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1.
Methods Mol Biol ; 2365: 185-202, 2021.
Article in English | MEDLINE | ID: mdl-34432245

ABSTRACT

The ubiquitin proteasome system (UPS) is a complex pathway that involves multiple enzymes and culminates in the formation of a polyubiquitin chain on a target protein. As its importance is becoming more evident in drug discovery, there is a renewed interest in understanding the role that polyubiquitin chains play. This has been a challenge, mostly due to the lack of experimental tools for detecting the polyubiquitinated forms of a protein of interest (POI). Tandem Ubiquitin Binding Entities (TUBEs) are engineered protein domains that bind specifically to polyubiquitin chains. These polyubiquitin affinity matrices are highly sensitive as they bind to polyubiquitin chains in the nanomolar range. They exist in two forms: pan-selective TUBEs and chain-selective TUBEs. The ability of TUBEs to be conjugated to different entities is truly what makes them unique. TUBEs are used in a wide variety of experiments such as in protein pulldowns to enrich for polyubiquitinated proteins. They are an alternative to ubiquitin antibodies in Western blots. Further, TUBEs are used as capture reagents for immobilizing polyubiquitinated proteins on a microtiter plate. The use of TUBEs as components of in vitro and cell-based assays presents the unique feature of confirming and assessing the polyubiquitination of a POI in response to inhibitors, activators, or PROTAC® molecules. Therefore, TUBEs not only play a big role in studying the UPS but also have a huge potential for speeding up the drug discovery process.


Subject(s)
Drug Discovery , Proteasome Endopeptidase Complex , Ubiquitin , Polyubiquitin/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Ubiquitin/metabolism
2.
Nat Immunol ; 20(9): 1196-1207, 2019 09.
Article in English | MEDLINE | ID: mdl-31406379

ABSTRACT

The response to systemic infection and injury requires the rapid adaptation of hematopoietic stem cells (HSCs), which proliferate and divert their differentiation toward the myeloid lineage. Significant interest has emerged in understanding the signals that trigger the emergency hematopoietic program. However, the mechanisms that halt this response of HSCs, which is critical to restore homeostasis, remain unknown. Here we reveal that the E3 ubiquitin ligase Speckle-type BTB-POZ protein (SPOP) restrains the inflammatory activation of HSCs. In the absence of Spop, systemic inflammation proceeded in an unresolved manner, and the sustained response in the HSCs resulted in a lethal phenotype reminiscent of hyper-inflammatory syndrome or sepsis. Our proteomic studies decipher that SPOP restricted inflammation by ubiquitinating the innate signal transducer myeloid differentiation primary response protein 88 (MYD88). These findings unearth an HSC-intrinsic post-translational mechanism that is essential for reestablishing homeostasis after emergency hematopoiesis.


Subject(s)
Inflammation/immunology , Leukocytosis/immunology , Myeloid Differentiation Factor 88/metabolism , Neutrophils/immunology , Nuclear Proteins/metabolism , Repressor Proteins/metabolism , Animals , Cell Line , Female , HEK293 Cells , Hematopoiesis/immunology , Humans , Male , Mice , Neutrophils/cytology , Ubiquitin-Protein Ligase Complexes , Ubiquitin-Protein Ligases/metabolism
3.
J Clin Invest ; 122(12): 4412-23, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23114596

ABSTRACT

ATR-X syndrome is a severe intellectual disability disorder caused by mutations in the ATRX gene. Many ancillary clinical features are attributed to CNS deficiencies, yet most patients have muscle hypotonia, delayed ambulation, or kyphosis, pointing to an underlying skeletal muscle defect. Here, we identified a cell-intrinsic requirement for Atrx in postnatal muscle growth and regeneration in mice. Mice with skeletal muscle-specific Atrx conditional knockout (Atrx cKO mice) were viable, but by 3 weeks of age presented hallmarks of underdeveloped musculature, including kyphosis, 20% reduction in body mass, and 34% reduction in muscle fiber caliber. Atrx cKO mice also demonstrated a marked regeneration deficit that was not due to fewer resident satellite cells or their inability to terminally differentiate. However, activation of Atrx-null satellite cells from isolated muscle fibers resulted in a 9-fold reduction in myoblast expansion, caused by delayed progression through mid to late S phase. While in S phase, Atrx colocalized specifically to late-replicating chromatin, and its loss resulted in rampant signs of genomic instability. These observations support a model in which Atrx maintains chromatin integrity during the rapid developmental growth of a tissue.


Subject(s)
DNA Helicases/genetics , Genomic Instability , Muscle Development , Nuclear Proteins/genetics , Animals , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/metabolism , Cell Differentiation , Cell Proliferation , Cells, Cultured , Chromatin/genetics , Chromatin/metabolism , DNA Damage , DNA Helicases/metabolism , DNA Helicases/physiology , DNA Replication , DNA-Binding Proteins/metabolism , Female , Histones/metabolism , Humans , Male , Mental Retardation, X-Linked/genetics , Mental Retardation, X-Linked/physiopathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mitosis , Muscle, Skeletal/pathology , Muscle, Skeletal/physiopathology , Nuclear Proteins/metabolism , Nuclear Proteins/physiology , Primary Cell Culture , Protein Serine-Threonine Kinases/metabolism , Rad51 Recombinase/metabolism , Regeneration/genetics , S Phase Cell Cycle Checkpoints , Satellite Cells, Skeletal Muscle/pathology , Satellite Cells, Skeletal Muscle/physiology , Telomere/genetics , Telomere/metabolism , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/metabolism , X-linked Nuclear Protein , alpha-Thalassemia/genetics , alpha-Thalassemia/physiopathology
4.
Anal Biochem ; 398(1): 45-51, 2010 Mar 01.
Article in English | MEDLINE | ID: mdl-19854148

ABSTRACT

alpha-Methylacyl-coenzyme A racemase (AMACR) catalyzes the epimerization of (2R)- and (2S)-methyl branched fatty acyl-coenzyme A (CoA) thioesters. AMACR is a biomarker for prostate cancer and a putative target for the development of therapeutic agents directed against the disease. To facilitate development of AMACR inhibitors, a continuous circular dichroism (CD)-based assay has been developed. The open reading frame encoding AMACR from Mycobacterium tuberculosis (MCR) was subcloned into a pET15b vector, and the enzyme was overexpressed and purified using metal ion affinity chromatography. The rates of MCR-catalyzed epimerization of either (2R)- or (2S)-ibuprofenoyl-CoA were determined by following the change in ellipticity at 279nm in the presence of octyl-beta-d-glucopyranoside (0.2%). MCR exhibited slightly higher affinity for (2R)-ibuprofenoyl-CoA (K(m)=48+/-5microM, k(cat)=291+/-30s(-1)), but turned over (2S)-ibuprofenoyl-CoA (K(m)=86+/-6microM, k(cat)=450+/-14s(-1)) slightly faster. MCR expressed as a fusion protein bearing an N-terminal His(6)-tag had a catalytic efficiency (k(cat)/K(m)) that was reduced 22% and 47% in the 2S-->2R and 2R-->2S directions, respectively, relative to untagged enzyme. The continuous CD-based assay offers an economical and efficient alternative method to the labor-intensive, fixed-time assays currently used to measure AMACR activity.


Subject(s)
Circular Dichroism/methods , Enzyme Assays/methods , Racemases and Epimerases/analysis , Acyl Coenzyme A/metabolism , Humans , Kinetics , Male , Prostatic Neoplasms/metabolism , Racemases and Epimerases/genetics , Racemases and Epimerases/metabolism , Recombinant Proteins/analysis , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Stereoisomerism
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