Specific inhibition of the chymotrypsin-like activity of the proteasome induces a bipolar morphology in neuroblastoma cells

AbstractBackground: Lactacystin inhibits cell proliferation and induces a distinctive, predominantly bipolar (two-neurite-bearing) morphology in Neuro 2A murine neuroblastoma cells. It binds with high specificity to the multicatalytic 20S proteasome and inhibits at least three of its peptidase activities (chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolyzing), each at a different rate, without inhibiting other known proteases. The chymotrypsin-like and trypsin-like activities of the proteasome are inhibited most rapidly, and irreversibly. In an effort to determine which of the peptidase activities needs to be inhibited for neurite outgrowth to occur, we treated Neuro 2A cells with peptide aldehydes that selectively inhibit different proteasome activities.Results: Treatment with peptide aldehydes ending in a hydrophobic residue, all of which inhibit the chymotrypsin-like activity, results in a bipolar morphology in Neuro 2A cells, whereas treatment with a peptide aidehyde inhibitor of the trypsin-like activity does not lead to a detectable change in morphology. One of the inhibitors that induces neurite outgrowth has been previously shown to inhibit the chymotrypsin-like activity of the proteasome without inhibiting the other apparently distinct peptidase activities that cleave after neutral residues, the so-called ‘branched chain amino acid preferring’ (BrAAP) and ‘small neutral amino acid preferring’ (SNAAP) activities, or the peptidylglutamyl-peptide hydrolyzing (PGPH) activity.Conclusions: The chymotrypsin-like activity appears to antagonize bipolar-type neurite outgrowth in Neuro 2A cells, while the trypsin-like, PGPH, BrAAP and SNAAP appear not to do so. Selective inhibition of a single peptidase activity, as opposed to general inhibition of the proteasome, appears sufficient to induce a specific cellular process. Selective inhibition might be useful in managing diseases where only one activity is involved without completely inhibiting the proteasome. It is also possible that endogenous regulators of the proteasome could affect cellular processes and that certain peptidase activities of the proteasome may have roles in specifying a given cell fate

Mode of inhibitory binding of epigallocatechin gallate to the ubiquitin-activating enzyme Uba1 via accelerated molecular dynamics

The green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) and some of its analogs potently inhibit the ubiquitin-activating enzyme Uba1. In an effort to understand the possible molecular basis of inhibitory activity of EGCG, we conducted a molecular docking and molecular dynamics simulation study. We found that EGCG and its two selected analogs, (-)-epicatechin-3-gallate (ECG) and (-)-epigallocatechin (EGC), bind favorably at two likely hot spots for small-molecule ligand binding on human Uba1. The compounds bind with energetics that mirror their experimental potency for inhibition of Uba1 similar to ubiquitin thioester formation. The binding of EGCG, ECG, and EGC at one of the hot spots, in particular, recapitulates the rank order of potency determined experimentally and suggests a possible mechanism for inhibition. A hinge-like conformational change of the second catalytic cysteine domain and the opposing ubiquitin-fold domain observed during accelerated molecular dynamics simulations of the EGCG-bound Uba1 complex that results in disruption of the ubiquitin-binding interfaces could explain the compounds' inhibitory activity. These results shed light on the possible molecular mechanism of EGCG and related catechins in the inhibition of Uba1

Journal Staff

Cells move along surfaces both as single cells and multi-cellular units. Recent research points toward pivotal roles for water flux through aquaporins (AQPs) in single cell migration. Their expression is known to facilitate this process by promoting rapid shape changes. However, little is known about the impact on migrating epithelial sheets during wound healing and epithelial renewal. Here, we investigate and compare the effects of AQP9 on single cell and epithelial sheet migration. To achieve this, MDCK-1 cells stably expressing AQP9 were subjected to migration assessment. We found that AQP9 facilitated cell locomotion at both the single and multi-cellular level. Furthermore, we identified major differences in the monolayer integrity and cell size upon expression of AQP9 during epithelial sheet migration, indicating a rapid volume-regulatory mechanism. We suggest a novel mechanism for epithelial wound healing based on AQP-induced swelling and expansion of the monolayer.Funding Agencies|Swedish Research Council for Medicine and Health|2007-34832009-66492010-3045|</p

Mode of Inhibitory Binding of Epigallocatechin Gallate to the Ubiquitin-Activating Enzyme Uba1 via Accelerated Molecular Dynamics

The green tea polyphenol (−)-epigallocatechin-3-gallate (EGCG) and some of its analogs potently inhibit the ubiquitin-activating enzyme Uba1. In an effort to understand the possible molecular basis of inhibitory activity of EGCG, we conducted a molecular docking and molecular dynamics simulation study. We found that EGCG and its two selected analogs, (−)-epicatechin-3-gallate (ECG) and (−)-epigallocatechin (EGC), bind favorably at two likely hot spots for small-molecule ligand binding on human Uba1. The compounds bind with energetics that mirror their experimental potency for inhibition of Uba1∼ubiquitin thioester formation. The binding of EGCG, ECG, and EGC at one of the hot spots, in particular, recapitulates the rank order of potency determined experimentally and suggests a possible mechanism for inhibition. A hinge-like conformational change of the second catalytic cysteine domain and the opposing ubiquitin-fold domain observed during accelerated molecular dynamics simulations of the EGCG-bound Uba1 complex that results in disruption of the ubiquitin-binding interfaces could explain the compounds' inhibitory activity. These results shed light on the possible molecular mechanism of EGCG and related catechins in the inhibition of Uba1

Light-Fueled Primitive Replication and Selection in Biomimetic Chemical Systems

The concept of chemically evolvable replicators is centralto abiogenesis.Chemical evolvability requires three essential components: energy-harvestingmechanisms for nonequilibrium dissipation, kinetically asymmetricreplication and decomposition pathways, and structure-dependent selectivetemplating in the autocatalytic cycles. We observed a UVA light-fueledchemical system displaying sequence-dependent replication and replicatordecomposition. The system was constructed with primitive peptidicfoldamer components. The photocatalytic formation-recombinationcycle of thiyl radicals was coupled with the molecular recognitionsteps in the replication cycles. Thiyl radical-mediated chain reactionwas responsible for the replicator death mechanism. The competingand kinetically asymmetric replication and decomposition processesled to light intensity-dependent selection far from equilibrium. Here,we show that this system can dynamically adapt to energy influx andseeding. The results highlight that mimicking chemical evolution isfeasible with primitive building blocks and simple chemical reactions

Ricin B chain targeted to the endoplasmic reticulum of tobacco protoplasts is degraded by a CDC48- and vacuole-independent mechanism

The B chain of ricin was expressed and delivered to the endoplasmic reticulum of tobacco protoplasts where it disappeared with time in a manner consistent with degradation. This turnover did not occur in the vacuoles or upon secretion. Indeed, several lines of evidence indicate that, in contrast to the turnover of endoplasmic reticulum-targeted ricin A chain in the cytosol, the bulk of expressed ricin B chain was degraded in the secretory pathway

A Series of Xanthenes Inhibiting Rad6 Function and Rad6–Rad18 Interaction in the PCNA Ubiquitination Cascade

Ubiquitination of proliferating cell nuclear antigen (PCNA) triggers pathways of DNA damage tolerance, including mutagenic translesion DNA synthesis, and comprises a cascade of reactions involving the E1 ubiquitin-activating enzyme Uba1, the E2 ubiquitin-conjugating enzyme Rad6, and the E3 ubiquitin ligase Rad18. We report here the discovery of a series of xanthenes that inhibit PCNA ubiquitination, Rad6∼ubiquitin thioester formation, and the Rad6–Rad18 interaction. Structure-activity relationship experiments across multiple assays reveal chemical and structural features important for different activities along the pathway to PCNA ubiquitination. The compounds that inhibit these processes are all a subset of the xanthen-3-ones we tested. These small molecules thus represent first-in-class probes of Rad6 function and the association of Rad6 and Rad18, the latter being a new inhibitory activity discovered for a small molecule, in the PCNA ubiquitination cascade and potential therapeutic agents to contain cancer progression