Publications by authors named "Pawel A Osmulski"

32 Publications

New Peptide-Based Pharmacophore Activates 20S Proteasome.

Molecules 2020 Mar 22;25(6). Epub 2020 Mar 22.

Department of Molecular Medicine, UT Health San Antonio, Texas, TX 78245, USA.

The proteasome is a pivotal element of controlled proteolysis, responsible for the catabolic arm of proteostasis. By inducing apoptosis, small molecule inhibitors of proteasome peptidolytic activities are successfully utilized in treatment of blood cancers. However, the clinical potential of proteasome activation remains relatively unexplored. In this work, we introduce short TAT peptides derived from HIV-1 Tat protein and modified with synthetic turn-stabilizing residues as proteasome agonists. Molecular docking and biochemical studies point to the α1/α2 pocket of the core proteasome α ring as the binding site of TAT peptides. We postulate that the TATs' pharmacophore consists of an N-terminal basic pocket-docking "activation anchor" connected via a β turn inducer to a C-terminal "specificity clamp" that binds on the proteasome α surface. By allosteric effects-including destabilization of the proteasomal gate-the compounds substantially augment activity of the core proteasome in vitro. Significantly, this activation is preserved in the lysates of cultured cells treated with the compounds. We propose that the proteasome-stimulating TAT pharmacophore provides an attractive lead for future clinical use.
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http://dx.doi.org/10.3390/molecules25061439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145288PMC
March 2020

Single-Cell Proteomic Profiling Identifies Combined AXL and JAK1 Inhibition as a Novel Therapeutic Strategy for Lung Cancer.

Cancer Res 2020 04 28;80(7):1551-1563. Epub 2020 Jan 28.

Department of Molecular Medicine, University of Texas Health Science Center, San Antonio, Texas.

Cytometry by time-of-flight (CyTOF) simultaneously measures multiple cellular proteins at the single-cell level and is used to assess intertumor and intratumor heterogeneity. This approach may be used to investigate the variability of individual tumor responses to treatments. Herein, we stratified lung tumor subpopulations based on AXL signaling as a potential targeting strategy. Integrative transcriptome analyses were used to investigate how TP-0903, an AXL kinase inhibitor, influences redundant oncogenic pathways in metastatic lung cancer cells. CyTOF profiling revealed that AXL inhibition suppressed SMAD4/TGFβ signaling and induced JAK1-STAT3 signaling to compensate for the loss of AXL. Interestingly, high JAK1-STAT3 was associated with increased levels of AXL in treatment-naïve tumors. Tumors with high AXL, TGFβ, and JAK1 signaling concomitantly displayed CD133-mediated cancer stemness and hybrid epithelial-to-mesenchymal transition features in advanced-stage patients, suggesting greater potential for distant dissemination. Diffusion pseudotime analysis revealed cell-fate trajectories among four different categories that were linked to clinicopathologic features for each patient. Patient-derived organoids (PDO) obtained from tumors with high AXL and JAK1 were sensitive to TP-0903 and ruxolitinib (JAK inhibitor) treatments, supporting the CyTOF findings. This study shows that single-cell proteomic profiling of treatment-naïve lung tumors, coupled with testing of PDOs, identifies continuous AXL, TGFβ, and JAK1-STAT3 signal activation in select tumors that may be targeted by combined AXL-JAK1 inhibition. SIGNIFICANCE: Single-cell proteomic profiling of clinical samples may facilitate the optimal selection of novel drug targets, interpretation of early-phase clinical trial data, and development of predictive biomarkers valuable for patient stratification.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-3183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7127959PMC
April 2020

Inhibitory Interplay of SULT2B1b Sulfotransferase with AKR1C3 Aldo-keto Reductase in Prostate Cancer.

Endocrinology 2020 02;161(2)

Department of Molecular Medicine, University of Texas Health San Antonio, San Antonio, Texas.

SULT2B1b (SULT2B) is a prostate-expressed hydroxysteroid sulfotransferase, which may regulate intracrine androgen homeostasis by mediating 3β-sulfation of dehydroepiandrosterone (DHEA), the precursor for 5α-dihydrotestosterone (DHT) biosynthesis. The aldo-keto reductase (AKR)1C3 regulates androgen receptor (AR) activity in castration-resistant prostate cancer (CRPC) by promoting tumor tissue androgen biosynthesis from adrenal DHEA and also by functioning as an AR-selective coactivator. Herein we report that SULT2B-depleted CRPC cells, arising from stable RNA interference or gene knockout (KO), are markedly upregulated for AKR1C3, activated for ERK1/2 survival signal, and induced for epithelial-to-mesenchymal (EMT)-like changes. EMT was evident from increased mesenchymal proteins and elevated EMT-inducing transcription factors SNAI1 and TWIST1 in immunoblot and single-cell mass cytometry analyses. SULT2B KO cells showed greater motility and invasion in vitro; growth escalation in xenograft study; and enhanced metastatic potential predicted on the basis of decreased cell stiffness and adhesion revealed from atomic force microscopy analysis. While AR and androgen levels were unchanged, AR activity was elevated, since PSA and FKBP5 mRNA induction by DHT-activated AR was several-fold higher in SULT2B-silenced cells. AKR1C3 silencing prevented ERK1/2 activation and SNAI1 induction in SULT2B-depleted cells. SULT2B was undetectable in nearly all CRPC metastases from 50 autopsy cases. Primary tumors showed variable and Gleason score (GS)-independent SULT2B levels. CRPC metastases lacking SULT2B expressed AKR1C3. Since AKR1C3 is frequently elevated in advanced prostate cancer, the inhibitory influence of SULT2B on AKR1C3 upregulation, ERK1/2 activation, EMT-like induction, and on cell motility and invasiveness may be clinically significant. Pathways regulating the inhibitory SULT2B-AKR1C3 axis may inform new avenue(s) for targeting SULT2B-deficient prostate cancer.
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http://dx.doi.org/10.1210/endocr/bqz042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7341717PMC
February 2020

Pipecolic esters as minimized templates for proteasome inhibition.

Org Biomol Chem 2019 03;17(10):2734-2746

Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.

Allosteric regulators of clinically important enzymes are gaining popularity as alternatives to competitive inhibitors. This is also the case for the proteasome, a major intracellular protease and a target of anti-cancer drugs. All clinically used proteasome inhibitors bind to the active sites in catalytic chamber and display a competitive mechanism. Unfortunately, inevitable resistance associated with this type of inhibition drives the search for non-competitive agents. The multisubunit and multicatalytic "proteolytic machine" such as the proteasome is occasionally found to be affected by agents with other primary targets. For example the immunosuppressive agent rapamycin has been shown to allosterically inhibit the proteasome albeit at levels far higher than its mTOR related efficacy. As part of an ongoing program to search for novel proteasome-targeting pharmacophores, we identified the binding domain of rapamycin as required for proteasome inhibition even without the macrocyclic context of the parent compound. By subsequent structure-activity relationship studies, we generated a pipecolic ester derivative compound 3 representing a new class of proteasome inhibitors. Compound 3 affects the core proteasome activities and proliferation of cancer cells with low micromolar/high nanomolar efficacy. Molecular modeling, atomic force microscopy imaging and biochemical data suggest that compound 3 binds into one of intersubunit pockets in the proteasomal α ring and destabilizes the α face and the gate. The α face is used as a docking area for proteasome-regulating protein modules and the gate is critical for controlling access to the catalytic chamber. Thus, the pipecolic ester template elicits a new and attractive mechanism for proteasome inhibition distinct from classical competitive drugs.
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http://dx.doi.org/10.1039/c9ob00122kDOI Listing
March 2019

Small Molecule Modulation of Proteasome Assembly.

Biochemistry 2018 07 27;57(28):4214-4224. Epub 2018 Jun 27.

Department of Chemistry , Michigan State University , East Lansing , Michigan 48824 , United States.

The 20S proteasome is the main protease that directly targets intrinsically disordered proteins (IDPs) for proteolytic degradation. Mutations, oxidative stress, or aging can induce the buildup of IDPs resulting in incorrect signaling or aggregation, associated with the pathogenesis of many cancers and neurodegenerative diseases. Drugs that facilitate 20S-mediated proteolysis therefore have many potential therapeutic applications. We report herein the modulation of proteasome assembly by the small molecule TCH-165, resulting in an increase in 20S levels. The increase in the level of free 20S corresponds to enhanced proteolysis of IDPs, including α-synuclein, tau, ornithine decarboxylase, and c-Fos, but not structured proteins. Clearance of ubiquitinated protein was largely maintained by single capped proteasome complexes (19S-20S), but accumulation occurs when all 19S capped proteasome complexes are depleted. This study illustrates the first example of a small molecule capable of targeting disordered proteins for degradation by regulating the dynamic equilibrium between different proteasome complexes.
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http://dx.doi.org/10.1021/acs.biochem.8b00579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6241286PMC
July 2018

Targeting Protein-Protein Interactions in the Ubiquitin-Proteasome Pathway.

Adv Protein Chem Struct Biol 2018 18;110:123-165. Epub 2017 Oct 18.

Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX, United States.

The ubiquitin-proteasome pathway (UPP) is a major venue for controlled intracellular protein degradation in Eukaryota. The machinery of several hundred proteins is involved in recognizing, tagging, transporting, and cleaving proteins, all in a highly regulated manner. Short-lived transcription factors, misfolded translation products, stress-damaged polypeptides, or worn-out long-lived proteins, all can be found among the substrates of UPP. Carefully choreographed protein-protein interactions (PPI) are involved in each step of the pathway. For many of the steps small-molecule inhibitors have been identified and often they directly or indirectly target PPI. The inhibitors may destabilize intracellular proteostasis and trigger apoptosis. So far this is the most explored option used as an anticancer strategy. Alternatively, substrate-specific polyubiquitination may be regulated for a precise intervention aimed at a particular metabolic pathway. This very attractive opportunity is moving close to clinical application. The best known drug target in UPP is the proteasome: the end point of the journey of a protein destined for degradation. The proteasome alone is a perfect object to study the mechanisms and roles of PPI on many levels. This giant protease is built from multisubunit modules and additionally utilizes a service from transient protein ligands, for example, delivering substrates. An elaborate set of PPI within the highest-order proteasome assembly is involved in substrate recognition and processing. Below we will outline PPI involved in the UPP and discuss the growing prospects for their utilization in pharmacological interventions.
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http://dx.doi.org/10.1016/bs.apcsb.2017.09.001DOI Listing
June 2019

TGF-β signal rewiring sustains epithelial-mesenchymal transition of circulating tumor cells in prostate cancer xenograft hosts.

Oncotarget 2016 Nov;7(47):77124-77137

Departments of Molecular Medicine Cancer Research and Therapy Center and School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA.

Activation of TGF-β signaling is known to promote epithelial-mesenchymal transition (EMT) for the development of metastatic castration-resistant prostate cancer (mCRPC). To determine whether targeting TGF-β signaling alone is sufficient to mitigate mCRPC, we used the CRISPR/Cas9 genome-editing approach to generate a dominant-negative mutation of the cognate receptor TGFBRII that attenuated TGF-β signaling in mCRPC cells. As a result, the delicate balance of oncogenic homeostasis is perturbed, profoundly uncoupling proliferative and metastatic potential of TGFBRII-edited tumor xenografts. This signaling disturbance triggered feedback rewiring by enhancing ERK signaling known to promote EMT-driven metastasis. Circulating tumor cells displaying upregulated EMT genes had elevated biophysical deformity and an increase in interactions with chaperone macrophages for facilitating metastatic extravasation. Treatment with an ERK inhibitor resulted in decreased aggressive features of CRPC cells in vitro. Therefore, combined targeting of TGF-β and its backup partner ERK represents an attractive strategy for treating mCRPC patients.
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http://dx.doi.org/10.18632/oncotarget.12808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5363574PMC
November 2016

AFM Imaging Reveals Topographic Diversity of Wild Type and Z Variant Polymers of Human α1-Proteinase Inhibitor.

PLoS One 2016 23;11(3):e0151902. Epub 2016 Mar 23.

Department of Molecular Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.

α1-Proteinase inhibitor (antitrypsin) is a canonical example of the serpin family member that binds and inhibits serine proteases. The natural metastability of serpins is crucial to carry out structural rearrangements necessary for biological activity. However, the enhanced metastability of the mutant Z variant of antitrypsin, in addition to folding defect, may substantially contribute to its polymerization, a process leading to incurable serpinopathy. The metastability also impedes structural studies on the polymers. There are no crystal structures of Z monomer or any kind of polymers larger than engineered wild type (WT) trimer. Our understanding of polymerization mechanisms is based on biochemical data using in vitro generated WT oligomers and molecular simulations. Here we applied atomic force microscopy (AFM) to compare topography of monomers, in vitro formed WT oligomers, and Z type polymers isolated from transgenic mouse liver. We found the AFM images of monomers closely resembled an antitrypsin outer shell modeled after the crystal structure. We confirmed that the Z variant demonstrated higher spontaneous propensity to dimerize than WT monomers. We also detected an unexpectedly broad range of different types of polymers with periodicity and topography depending on the applied method of polymerization. Short linear oligomers of unit arrangement similar to the Z polymers were especially abundant in heat-treated WT preparations. Long linear polymers were a prominent and unique component of liver extracts. However, the liver preparations contained also multiple types of oligomers of topographies undistinguishable from those found in WT samples polymerized with heat, low pH or guanidine hydrochloride treatments. In conclusion, we established that AFM is an excellent technique to assess morphological diversity of antitrypsin polymers, which is important for etiology of serpinopathies. These data also support previous, but controversial models of in vivo polymerization showing a surprising diversity of polymer topography.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0151902PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4805282PMC
August 2016

Interplay between Structure and Charge as a Key to Allosteric Modulation of Human 20S Proteasome by the Basic Fragment of HIV-1 Tat Protein.

PLoS One 2015 17;10(11):e0143038. Epub 2015 Nov 17.

Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdańsk, Gdańsk, Poland.

The proteasome is a giant protease responsible for degradation of the majority of cytosolic proteins. Competitive inhibitors of the proteasome are used against aggressive blood cancers. However, broadening the use of proteasome-targeting drugs requires new mechanistic approaches to the enzyme's inhibition. In our previous studies we described Tat1 peptide, an allosteric inhibitor of the proteasome derived from a fragment of the basic domain of HIV-Tat1 protein. Here, we attempted to dissect the structural determinants of the proteasome inhibition by Tat1. Single- and multiple- alanine walking scans were performed. Tat1 analogs with stabilized beta-turn conformation at positions 4-5 and 8-9, pointed out by the molecular dynamics modeling and the alanine scan, were synthesized. Structure of Tat1 analogs were analyzed by circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopy studies, supplemented by molecular dynamics simulations. Biological activity tests and structural studies revealed that high flexibility and exposed positive charge are hallmarks of Tat1 peptide. Interestingly, stabilization of a beta-turn at the 8-9 position was necessary to significantly improve the inhibitory potency.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143038PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4648528PMC
June 2016

Targeting Protein-Protein Interactions in the Proteasome Super-Assemblies.

Curr Top Med Chem 2015 ;15(20):2056-67

Department of Molecular Medicine, Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, San Antonio, TX, 78245; USA.

Protein-protein interactions (PPI) are at the center of molecular mechanisms of life. The protein ligands convene for regulation of biological function: adding, enhancing or inhibiting activity, for assistance in structural integrity or to enable subsequent PPI. All these general roles of PPI are represented in the proteasome, the giant proteolytic factory universally present in human cells. The proteasome is a renowned target for anti-cancer drugs and a considered target for drugs curbing inflammation. The essential function of the proteasome, the degradation of a majority of intracellular proteins via the ubiquitin-proteasome pathway, relies on proper interactions between multiple subunits of the enzyme and between multiple modules forming distinct super-assemblies covered by the "proteasome" name. The interface regions between constitutive, alternative or transient protein components of the proteasome provide a rich platform for design of drugs with potentially very diverse actions. Still, the resource remains largely untapped since all proteasome-targeting drugs used so far in humans are classical competitive inhibitors blocking catalytic centers. In this review, we will discuss the opportunities and challenges of targeting PPI in the hub enzyme for intracellular protein catabolism, the proteasome.
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http://dx.doi.org/10.2174/1568026615666150519103206DOI Listing
April 2016

A cytosolic protein factor from the naked mole-rat activates proteasomes of other species and protects these from inhibition.

Biochim Biophys Acta 2014 Nov 10;1842(11):2060-72. Epub 2014 Jul 10.

Sam and Ann Barshop Institute for Aging and Longevity Studies, University of Texas Health Science Center at San Antonio, 15355 Lambda Dr., San Antonio, TX 78245, USA; Department of Physiology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA; Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA. Electronic address:

The naked mole-rat maintains robust proteostasis and high levels of proteasome-mediated proteolysis for most of its exceptional (~31years) life span. Here, we report that the highly active proteasome from the naked mole-rat liver resists attenuation by a diverse suite of proteasome-specific small molecule inhibitors. Moreover, mouse, human, and yeast proteasomes exposed to the proteasome-depleted, naked mole-rat cytosolic fractions, recapitulate the observed inhibition resistance, and mammalian proteasomes also show increased activity. Gel filtration coupled with mass spectrometry and atomic force microscopy indicates that these traits are supported by a protein factor that resides in the cytosol. This factor interacts with the proteasome and modulates its activity. Although Heat shock protein 72 kDa (HSP72) and Heat shock protein 40 kDa (Homolog of bacterial DNAJ1) (HSP40(Hdj1)) are among the constituents of this factor, the observed phenomenon, such as increasing peptidase activity and protecting against inhibition cannot be reconciled with any known chaperone functions. This novel function may contribute to the exceptional protein homeostasis in the naked mole-rat and allow it to successfully defy aging.
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http://dx.doi.org/10.1016/j.bbadis.2014.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4829350PMC
November 2014

Dissecting a role of a charge and conformation of Tat2 peptide in allosteric regulation of 20S proteasome.

J Pept Sci 2014 Aug 13;20(8):649-56. Epub 2014 May 13.

Department of Medicinal Chemistry, Faculty of Chemistry, University of Gdańsk, Wita Stwosza 63, 80-308, Gdańsk, Poland.

Proteasome is a 'proteolytic factory' that constitutes an essential part of the ubiquitin-proteasome pathway. The involvement of proteasome in regulation of all major aspects of cellular physiology makes it an attractive drug target. So far, only inhibitors of the proteasome entered the clinic as anti-cancer drugs. However, proteasome regulators may also be useful for treatment of inflammatory and neurodegenerative diseases. We established in our previous studies that the peptide Tat2, comprising the basic domain of HIV-1 Tat protein: R(49) KKRRQRR(56) , supplemented with Q(66) DPI(69) fragment, inhibits the 20S proteasome in a noncompetitive manner. Mechanism of Tat2 likely involves allosteric regulation because it competes with the proteasome natural 11S activator for binding to the enzyme noncatalytic subunits. In this study, we performed alanine walking coupled with biological activity measurements and FTIR and CD spectroscopy to dissect contribution of a charge and conformation of Tat2 to its capability to influence peptidase activity of the proteasome. In solution, Tat2 and most of its analogs with a single Ala substitution preferentially adopted a conformation containing PPII/turn structural motifs. Replacing either Asp10 or two or more adjacent Arg/Lys residues induced a random coil conformation, probably by disrupting ionic interactions responsible for stabilization of the peptides ordered structure. The random coil Tat2 analogs lost their capability to activate the latent 20S proteasome. In contrast, inhibitory properties of the peptides more significantly depended on their positive charge. The data provide valuable clues for the future optimization of the Tat2-based proteasome regulators.
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http://dx.doi.org/10.1002/psc.2642DOI Listing
August 2014

Harnessing proteasome dynamics and allostery in drug design.

Antioxid Redox Signal 2014 Dec 21;21(17):2286-301. Epub 2014 Feb 21.

Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio , San Antonio, Texas.

Significance: The proteasome is the essential protease that is responsible for regulated cleavage of the bulk of intracellular proteins. Its central role in cellular physiology has been exploited in therapies against aggressive cancers where proteasome-specific competitive inhibitors that block proteasome active centers are very effectively used. However, drugs regulating this essential protease are likely to have broader clinical usefulness. The non-catalytic sites of the proteasome emerge as an attractive alternative target in search of highly specific and diverse proteasome regulators.

Recent Advances: Crystallographic models of the proteasome leave the false impression of fixed structures with minimal molecular dynamics lacking long-distance allosteric signaling. However, accumulating biochemical and structural observations strongly support the notion that the proteasome is regulated by precise allosteric interactions arising from protein dynamics, encouraging the active search for allosteric regulators. Here, we discuss properties of several promising compounds that affect substrate gating and processing in antechambers, and interactions of the catalytic core with regulatory proteins.

Critical Issues: Given the structural complexity of proteasome assemblies, it is a painstaking process to better understand their allosteric regulation and molecular dynamics. Here, we discuss the challenges and achievements in this field. We place special emphasis on the role of atomic force microscopy imaging in probing the allostery and dynamics of the proteasome, and in dissecting the mechanisms involving small-molecule allosteric regulators.

Future Directions: New small-molecule allosteric regulators may become a next generation of drugs targeting the proteasome, which is critical to the development of new therapies in cancers and other diseases.
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http://dx.doi.org/10.1089/ars.2013.5816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4241894PMC
December 2014

Rapamycin allosterically inhibits the proteasome.

Mol Pharmacol 2013 Jul 25;84(1):104-13. Epub 2013 Apr 25.

University of Texas Health Science Center at San Antonio, Department of Molecular Medicine, Institute of Biotechnology, 15355 Lambda Drive, San Antonio, TX 78245, USA.

Rapamycin is a canonical allosteric inhibitor of the mammalian tarpet of rapamycin (mTOR) kinase with immunosuppressive and proapoptotic activities. We found that in vitro rapamycin also regulates the proteasome, which is an essential intracellular protease of the ubiquitin-proteasome pathway. Rapamycin inhibits proteinase and selected peptidase activities of the catalytic core proteasome at low micromolar concentrations. Moreover, the drug interferes with binding of the 19S cap essential for processing of polyubiquitinylated substrates and with the PA200 proteasome activator to the 20S catalytic core proteasome. These protein complexes are known to bind to specific grooves on the α face region of the 20S core. Treatment with rapamycin affects the conformational dynamics of the proteasomal gate, which is centrally positioned within the α face and allosterically regulated element responsible for the intake of substrates. We showed that rapamycin shares all the proteasome targeting properties not only with other two-domain, closed-ring analogs (rapalogs) but also with its single domain mimics and seco-rapamycin, which is the first in vivo open-ring metabolite of rapamycin that does not affect mTOR. We hypothesize that rapamycin and related compounds bind to the α face and allosterically impact proteasome function. This article discusses the implications of our findings for the mechanism of in vivo actions of rapamycin and for the design of novel allosteric drugs targeting the proteasome.
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http://dx.doi.org/10.1124/mol.112.083873DOI Listing
July 2013

Amyloid beta and the longest-lived rodent: the naked mole-rat as a model for natural protection from Alzheimer's disease.

Neurobiol Aging 2013 Oct 22;34(10):2352-60. Epub 2013 Apr 22.

Department of Physiology, University of Texas Health Science Center, San Antonio, TX 78229, USA.

Amyloid beta (Aβ) is implicated in Alzheimer's disease (AD) as an integral component of both neural toxicity and plaque formation. Brains of the longest-lived rodents, naked mole-rats (NMRs) approximately 32 years of age, had levels of Aβ similar to those of the 3xTg-AD mouse model of AD. Interestingly, there was no evidence of extracellular plaques, nor was there an age-related increase in Aβ levels in the individuals examined (2-20+ years). The NMR Aβ peptide showed greater homology to the human sequence than to the mouse sequence, differing by only 1 amino acid from the former. This subtle difference led to interspecies differences in aggregation propensity but not neurotoxicity; NMR Aβ was less prone to aggregation than human Aβ. Nevertheless, both NMR and human Aβ were equally toxic to mouse hippocampal neurons, suggesting that Aβ neurotoxicity and aggregation properties were not coupled. Understanding how NMRs acquire and tolerate high levels of Aβ with no plaque formation could provide useful insights into AD, and may elucidate protective mechanisms that delay AD progression.
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http://dx.doi.org/10.1016/j.neurobiolaging.2013.03.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4496028PMC
October 2013

The proteasome in health and disease.

Curr Pharm Des 2013 ;19(6):1010-28

Faculty of Chemistry, University of Gdansk, Sobieskiego 18, Gdansk 80-952, Poland.

The giant proteolytic factory called the proteasome came a long way from a biochemical curio to a major regulator of cellular physiology and a renowned drug target within the ubiquitin proteasome pathway (UPP). Thanks to availability of highly specific inhibitors of the proteasome, in less than twenty years it was possible to identify major transcription factors, cyclins, and products of oncogenes as crucial substrates for the UPP. Nine years passed since the FDA speedily approved bortezomib, the inhibitor of proteasome, for treatment of multiple myeloma. One year after its approval, the field was honored by awarding the Nobel Prize to Hershko, Ciechanover and Rose for introducing the concept of controlled proteolysis of ubiquitin-tagged substrates, with proteasome as the intracellular recycling facility. Taking into consideration the universal involvement of the proteasome in the life of all cells in human body, it comes to no surprise that the enzyme is deeply implicated in etiology, progression, diagnosis or cure of multiple diseases. Below we discuss some aspects of the involvement: from direct causative links to changes in proteasome properties that correlate with pathological conditions. We start with diseases collectively known as cancer, and with immune system-related pathologies. Here, the proteasome inhibitors are either already used in clinics, or undergo advanced preclinical screening. Then, we will continue with cardiovascular disorders, followed by aging. Changes of the proteasome make-up during aging may be a priming factor for neurodegenerative diseases, described last. We discuss the potential for proteasome regulation: inhibition, activation or specificity modulation, to successfully enter the clinical setting.
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July 2013

The growth-suppressive function of the polycomb group protein polyhomeotic is mediated by polymerization of its sterile alpha motif (SAM) domain.

J Biol Chem 2012 Mar 24;287(12):8702-13. Epub 2012 Jan 24.

Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3990, USA.

Polyhomeotic (Ph), a member of the Polycomb Group (PcG), is a gene silencer critical for proper development. We present a previously unrecognized way of controlling Ph function through modulation of its sterile alpha motif (SAM) polymerization leading to the identification of a novel target for tuning the activities of proteins. SAM domain containing proteins have been shown to require SAM polymerization for proper function. However, the role of the Ph SAM polymer in PcG-mediated gene silencing was uncertain. Here, we first show that Ph SAM polymerization is indeed required for its gene silencing function. Interestingly, the unstructured linker sequence N-terminal to Ph SAM can shorten the length of polymers compared with when Ph SAM is individually isolated. Substituting the native linker with a random, unstructured sequence (RLink) can still limit polymerization, but not as well as the native linker. Consequently, the increased polymeric Ph RLink exhibits better gene silencing ability. In the Drosophila wing disc, Ph RLink expression suppresses growth compared with no effect for wild-type Ph, and opposite to the overgrowth phenotype observed for polymer-deficient Ph mutants. These data provide the first demonstration that the inherent activity of a protein containing a polymeric SAM can be enhanced by increasing SAM polymerization. Because the SAM linker had not been previously considered important for the function of SAM-containing proteins, our finding opens numerous opportunities to manipulate linker sequences of hundreds of polymeric SAM proteins to regulate a diverse array of intracellular functions.
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http://dx.doi.org/10.1074/jbc.M111.336115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308824PMC
March 2012

Atomic force microscopy of proteasome assemblies.

Methods Mol Biol 2011 ;736:117-32

Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.

The proteasome is the essential prime protease in all eukaryotes. The large, multisubunit, modular, and multifunctional enzyme is responsible for the majority of regulated intracellular protein degradation. It constitutes a part of the multienzyme ubiquitin-proteasome pathway, which is broadly implicated in recognition, tagging, and cleavage of proteins. The name "proteasome" refers to several types of protein assemblies sharing a common catalytic core particle. Additional protein modules attach to the core, regulate its activities, and broaden its functional capabilities. The structure of proteasomes has been studied extensively with multiple methods. The crystal structure of the core particle was solved for several species. However, only a single structure of the core particle decorated with PA26 activator has been determined. NMR spectroscopy was successfully applied to probe a much -simpler, archaebacterial type of the core particle. In turn, electron microscopy was very effective in exploring the spatial arrangement of many classes of assemblies. Still, the makeup of higher-order -complexes is not well established. Besides, the crystal structure provided very limited information on proteasome molecular dynamics. Atomic force microscopy (AFM) is an ideal technique to address questions that are unanswered by other approaches. For example, AFM is perfectly suited to study allosteric regulation of proteasome, the role of protein dynamics in enzymatic catalysis, and the spatial organization of modules and subunits in assemblies. Here, we present a method that probes the conformational diversity and dynamics of yeast core particle using the oscillating mode AFM in liquid. We are taking advantage of the observation that the tube-shaped core particle is equipped with a swinging gate leading to the catalytic chamber. We demonstrate how to identify distinct gate conformations in AFM images and how to characterize the gate dynamics controlled with ligands and disturbed by mutations.
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http://dx.doi.org/10.1007/978-1-61779-105-5_9DOI Listing
October 2011

Molecular mechanisms of proteasome plasticity in aging.

Mech Ageing Dev 2010 Feb 18;131(2):144-55. Epub 2010 Jan 18.

Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center, 15355 Lambda Drive, San Antonio, TX 78245, United States of America.

The ubiquitin-proteasome pathway plays a crucial role in regulation of intracellular protein turnover. Proteasome, the central protease of the pathway, encompasses multi-subunit assemblies sharing a common catalytic core supplemented by regulatory modules and localizing to different subcellular compartments. To better comprehend age-related functions of the proteasome we surveyed content, composition and catalytic properties of the enzyme in cytosolic, microsomal and nuclear fractions obtained from mouse livers subjected to organismal aging. We found that during aging subunit composition and subcellular distribution of proteasomes changed without substantial alterations in the total level of core complexes. We observed that the general decline in proteasomes functions was limited to nuclear and cytosolic compartments. Surprisingly, the observed changes in activity and specificity were linked to the amount of the activator module and distinct composition of the catalytic subunits. In contrast, activity, specificity and composition of the microsomal-associated proteasomes remained mostly unaffected by aging; however their relative contribution to the total activity was substantially elevated. Unexpectedly, the nuclear proteasomes were affected most profoundly by aging possibly triggering significant changes in cellular signaling and transcription. Collectively, the data indicate an age-related refocusing of proteasome from the compartment-specific functions towards general protein maintenance.
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http://dx.doi.org/10.1016/j.mad.2010.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2849732PMC
February 2010

AFM of biological complexes: what can we learn?

Curr Opin Colloid Interface Sci 2008 Oct;13(5):351-367

Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX, 78245.

The term "biological complexes" broadly encompasses particles as diverse as multisubunit enzymes, viral capsids, transport cages, molecular nets, ribosomes, nucleosomes, biological membrane components and amyloids. The complexes represent a broad range of stability and composition. Atomic force microscopy offers a wealth of structural and functional data about such assemblies. For this review, we choose to comment on the significance of AFM to study various aspects of biology of selected nonmembrane protein assemblies. Such particles are large enough to reveal many structural details under the AFM probe. Importantly, the specific advantages of the method allow for gathering dynamic information about their formation, stability or allosteric structural changes critical for their function. Some of them have already found their way to nanomedical or nanotechnological applications. Here we present examples of studies where the AFM provided pioneering information about the biology of complexes, and examples of studies where the simplicity of the method is used toward the development of potential diagnostic applications.
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http://dx.doi.org/10.1016/j.cocis.2008.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2630216PMC
October 2008

A tetrahedral transition state at the active sites of the 20S proteasome is coupled to opening of the alpha-ring channel.

Structure 2009 Aug;17(8):1137-47

Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78245, USA.

Intrinsic conformational transitions contribute to the catalytic action of many enzymes. Here we use a single-molecule approach to demonstrate how such transitions are linked to the catalytic sites of the eukaryotic proteasome, an essential protease of the ubiquitin pathway. The active sites of the cylindrical proteasomal core particle are located in a central chamber accessible through gated entry channels. By using atomic force microscopy, we found continual alternation between open and closed gate conformations. We analyzed the relative abundance of these conformers in wild-type and mutated yeast core particles upon exposure to substrates or inhibitors. Our data indicate that the dynamic gate can be opened by allosteric coupling to a tetrahedral transition state at any of the working active centers. The results point to the N(alpha)-amine of the N-terminal active site threonyl residue as the major effector group responsible for triggering the essential conformational switch.
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http://dx.doi.org/10.1016/j.str.2009.06.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2746003PMC
August 2009

Atomic force microscopy as a tool to study the proteasome assemblies.

Methods Cell Biol 2008 ;90:39-60

Department of Molecular Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245, USA.

Proteasome is an exceptional enzyme because of its essential physiological role, multiple activities, and structural complexity. It is, in fact, a family of enzymes sharing a common catalytic core and equipped with distinct protein attachments regulating the core and adding to its new functional capabilities. As a drug target and a major regulator of cellular processes, proteasome is extensively studied with tools of structural, biochemical, and molecular biology. Atomic force microscopy (AFM) besides X-ray crystallography and electron microscopy is one of the most attractive methods to study proteasome. The noninvasive nature of this method is particularly well suited for investigating the structure-function relationship within the core particle (CP) as well as in higher-order assemblies. Here we review, from the methodological point of view, AFM-based studies on the proteasome. First, we will present the application of height distribution analysis of proteasome complexes to dissect the subunit organization in the base of the regulatory particle (RP). The RP is considered the most physiologically important among all the attachments of the CP; however, its structure remains enigmatic. Then, we will outline the use of AFM imaging to research on structural dynamics of the proteasome, a phenomenon which is starting to gain a broad interest. We will finish with a brief presentation of nanotechnological studies performed using ordered proteasomes and nanolithography carried out with the particles. The presented AFM research offers a unique and often unexpected insight into the structure and function of the proteasome.
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http://dx.doi.org/10.1016/S0091-679X(08)00803-0DOI Listing
March 2009

The central unit within the 19S regulatory particle of the proteasome.

Nat Struct Mol Biol 2008 Jun 30;15(6):573-80. Epub 2008 May 30.

Department of Biology, Technion-Israel Institute of Technology, 32000 Haifa, Israel.

The 26S proteasome is a multisubunit enzyme composed of a cylindrical catalytic core (20S) and a regulatory particle (19S) that together perform the essential degradation of cellular proteins tagged by ubiquitin. To date, however, substrate trajectory within the complex remains elusive. Here we describe a previously unknown functional unit within the 19S, comprising two subunits, Rpn1 and Rpn2. These toroids physically link the site of substrate recruitment with the site of proteolysis. Rpn2 interfaces with the 20S, whereas Rpn1 sits atop Rpn2, serving as a docking site for a substrate-recruitment factor. The 19S ATPases encircle the Rpn1-Rpn2 stack, covering the remainder of the 20S surface. Both Rpn1-Rpn2 and the ATPases are required for substrate translocation and gating of the proteolytic channel. Similar pairing of units is found in unfoldases and nuclear transporters, exposing common features of these protein nanomachines.
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http://dx.doi.org/10.1038/nsmb.1427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2481239PMC
June 2008

Allosteric regulators of the proteasome: potential drugs and a novel approach for drug design.

Curr Med Chem 2006 ;13(2):155-65

Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245, USA.

The proteasome recently gained an exceptional attention as a novel drug target, therefore its inhibitors became important subjects for rational drug design. A synthetic competitive inhibitor Velcade was lately approved in a fast-track process to treat multiple myeloma and is tested with other types of cancers. The proteasome is a major proteolytic assembly in eukaryotic cells responsible for the degradation of most intracellular proteins, including proteins crucial to cell cycle regulation and apoptosis. The ubiquitin-proteasome pathway has been implicated in many diseases such as cancer, autoimmune diseases, inflammation, and stroke. The activity of the proteasome can be blocked for therapeutic purposes with competitive inhibitors like Velcade, which trigger apoptosis in target cells. However, much more versatile outcomes and a true control of the proteasome can be achieved with allosteric regulators. Certain natural proteins and peptides bind to the catalytic core of the proteasome and allosterically induce a wide array of effects ranging from changes in product size to substrate-specific inhibition. Designing small synthetic compounds allosterically interacting with the proteasome represents a novel approach that has enormous potential for the treatment of a wide range of diseases. Below we provide a review of current knowledge about proteasomal allosteric ligands.
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http://dx.doi.org/10.2174/092986706775197926DOI Listing
April 2006

Highbrow proteasome in high-throughput technology.

Expert Rev Proteomics 2006 Feb;3(1):115-27

Institute of Biotechnology, Department of Molecular Medicine, The University of Texas Health Science Center, San Antonio, TX 78245, USA.

Proteasome is a major protease of the ubiquitin-proteasome pathway involved in the regulation of practically all intracellular biochemical processes. The enzyme core is created by a heteromultimer of complex architecture built with multiple subunits arranged into a tube-like structure. The multiple active sites of diverse peptidase specificity are hidden inside the tube. Access to the interior is guarded by a gate formed by the N-termini of specialized subunits and by the attachment of additional multisubunit protein complexes controlling the enzymatic capabilities of the core. Proteasome, due to its Byzantine molecular architecture and equally sophisticated enzymatic mechanism, is by itself a fascinating biophysical object. Recently, the position of the protease advanced from an academically remarkable protein processor to a providential anticancer drug target and futuristic nanomachine. Proteomics studies actively shape our current understanding of the protease and direct the future applications of the proteasome in medicine.
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http://dx.doi.org/10.1586/14789450.3.1.115DOI Listing
February 2006

Characterization of noncompetitive regulators of proteasome activity.

Methods Enzymol 2005 ;398:425-38

Department of Molecular Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX 78245-3207, USA.

The success of bortezomib, a competitive proteasome inhibitor and a drug approved to treat multiple myeloma, spurred interest in compounds targeting catalytic sites of the enzyme. The aim of this chapter, however, is to focus attention on the small molecule, natural or synthetic compounds binding far away from the catalytic centers, yet modifying the performance of the proteasome. Defining allostery broadly as any kind of ligand-induced, long-distance transfer of conformational signals within a molecule, most such compounds are allosteric effectors capable of regulating the proteasome in vitro and in vivo in a manner more diverse and precise than competitive inhibitors. Proline- and arginine-rich peptides (PR peptides) are examples of such compounds and are currently being considered as potential drugs with anti-inflammatory and proangiogenic activities. This chapter describes a set of methods useful for characterizing the effects of such inhibitors on the proteasome.
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http://dx.doi.org/10.1016/S0076-6879(05)98035-XDOI Listing
December 2005

Atomic force microscopy of the proteasome.

Methods Enzymol 2005 ;398:414-25

Department of Molecular Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX 78245-3207, USA.

The proteasome should be an ideal molecule for studies on large enzymatic complexes, given its multisubunit and modular structure, compartmentalized design, numerous activities, and its own means of regulation. Considering the recent increased interest in the ubiquitin-proteasome pathway, it is surprising that biophysical approaches to study this enzymatic assembly are applied with limited frequency. Methods including atomic force microscopy, fluorescence spectroscopy, surface plasmon resonance, and high-pressure procedures all have gained popularity in characterization of the proteasome. These methods provide significant and often unexpected insight regarding the structure and function of the enzyme. This chapter describes the use of atomic force microscopy for dynamic structural studies of the proteasome.
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http://dx.doi.org/10.1016/S0076-6879(05)98034-8DOI Listing
December 2005

Small-molecule inhibitors of proteasome activity.

Methods Mol Biol 2005 ;301:3-22

Institute of Biotechnology, University of Texas Health Science Center at San Antonio, USA.

The fast-track approval of a proteasome inhibitor, PS-341, to treat multiple myeloma spurred a wave of interest in both the proteasome itself and small-molecule compounds blocking its activities. Besides being candidates for drugs against cancer, autoimmune diseases, inflammation, or stroke, specific proteasome inhibitors are indispensable tools for biochemical and cell biology investigations of the proteasome and proteasome-ubiquitin system. Numerous synthetic peptide derivatives, such as boronates, epoxides, aldehydes, vinyl sulfones, cyclic peptides, and lactones, block the N-terminal threonine-type active centers of the enzyme, halting the cleavage of proteasomal protein substrates both in vitro and in vivo. Because some of the proteasomal inhibitors exhibit a high specificity toward only one particular type of an active center of the proteasome, they constitute valuable probes for testing the mechanism of proteolysis catalyzed by the enzyme. In this chapter we discuss the most common applications of available proteasome inhibitors. In addition to the best-known competitive inhibitors, we also describe the benefits from the use of allosteric inhibitors, which induce distinct but less understood in vitro and in vivo effects on the proteasomal machinery. Finally, we present the application of the basic biochemical procedures to decipher the mechanism of interactions of a novel compound with the proteasome.
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http://dx.doi.org/10.1385/1-59259-895-1:003DOI Listing
June 2005

Atomic force microscopic analysis of the binding of the Schizosaccharomyces pombe origin recognition complex and the spOrc4 protein with origin DNA.

Proc Natl Acad Sci U S A 2004 Dec 14;101(52):17952-7. Epub 2004 Dec 14.

Institute of Biotechnology, University of Texas Health Science Center, 15355 Lambda Drive, San Antonio, TX 78245, USA.

In eukaryotes, the initiation of DNA replication requires the interaction between origin sequences and the origin recognition complex (ORC), which is highly conserved. In this report, atomic force microscopy (AFM) was used to examine the binding of Schizosaccharomyces pombe (sp) ORC and the spOrc4 protein with the sp autonomously replicating sequence 1 (ars1). AFM imaging revealed that spORC binding to ars1 occurred solely through spOrc4p and depended on the N-terminal AT-hook domains present in spOrc4p. At high molar ratios of spORC (or spOrc4p alone) to DNA (6:1), all of the input ars1 was bound in a one protein complex to one plasmid manner. Restriction digestion and AFM analysis of protein-DNA fragments revealed the presence of two binding sites in ars1. One site mapped to a region centered at nucleotide 838 of ars1 previously detected by DNase I protection that was reported to be essential for the autonomously replicating sequence activity of ars1. The second site mapped to a previously uncharacterized region centered at nucleotide 1148. AFM showed that the length of the DNA fragment complexed with either spORC or spOrc4p was shortened by approximately 140 bp, suggesting the wrapping of two turns of the DNA around the spOrc4p alone as well as the spOrc4p in spORC. We also show that treatment of the spORC (spOrc4p)-ars1 complex with topoisomerase I induced a negative shift in the topoisomer distribution. These findings suggest that the binding of spORC to origin DNA alters the structure of the DNA. Thus, in the case of spORC, due to its unusual spOrc4p, at least two factors are likely to influence ars1 activation. These include the selective binding of the complex to A- and T-rich regions and the alteration of the DNA structure due to its wrapping around spOrc4p.
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http://dx.doi.org/10.1073/pnas.0408369102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC539809PMC
December 2004

DNA-looping by RXR tetramers permits transcriptional regulation "at a distance".

J Mol Biol 2004 Oct;343(2):327-38

Division of Nutritional Sciences, Savage Hall, Cornell University, Ithaca, NY 14853, USA.

RXR, a member of the superfamily of nuclear hormone receptors, regulates gene transcription in response to 9-cis-retinoic acid. We previously showed that, among nuclear receptors, RXR is unique in that it self-associates into homotetramers, and that these tetramers dissociate rapidly upon ligation. Here, we report that binding of RXR tetramers to DNA containing two RXR response elements results in a dramatic DNA-looping. RXR can thus juxtapose distant DNA sequences, enabling transcriptional regulation by far-upstream factors. We show that RXR functions as a DNA architectural factor and that, while this activity is regulated by 9-cis-retinoic acid, it is distinct from and independent of the receptor's intrinsic transcriptional activity. The data establish RXR as the first identified architectural factor whose activity is regulated by a small ligand, and demonstrate a novel mechanism of transcriptional regulation by retinoids.
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http://dx.doi.org/10.1016/j.jmb.2004.08.070DOI Listing
October 2004