10 research outputs found
Development of a Capillary Electrophoresis Platform for Identifying Inhibitors of Protein–Protein Interactions
Methods for identifying chemical
inhibitors of protein–protein
interactions (PPIs) are often prone to discovery of false positives,
particularly those caused by molecules that induce protein aggregation.
Thus, there is interest in developing new platforms that might allow
earlier identification of these problematic compounds. Capillary electrophoresis
(CE) has been evaluated as a method to screen for PPI inhibitors using
the challenging system of Hsp70 interacting with its co-chaperone
Bag3. In the method, Hsp70 is labeled with a fluorophore, mixed with
Bag3, and the resulting bound and free Hsp70 are separated and detected
by CE with laser-induced fluorescence detection. The method used a
chemically modified CE capillary to prevent protein adsorption. Inhibitors
of the Hsp70–Bag3 interaction were detected by observing a
reduction in the bound-to-free ratio. The method was used to screen
a library of 3443 compounds, and the results were compared to those
from a flow cytometry protein interaction assay. CE was found to produce
a lower hit rate with more compounds that were reconfirmed in subsequent
testing, suggesting greater specificity. This finding was attributed
to the use of electropherograms to detect artifacts such as aggregators
and to differences in protein modifications required to perform the
different assays. Increases in throughput are required to make the
CE method suitable for primary screens, but at the current stage of
development it is attractive as a secondary screen to test hits found
by higher-throughput methods
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Immunoproteasome functions explained by divergence in cleavage specificity and regulation.
The immunoproteasome (iP) has been proposed to perform specialized roles in MHC class I antigen presentation, cytokine modulation, and T cell differentiation and has emerged as a promising therapeutic target for autoimmune disorders and cancer. However, divergence in function between the iP and the constitutive proteasome (cP) has been unclear. A global peptide library-based screening strategy revealed that the proteasomes have overlapping but distinct substrate specificities. Differing iP specificity alters the quantity of production of certain MHC I epitopes but does not appear to be preferentially suited for antigen presentation. Furthermore, iP specificity was found to have likely arisen through genetic drift from the ancestral cP. Specificity differences were exploited to develop isoform-selective substrates. Cellular profiling using these substrates revealed that divergence in regulation of the iP balances its relative contribution to proteasome capacity in immune cells, resulting in selective recovery from inhibition. These findings have implications for iP-targeted therapeutic development
The immunoproteasome β5i subunit is a key contributor to ictogenesis in a rat model of chronic epilepsy
The proteasome is the core of the ubiquitin-proteasome system and is involved in synaptic protein metabolism. The incorporation of three inducible immuno-subunits into the proteasome results in the generation of the so-called immunoproteasome, which is endowed of pathophysiological functions related to immunity and inflammation. In healthy human brain, the expression of the key catalytic b5i subunit of the immunoproteasome is almost absent, while it is induced in the epileptogenic foci surgically resected from patients with pharmaco-resistant seizures, including temporal lobe epilepsy.
We show here that the b5i immuno-subunit is induced in experimental epilepsy, and its selective pharmacological inhibition significantly prevents, or delays, 4-aminopyridine-induced seizure-like events in acute rat hippocampal/entorhinal cortex slices. These effects are stronger in slices from epileptic vs normal rats, likely due to the more prominent b5i subunit expression in neurons and glia cells of diseased tissue. b5i subunit is transcriptionally induced in epileptogenic tissue likely by Toll-like receptor 4 signaling
activation, and independently on promoter methylation.
The recent availability of selective b5i subunit inhibitors opens up novel therapeutic opportunities for seizure inhibition in drug-resistant epilepsies
Paradoxical resistance of multiple myeloma to proteasome inhibitors by decreased levels of 19S proteasomal subunits
© 2015 eLife Sciences Publications Ltd. All rights reserved.Hallmarks of cancer, including rapid growth and aneuploidy, can result in non-oncogene addiction to the proteostasis network that can be exploited clinically. The defining example is the exquisit
Biochemical and Structural Characterization of Germicidin Synthase: Analysis of a Type III Polyketide Synthase That Employs Acyl-ACP as a Starter Unit Donor
Germicidin synthase (Gcs) from <i>Streptomyces coelicolor</i> is a type III polyketide synthase (PKS) with broad substrate flexibility
for acyl groups linked through a thioester bond to either coenzyme
A (CoA) or acyl carrier protein (ACP). Germicidin synthesis was reconstituted <i>in vitro</i> by coupling Gcs with fatty acid biosynthesis. Since
Gcs has broad substrate flexibility, we directly compared the kinetic
properties of Gcs with both acyl-ACP and acyl-CoA. The catalytic efficiency
of Gcs for acyl-ACP was 10-fold higher than for acyl-CoA, suggesting
a strong preference toward carrier protein starter unit transfer.
The 2.9 Ă… germicidin synthase crystal structure revealed canonical
type III PKS architecture along with an unusual helical bundle of
unknown function that appears to extend the dimerization interface.
A pair of arginine residues adjacent to the active site affect catalytic
activity but not ACP binding. This investigation provides new and
surprising information about the interactions between type III PKSs
and ACPs that will facilitate the construction of engineered systems
for production of novel polyketides
Interrogating the Molecular Basis for Multiple Macrolactone Ring Formation by the Pikromycin Polyketide Synthase
Avadomide monotherapy in relapsed/refractory DLBCL: safety, efficacy, and a predictive gene classifier.
Treatment options for relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) are limited, with no standard of care; prognosis is poor, with 4- to 6-month median survival. Avadomide (CC-122) is a cereblon-modulating agent with immunomodulatory and direct antitumor activities. This phase 1 dose-expansion study assessed safety and clinical activity of avadomide monotherapy in patients with de novo R/R DLBCL and transformed lymphoma. Additionally, a novel gene expression classifier, which identifies tumors with a high immune cell infiltration, was shown to enrich for response to avadomide in R/R DLBCL. Ninety-seven patients with R/R DLBCL, including 12 patients with transformed lymphoma, received 3 to 5 mg avadomide administered on continuous or intermittent schedules until unacceptable toxicity, disease progression, or withdrawal. Eighty-two patients (85%) experienced ≥1 grade 3/4 treatment-emergent adverse events (AEs), most commonly neutropenia (51%), infections (24%), anemia (12%), and febrile neutropenia (10%). Discontinuations because of AEs occurred in 10% of patients. Introduction of an intermittent 5/7-day schedule improved tolerability and reduced frequency and severity of neutropenia, febrile neutropenia, and infections. Among 84 patients with de novo R/R DLBCL, overall response rate (ORR) was 29%, including 11% complete response (CR). Responses were cell-of-origin independent. Classifier-positive DLBCL patients (de novo) had an ORR of 44%, median progression-free survival (mPFS) of 6 months, and 16% CR vs an ORR of 19%, mPFS of 1.5 months, and 5% CR in classifier-negative patients (P = .0096). Avadomide is being evaluated in combination with other antilymphoma agents. This trial was registered at www.clinicaltrials.gov as #NCT01421524
Development of a Capillary Electrophoresis Platform for Identifying Inhibitors of Protein–Protein Interactions
Methods for identifying chemical inhibitors of protein-protein interactions (PPIs) are often prone to discovery of false positives, particularly those caused by molecules that induce protein aggregation. Thus, there is interest in developing new platforms that might allow earlier identification of these problematic compounds. Capillary electrophoresis (CE) has been evaluated as a method to screen for PPI inhibitors using the challenging system of Hsp70 interacting with its co-chaperone Bag3. In the method, Hsp70 is labeled with a fluorophore, mixed with Bag3, and the resulting bound and free Hsp70 separated and detected by CE with laser-induced fluorescence detection. The method used a chemically modified CE capillary to prevent protein adsorption. Inhibitors of the Hsp70-Bag3 interaction were detected by observing a reduction in the bound to free ratio. The method was used to screen a library of 3,443 compounds and results compared to those from a flow cytometry protein interaction assay. CE was found to produce a lower hit rate with more compounds that reconfirmed in subsequent testing suggesting greater specificity. This finding was attributed to use of electropherograms to detect artifacts such as aggregators and to differences in protein modifications required to perform the different assays. Increases in throughput are required to make the CE method suitable for primary screens but at the current stage of development it is attractive as a secondary screen to test hits found by higher throughput methods