Detection of sequential polyubiquitylation on a millisecond timescale

The pathway by which ubiquitin chains are generated on substrate through a cascade of enzymes consisting of an E1, E2 and E3 remains unclear. Multiple distinct models involving chain assembly on E2 or substrate have been proposed. However, the speed and complexity of the reaction have precluded direct experimental tests to distinguish between potential pathways. Here we introduce new theoretical and experimental methodologies to address both limitations. A quantitative framework based on product distribution predicts that the really interesting new gene (RING) E3 enzymes SCF^(Cdc4) and SCF^(β-TrCP) work with the E2 Cdc34 to build polyubiquitin chains on substrates by sequential transfers of single ubiquitins. Measurements with millisecond time resolution directly demonstrate that substrate polyubiquitylation proceeds sequentially. Our results present an unprecedented glimpse into the mechanism of RING ubiquitin ligases and illuminate the quantitative parameters that underlie the rate and pattern of ubiquitin chain assembly

The Acidic Tail of the Cdc34 Ubiquitin-conjugating Enzyme Functions in Both Binding to and Catalysis with Ubiquitin Ligase SCFC^(dc4*)

Ubiquitin ligases, together with their cognate ubiquitin-conjugating enzymes, are responsible for the ubiquitylation of proteins, a process that regulates a myriad of eukaryotic cellular functions. The first cullin-RING ligase discovered, yeast SCF^(Cdc4), functions with the conjugating enzyme Cdc34 to regulate the cell cycle. Cdc34 orthologs are notable for their highly acidic C-terminal extension. Here we confirm that the Cdc34 acidic C-terminal tail has a role in Cdc34 binding to SCF^(Cdc4) and makes a major contribution to the submicromolar K_m of Cdc34 for SCF^(Cdc4). Moreover, we demonstrate that a key functional property of the tail is its acidity. Our analysis also uncovers an unexpected new function for the acidic tail in promoting catalysis. We demonstrate that SCF is functional when Cdc34 is fused to the C terminus of Cul1 and that this fusion retains partial function even when the acidic tail has been deleted. The Cdc34-SCF fusion proteins that lack the acidic tail must interact in a fundamentally different manner than unfused SCF and wild type Cdc34, demonstrating that distinct mechanisms of E2 recruitment to E3, as is seen in nature, can sustain substrate ubiquitylation. Finally, a search of the yeast proteome uncovered scores of proteins containing highly acidic stretches of amino acids, hinting that electrostatic interactions may be a common mechanism for facilitating protein assembly

Rapid E2-E3 Assembly and Disassembly Enable Processive Ubiquitylation of Cullin-RING Ubiquitin Ligase Substrates

Degradation by the ubiquitin-proteasome system requires assembly of a polyubiquitin chain upon substrate. However, the structural and mechanistic features that enable template-independent processive chain synthesis are unknown. We show that chain assembly by ubiquitin ligase SCF and ubiquitin-conjugating enzyme Cdc34 is facilitated by the unusual nature of Cdc34-SCF transactions: Cdc34 binds SCF with nanomolar affinity, nevertheless the complex is extremely dynamic. These properties are enabled by rapid association driven by electrostatic interactions between the acidic tail of Cdc34 and a basic ‘canyon’ in the Cul1 subunit of SCF. Ab initio docking between Cdc34 and Cul1 predicts intimate contact between the tail and the basic canyon, an arrangement confirmed by crosslinking and kinetic analysis of mutants. Basic canyon residues are conserved in both Cul1 paralogs and orthologs, suggesting that the same mechanism underlies processivity for all cullin-RING ubiquitin ligases. We discuss different strategies by which processive ubiquitin chain synthesis may be achieved

The San1 Ubiquitin Ligase Avidly Recognizes Misfolded Proteins Through Multiple Substrate Binding Sites

Cellular homeostasis depends on robust protein quality control (PQC) pathways that discern misfolded proteins from functional ones in the cell. One major branch of PQC involves the controlled degradation of misfolded proteins by the ubiquitin-proteasome system. Here ubiquitin ligases must recognize and bind to misfolded proteins with sufficient energy to form a complex and with an adequate half-life to achieve poly-ubiquitin chain formation, the signal for protein degradation, prior to its dissociation from the ligase. It is not well understood how PQC ubiquitin ligases accomplish these tasks. Employing a fully reconstituted enzyme and substrate system to perform quantitative biochemical experiments, we demonstrate that the yeast PQC ubiquitin ligase San1 contains multiple substrate binding sites along its polypeptide chain that appear to display specificity for unique misfolded proteins. The results are consistent with a model where these substrate binding sites enable San1 to bind to misfolded substrates avidly, resulting in high affinity ubiquitin ligase-substrate complexes

Mechanism of Lysine 48 Selectivity during Polyubiquitin Chain Formation by the Ube2R1/2 Ubiquitin-Conjugating Enzyme

Lysine selectivity is of critical importance during polyubiquitin chain formation because the identity of the lysine controls the biological outcome. Ubiquitins are covalently linked in polyubiquitin chains through one of seven lysine residues on its surface and the C terminus of adjacent protomers. Lys 48-linked polyubiquitin chains signal for protein degradation; however, the structural basis for Lys 48 selectivity remains largely unknown. The ubiquitin-conjugating enzyme Ube2R1/2 has exquisite specificity for Lys 48, and computational docking of Ube2R1/2 and ubiquitin predicts that Lys 48 is guided to the active site through a key electrostatic interaction between Arg 54 on ubiquitin and Asp 143 on Ube2R1/2. The validity of this interaction was confirmed through biochemical experiments. Since structural examples involving Arg 54 in protein-ubiquitin complexes are exceedingly rare, these results provide additional insight into how ubiquitin-protein complexes can be stabilized. We discuss how these findings relate to how other ubiquitin-conjugating enzymes direct the lysine specificity of polyubiquitin chains

Mechanism of Lysine 48 Selectivity during Polyubiquitin Chain Formation by the Ube2R1/2 Ubiquitin-Conjugating Enzyme

Lysine selectivity is of critical importance during polyubiquitin chain formation because the identity of the lysine controls the biological outcome. Ubiquitins are covalently linked in polyubiquitin chains through one of seven lysine residues on its surface and the C terminus of adjacent protomers. Lys 48-linked polyubiquitin chains signal for protein degradation; however, the structural basis for Lys 48 selectivity remains largely unknown. The ubiquitin-conjugating enzyme Ube2R1/2 has exquisite specificity for Lys 48, and computational docking of Ube2R1/2 and ubiquitin predicts that Lys 48 is guided to the active site through a key electrostatic interaction between Arg 54 on ubiquitin and Asp 143 on Ube2R1/2. The validity of this interaction was confirmed through biochemical experiments. Since structural examples involving Arg 54 in protein-ubiquitin complexes are exceedingly rare, these results provide additional insight into how ubiquitin-protein complexes can be stabilized. We discuss how these findings relate to how other ubiquitin-conjugating enzymes direct the lysine specificity of polyubiquitin chains

Robust cullin-RING ligase function is established by a multiplicity of poly-ubiquitylation pathways.

Funder: Max-Planck-Gesellschaft; FundRef: http://dx.doi.org/10.13039/501100004189Funder: Canada Research Chairs; FundRef: http://dx.doi.org/10.13039/501100001804The cullin-RING ligases (CRLs) form the major family of E3 ubiquitin ligases. The prototypic CRLs in yeast, called SCF enzymes, employ a single E2 enzyme, Cdc34, to build poly-ubiquitin chains required for degradation. In contrast, six different human E2 and E3 enzyme activities, including Cdc34 orthologs UBE2R1 and UBE2R2, appear to mediate SCF-catalyzed substrate polyubiquitylation in vitro. The combinatorial interplay of these enzymes raises questions about genetic buffering of SCFs in human cells and challenges the dogma that E3s alone determine substrate specificity. To enable the quantitative comparisons of SCF-dependent ubiquitylation reactions with physiological enzyme concentrations, mass spectrometry was employed to estimate E2 and E3 levels in cells. In combination with UBE2R1/2, the E2 UBE2D3 and the E3 ARIH1 both promoted SCF-mediated polyubiquitylation in a substrate-specific fashion. Unexpectedly, UBE2R2 alone had negligible ubiquitylation activity at physiological concentrations and the ablation of UBE2R1/2 had no effect on the stability of SCF substrates in cells. A genome-wide CRISPR screen revealed that an additional E2 enzyme, UBE2G1, buffers against the loss of UBE2R1/2. UBE2G1 had robust in vitro chain extension activity with SCF, and UBE2G1 knockdown in cells lacking UBE2R1/2 resulted in stabilization of the SCF substrates p27 and CYCLIN E as well as the CUL2-RING ligase substrate HIF1α. The results demonstrate the human SCF enzyme system is diversified by association with multiple catalytic enzyme partners

Central Neuroplasticity and Decreased Heart Rate Variability after Particulate Matter Exposure in Mice

BackgroundEpidemiologic studies show that exposure to fine particulate matter [aerodynamic diameter < or = 2.5 microm (PM(2.5))] increases the total daily cardiovascular mortality. Impaired cardiac autonomic function, which manifests as reduced heart rate variability (HRV), may be one of the underlying causes. However, the cellular mechanism(s) by which PM(2.5) exposure induces decreased HRV is not known.ObjectivesWe tested the hypothesis that exposure to PM(2.5) impairs HRV by decreasing the excitability of the cardiac vagal neurons in the nucleus ambiguus. We also determined the effect of iron on PM-exposure-induced decrease in HRV.MethodsWe measured 24-hr HRV in time domains from electrocardiogram telemetry recordings obtained in conscious, freely moving mice after 3 days of exposure to PM(2.5) in the form of soot only or iron-soot. In parallel studies, we determined the intrinsic properties of identified cardiac vagal neurons, retrogradely labeled with a fluorescent dye applied to the sinoatrial node.ResultsSoot-only exposure decreased short-term HRV (root mean square of successive difference). With the addition of iron, all HRV parameters were significantly reduced. In nonexposed mice, vagal blockade significantly reduced all HRV parameters, suggesting that HRV is, in part, under vagal regulation in mice. Iron-soot exposure had no significant effect on resting membrane potential but decreased spiking responses of the identified cardiac vagal neurons to depolarizations (p < 0.05). The decreased spiking response was accompanied with a higher minimal depolarizing current required to evoke spikes and a lower peak discharge frequency.ConclusionsThe data suggest that PM-induced neuroplasticity of cardiac vagal neurons may be one mechanism contributing to the cardiovascular consequences associated with PM(2.5) exposure seen in humans

SUDDEN CARDIAC DEATH: ROLE OF LEFT VENTRICULAR DYSFUNCTION

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72037/1/j.1749-6632.1982.tb55219.x.pd

Phosfinder: a web server for the identification of phosphate-binding sites on protein structures

Phosfinder is a web server for the identification of phosphate binding sites in protein structures. Phosfinder uses a structural comparison algorithm to scan a query structure against a set of known 3D phosphate binding motifs. Whenever a structural similarity between the query protein and a phosphate binding motif is detected, the phosphate bound by the known motif is added to the protein structure thus representing a putative phosphate binding site. Predicted binding sites are then evaluated according to (i) their position with respect to the query protein solvent-excluded surface and (ii) the conservation of the binding residues in the protein family. The server accepts as input either the PDB code of the protein to be analyzed or a user-submitted structure in PDB format. All the search parameters are user modifiable. Phosfinder outputs a list of predicted binding sites with detailed information about their structural similarity with known phosphate binding motifs, and the conservation of the residues involved. A graphical applet allows the user to visualize the predicted binding sites on the query protein structure. The results on a set of 52 apo/holo structure pairs show that the performance of our method is largely unaffected by ligand-induced conformational changes. Phosfinder is available at http://phosfinder.bio.uniroma2.it