good agreement with experimental measurements, and provides a plausible explanation for pheromone-induced cell morphology.
Cerebral cavernous malformations (CCM) are neurovascular dysplasias affecting up to 0.5% of the population. Mutations in the CCM2 gene are associated with acquisition of CCM. We identify a previously uncharacterized domain at the C-terminus of CCM2 and determine its 1.9 Å resolution crystal structure. Because this domain is structurally homologous to the N-terminal domain of harmonin, we name it the CCM2 harmonin-homology domain or HHD. CCM2 HHD is observed in two conformations, and we employ analytical ultracentrifugation to test its oligomerization. Additionally, CCM2 HHD contains an unusually long 13-residue 310 helix. This study provides the first structural characterization of CCM2.
Protein-protein interaction; Signal transduction; Cerebral cavernous malformation; X-ray crystallography; harmonin-homology domain
We have derived structures of intact calmodulin(CaM)-free and CaM-bound endothelial nitric oxide synthase (eNOS) by reconstruction from cryo-electron micrographs. The CaM-free reconstruction is well fitted by the oxygenase domain dimer, but the reductase domains are not visible, suggesting they are mobile and thus delocalized. Additional protein is visible in the CaM-bound reconstruction, concentrated in volumes near two basic patches on each oxygenase domain. One of these corresponds with a presumptive docking site for the reductase domain FMN-binding module. The other is proposed to correspond with a docking site for CaM. A model is suggested in which CaM binding and docking position the reductase domains near the oxygenase domains and promote docking of the FMN-binding modules required for electron transfer.
nitric oxide synthase; calmodulin; enzyme regulation; enzyme structure
SnoN (Ski-novel protein) plays an important role in embryonic development, tumorigenesis and aging. Past studies largely focused on its roles in tumorigenesis. Recent studies of its expression patterns and functions in mouse models and mammalian cells have revealed that SnoN interacts with multiple signaling molecules at different cellular levels to modulate the activities of several signaling pathways in a tissue context and developmental stage dependent manner. These studies suggest that SnoN may have broad functions in the embryonic development and tissue morphogenesis.
SnoN; Embryonic development; Morphogenesis
Clathrin is a trimeric protein involved in receptor-mediated-endocytosis, but can function as a non-trimer outside of endocytosis. We have discovered that the subcellular distribution of a clathrin cysteine mutant we previously studied is altered and a proportion is also localized to nuclear spaces. MALS shows C1573A hub is a mixture of trimer-like and detrimerized molecules. The X-ray structure of the trimerization domain reveals that that without light chains, a helix harboring cysteine-1573 is reoriented. We propose clathrin has a detrimerization switch, which suggests clathrin topology can be altered naturally for new functions.
Clathrin; endocytosis; clathrin monomer; clathrin dimer; detrimerization; p53 transactivation; cancer
Synaptobrevin 2 (Syb2), Syntaxin (Sx1A), and SNAP-25, generate a force to induce fusion pore formation. The v-SNARE, Syb2, is anchored to the vesicle membrane by a single transmembrane domain. Here we show that 2 tryptophans (W89/W90) located in the juxtamembrane domain of Syb2, which stabilize the transmembrane (TM) domain position, control the ratio of spontaneous vs. stimulated membrane fusion events in chromaffin cells. Changing the 2 hydrophobic tryptophans to neutral alanines promotes spontaneous membrane fusion, faster transmitter release kinetics and complete release from individual vesicles. The results indicate that the two tryptophans act as a fusion clamp making fusion stimulus-dependent.
exocytosis; membrane fusion; synaptobrevin; snare complex; amperometry
SnoN/SkiL (TGFβ regulator) is dysregulated in ovarian cancer, a disease associated with acquired drug-resistance. Arsenic trioxide (As2O3used in treating APL) induces SnoN to oppose the apoptotic response in ovarian cancer cells. We now report that As2O3 increases phosphorylation of EGFR/p66ShcA and EGFR degradation. As2O3 activates Src(Y416) whose activity (inhibited by PP2) modulates EGFR activation, its interaction with Shc/Grb2, and p-AKT. Inhibition of PI3K reduces SnoN and cell survival. Although EGFR or MAPK1 siRNA did not alter SnoN expression, As2O3–induced cleaved PARP was reduced together with increased XIAP. Collectively, As2O3 mediates an initial rise in pY-Src(416) to regulate the PI3K/AKT pathway which increases SnoN and cell survival; these early events may counter the cell death response associated with increased pY-EGFR/MAPK activation.
ovarian cancer; SnoN/SkiL; epidermal growth factor receptor (EGFR); Src tyrosine kinase; PI3K/AKT; As2O3
Crk and CrkL adaptors play essential neuronal positioning roles downstream of Reelininduced Dab1 tyrosine phosphorylation. Recently we identified Cin85 to be a CrkL-SH3 binding partner from embryonic murine brain while others found Cin85 binds directly to Dab1. Here using mass spectrometry, biochemical and mutational analyses we show that Dab1 suppresses Cin85 phosphorylation at Ser587. Furthermore a Cin85 Ser587 phosphomimetic disrupts the Dab1-Cin85 complex without affecting the Cin85-CapZ complex. These data provide an early glimpse into how Cin85 phosphorylation might alter the composition of its scaffolding partners to regulate its diverse roles including vesicular trafficking, receptor endocytosis and actin remodeling.
Dab1; Cin85; Reelin; phosphorylation; CrkL; kinase
•The structure of CTNNBL1 includes an abbreviated armadillo domain.•CTNNBL1 is a novel NLS binding protein with a unique carboxy-terminal structure.•Structure-based mutagenesis of CTNNBL1 shows NLS binding distinct from karyopherins.
CTNNBL1 is a spliceosome-associated protein that binds nuclear localization signals (NLSs) in splice factors CDC5L and Prp31 as well as the antibody diversifying enzyme AID. Here, crystal structures of human CTNNBL1 reveal a distinct structure from its closest homologue karyopherin-α. CTNNBL1 comprises a HEAT-like domain (including a nuclear export signal), a central armadillo domain, and a coiled-coil C-terminal domain. Structure-guided mutations of the region homologous to the karyopherin-α NLS-binding site fail to disrupt CTNNBL1–NLS interactions. Our results identify CTNNBL1 as a unique selective NLS-binding protein with striking differences from karyopherin-αs.
Structured summary of protein interactions
CTNNBL1binds to CDC5L NLS3 by pull down (View interaction)
CTNNBL1 and CDC5L NLS3bind by isothermal titration calorimetry (View interaction)
CTNNBL1 and Prp31 NLSbind by isothermal titration calorimetry (View interaction)
Splicing; Nuclear import; Armadillo domain
The CRISPR system is an adaptive RNA-based microbial immune system against invasive genetic elements. CasB is an essential protein component in Type I-E Cascade. Here, we characterize CasB proteins from three different organisms as nonspecific nucleic acid binding proteins. The T. fusca CasB crystal structure reveals conserved positive surface charges, which we show are important for its nucleic acid binding function. EM docking reveals that CasB dimerization aligns individual nucleic acid binding surfaces into a curved, elongated binding surface inside Type I-E Cascade, consistent with the putative functions of CasB in ds-DNA recruitment and crRNA-DNA duplex formation steps.
Structured summary of protein interactions
TthCasB and TthCasB
bind by x-ray crystallography (View interaction)
TfuCasB1 and TfuCasB1
bind by molecular sieving (View Interaction: 1, 2)
CRISPR; Cas; CasB; nucleic acid binding; Cascade
Sub-micron scale signaling domains induced in the plasma membrane of cells are thought to play important roles in signal transduction. In T cells, agonist MHC-peptide complexes induce small diffraction-limited domains enriched in T cell receptor (TCR) and signaling molecules. These microclusters serve as transient platforms for signal initiation and are required for sustained signaling in T cells, although each microcluster functions for only a couple of minutes. How they are formed, and what mechanisms promote and regulate signaling within TCR microclusters is largely unknown, although it is clear that TCR engagement and dynamic reorganization of cortical actin are involved. Here, we review current understanding of signaling within microclusters in T cells, and speculate on how these structures may form, initiate biochemical signals, and serve as sites of both signal integration and amplification, while also facilitating appropriate termination of TCR and related signaling.
T cell; signaling; microcluster; microdomain; triggering; immunological synapse
MauG catalyzes posttranslational modifications of methylamine dehydrogenase to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. MauG possesses a five-coordinate high-spin and a six-coordinate low-spin ferric heme, the latter with His-Tyr ligation. Replacement of this tyrosine with lysine generates a MauG variant with only high-spin ferric heme and altered spectroscopic and redox properties. Y294K MauG cannot stabilize the bis-Fe(IV) redox state required for TTQ biosynthesis but instead forms a compound I-like species on reaction with peroxide. The results clarify the role of Tyr ligation of the five-coordinate heme in determining the physical and redox properties and reactivity of MauG.
The Fanconi anemia (FA) pathway maintains genome stability through co-ordination of DNA repair of interstrand crosslinks (ICLs). Disruption of the FA pathway yields hypersensitivity to interstrand crosslinking agents, bone marrow failure and cancer predisposition. Early steps in DNA damage dependent activation of the pathway are governed by monoubiquitylation of FANCD2 and FANCI by the intrinsic FA E3 ubiquitin ligase, FANCL. Downstream FA pathway components and associated factors such as FAN1 and SLX4 exhibit ubiquitin-binding motifs that are important for their DNA repair function, underscoring the importance of ubiquitylation in FA pathway mediated repair. Importantly, ubiquitylation provides the foundations for cross-talk between repair pathways, which in concert with the FA pathway, resolve interstrand crosslink damage and maintain genomic stability.
Monoubiquitylation; FANCL; FANCD2; FANCI; SLX4; FAN1
O-Aryloxycarbonyl hydroxamates and 1,3,4-oxathiazol-2-ones have been identified as covalent inhibitors of β-lactamases and proteasomes, respectively. The products of these inhibition reactions are remarkably similar, involving carbonyl cross-linking of the active sites. We have cross-checked these inhibitors, showing that the former inhibit proteasomes and the latter β-lactamases, to form the same inactive carbonyl adducts. These results are discussed in terms of similarities of the active site structures and catalytic mechanisms. It is likely that a mechanistic imperative has led to convergent evolution of these enzyme active sites, of a β-lactam-recognizing enzyme and a N-terminal protease belonging to different amidohydrolase superfamilies.
Enzyme; β-Lactamase; Proteasome; Inhibitors; Convergent Evolution
•TRAK2, a kinesin adaptor protein, binds the cargo binding domain of the kinesin-1 motor, KIF5A.•Three KIF5A regions were found to contribute to the TRAK2 binding site.•KIF5A discriminates between TRAK1 and TRAK2 with respect to binding specificity.•These data yield insights into kinesin/kinesin adaptor protein interactions.
Understanding specific cargo distribution in differentiated cells is a major challenge. Trafficking kinesin proteins (TRAKs) are kinesin adaptors. They bind the cargo binding domain of kinesin-1 motor proteins forming a link between the motor and their cargoes. To refine the TRAK1/2 binding sites within the kinesin-1 cargo domain, rationally designed C-terminal truncations of KIF5A and KIF5C were generated and their co-association with TRAK1/2 determined by quantitative co-immunoprecipitations following co-expression in mammalian cells. Three contributory regions forming the TRAK2 binding site within KIF5A and KIF5C cargo binding domains were delineated. Differences were found between TRAK1/2 with respect to association with KIF5A.
Structured summary of protein interactions
TRAK2physically interacts with KIF5C by anti tag coimmunoprecipitation (View interaction)
TRAK1physically interacts with KIF5A by anti tag coimmunoprecipitation (View interaction)
TRAK2physically interacts with KIF5A by anti tag coimmunoprecipitation (1, 2)
FEZ1, fasciculation and elongation protein-ζ; FRET, Forster resonance energy transfer; GRIP, glutamate receptor-interacting protein 1; HAP1, Huntingtin-associated protein; HEK, human embryonic kidney; MADD, adaptor protein mitogen-activated protein kinase-activating death domain; TRAK, trafficking kinesin protein; Kinesin; Kinesin adaptor protein; TRAK; Motor protein; Intracellular transport; Mitochondrial trafficking
hCINAP is an atypical nucleoplasmic enzyme, combining structural features of adenylate kinases and ATPases, which exhibits dual enzymatic activity. It interacts with the Cajal Body marker coilin and its level of expression and enzymatic activity influence Cajal Body numbers. Here we show that upon specific transcriptional inhibition of RNA pol.II, hCINAP segregates in perinuclear caps identified as Dark Nucleolar Caps (DNCs). These are distinct from perinucleolar caps where coilin and fibrillarin (both Cajal Body components) accumulate. In DNCs, hCINAP co-localizes with Paraspeckle Protein (PSP1) and also co-segregates with PSP1, and not coilin, in nuclear and nucleolar foci upon UV irradiation.
Nuclear organization; Cajal Body; Coilin; Paraspeckle Protein 1; Paraspeckle
ShK, a 35-residue peptide from a sea anemone, is a potent blocker of potassium channels. Here we describe a new ShK analogue with an additional C-terminus Lys residue and amide. ShK-K-amide is a potent blocker of Kv1.3 and, in contrast to ShK and ShK-amide, is selective for Kv1.3. To understand this selectivity, we created complexes of ShK-K-amide with Kv1.3 and Kv1.1 using docking and molecular dynamics simulations, then performed umbrella sampling simulations to construct the potential of mean force of the ligand and calculate the corresponding binding free energy for the most stable configuration. The results agree well with experimental data.
ShK; C-terminal amide; potassium channel; electrophysiology; potential of mean force; umbrella sampling
NSMase2 is associated to the plasma membrane, whereas ASMase is predominantly lysosomal; both hydrolyze sphingomyelin (SM) to ceramide and phosphocholine. Although SM accumulated in both ASMase-/- and fro/fro (NSMase2-/-) fibroblasts, the reduction of ceramides was more dramatic in fro/fro cells. ASMase mRNA, protein and enzyme activity were substantially elevated in fro/fro fibroblasts. In contrast, NSMase2 activity was unaffected in ASMase-/- fibroblasts. ASMase-/- cells showed normal cell cycling whereas fro/fro cells grew slowly and were arrested in G1/G0 and could be corrected by transfection with smpd3 gene. This suggests two distinct subcellular pathways for SM catabolism with distinct functions.
NSMase2; ASMase; coordination
During oxidative stress in E. coli, the SufABCDSE stress response pathway mediates iron-sulfur (Fe-S) cluster biogenesis rather than the Isc pathway. To determine why the Suf pathway is favored under stress conditions, the stress response SufS-SufE sulfur transfer pathway and the basal housekeeping IscS-IscU pathway were directly compared. We found that SufS-SufE cysteine desulfurase activity is significantly higher than IscS-IscU at physiological cysteine concentrations and after exposure to H2O2. Mass spectrometry analysis demonstrated that IscS-IscU is more susceptible than SufS-SufE to oxidative modification by H2O2. These important results provide biochemical insight into the stress resistance of the Suf pathway.
Allosteric activation of antithrombin as a rapid inhibitor of factors IXa and Xa requires binding of a high-affinity heparin pentasaccharide. The currently-accepted mechanism involves removal of a constraint on the antithrombin reactive center loop (RCL) so that the proteinase can simultaneously engage both the P1 arginine and an exosite at Y253. Recent results suggest that this mechanism is incorrect in that activation can be achieved without loop expulsion, while the exosite can be engaged in both low and high activity states. We propose a quite different mechanism in which heparin activates antithrombin by mitigating an unfavorable surface interaction, by altering its nature, and by moving the attached proteinase away from the site of the unfavorable interaction through RCL expulsion.
antithrombin; heparin activation; proteinase inhibition; factor Xa; factor IXa
Aminoacylase 3 (AA3) mediates deacetylation of N-acetyl aromatic amino acids and mercapturic acids. Deacetylation of mercapturic acids of exo- and endobiotics are likely involved in their toxicity. AA3 is predominantly expressed in kidney, and to a lesser extent in liver, brain, and blood. AA3 has been recently reported to interact with the hepatitis C virus core protein (HCVCP) in the yeast two-hybrid system. Here we demonstrate that AA3 directly binds to HCVCP (Kd~10 μM) that may by implicated in HCV pathogenesis. AA3 also revealed a weak endopeptidase activity towards the N-terminus of HCVCP.
aminoacylase 3; hepatitis C virus core protein; endopeptidase
Escherichia coli DNA damage inducible protein DinG is a superfamily II DNA helicase and is closely related to human DNA helicase XPD. Here, we report that E. coli single-stranded DNA binding protein (SSB) is able to form a stable protein complex with DinG and to stimulate the DinG DNA helicase activity. An SSB mutant that retains the single-stranded DNA binding activity but fails to form a protein complex with DinG becomes a potent inhibitor for the DinG DNA helicase, suggesting that E. coli wild-type SSB stimulates the DinG DNA helicase via specific protein-protein interaction.
DNA damage; DinG; XPD; SSB; DNA helicase; iron-sulfur protein
Over 300 amino acids are found in proteins in nature, yet typically only 20 are genetically encoded. Reassigning stop codons and use of quadruplet codons emerged as the main avenues for genetically encoding non-canonical amino acids (NCAAs). Canonical aminoacyl-tRNAs with near-cognate anticodons also read these codons to some extent. This background suppression leads to ‘statistical protein’ that contains some natural amino acid(s) at a site intended for NCAA. We characterize near-cognate suppression of amber, opal and a quadruplet codon in common Escherichia coli laboratory strains and find that the PylRS/tRNAPyl orthogonal pair cannot completely outcompete contamination by natural amino acids.
4-base codon; Boc-lysine; orthogonal translation systems; pyrrolysyl-tRNA synthetase; tRNA; synthetic biology
The repair of DNA double-strand breaks (DSBs) requires remodeling of the local chromatin architecture to allow the repair machinery to access sites of damage. Here, we report that the histone variant macroH2A1.1 is recruited to DSBs. Cells lacking macroH2A1 have defective recruitment of 53BP1, defective activation of chk2 kinase and increased radiosensitivity. Importantly, macroH2A1.1 is not incorporated into nucleosomes at DSBs, but instead associates with the chromatin through a mechanism which requires PARP1 activity. These results reveal an unusual mechanism involving a direct association of macroH2A1.1 with PARylated chromatin which is critical for retaining 53BP1 at sites of damage.
DNA repair; macroH2A1; ionizing radiation; 53BP1; nucleosome