Publications by authors named "Maria Gaczynska"

43 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c9ob00122kDOI Listing
March 2019

Proline- and Arginine-Rich Peptides as Flexible Allosteric Modulators of Human Proteasome Activity.

J Med Chem 2019 01 3;62(1):359-370. Epub 2018 Dec 3.

Department of Biomedical Chemistry, Faculty of Chemistry , University of Gdansk , Wita Stwosza 63 , 80-308 Gdansk , Poland.

Proline- and arginine-rich peptide PR11 is an allosteric inhibitor of 20S proteasome. We modified its sequence inter alia by introducing HbYX, RYX, or RHbX C-terminal extensions (Hb, hydrophobic moiety; R, arginine; Y, tyrosine; X, any residue). Consequently, we were able to improve inhibitory potency or to convert inhibitors into strong activators: the former with an aromatic penultimate Hb residue and the latter with the HbYX motif. The PR peptide activator stimulated 20S proteasome in vitro to efficiently degrade protein substrates, such as α-synuclein and enolase, but also activated proteasome in cultured fibroblasts. The positive and negative PR modulators differently influenced the proteasome conformational dynamics and affected opening of the substrate entry pore. The resolved crystal structure showed PR inhibitor bound far from the active sites, at the proteasome outer face, in the pocket used by natural activators. Our studies indicate the opportunity to tune proteasome activity by allosteric regulators based on PR peptide scaffold.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.8b01025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796967PMC
January 2019

Adipokines Deregulate Cellular Communication via Epigenetic Repression of Loci in Obese Endometrial Cancer.

Cancer Res 2019 01 2;79(1):196-208. Epub 2018 Nov 2.

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

Emerging evidence indicates that adipose stromal cells (ASC) are recruited to enhance cancer development. In this study, we examined the role these adipocyte progenitors play relating to intercellular communication in obesity-associated endometrial cancer. This is particularly relevant given that gap junctions have been implicated in tumor suppression. Examining the effects of ASCs on the transcriptome of endometrial epithelial cells (EEC) in an coculture system revealed transcriptional repression of (encoding the gap junction protein Cx43) and other genes related to intercellular communication. This repression was recapitulated in an obesity mouse model of endometrial cancer. Furthermore, inhibition of plasminogen activator inhibitor 1 (PAI-1), which was the most abundant ASC adipokine, led to reversal of cellular distribution associated with the repression profile, suggesting that PAI-1 may mediate actions of ASC on transcriptional regulation in EEC. In an endometrial cancer cohort ( = 141), DNA hypermethylation of and related loci and was observed in primary endometrial endometrioid tumors and was associated with obesity. Pharmacologic reversal of DNA methylation enhanced gap-junction intercellular communication and cell-cell interactions . Restoring Cx43 expression in endometrial cancer cells reduced cellular migration; conversely, depletion of Cx43 increased cell migration in immortalized normal EEC. Our data suggest that persistent repression by ASC adipokines leads to promoter hypermethylation of and related genes in the endometrium, triggering long-term silencing of these loci in endometrial tumors of obese patients. SIGNIFICANCE: Studies reveal that adipose-derived stem cells in endometrial cancer pathogenesis influence epigenetic repression of gap junction loci, which suggests targeting of gap junction activity as a preventive strategy for obesity-associated endometrial cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-18-1615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6705596PMC
January 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/bs.apcsb.2017.09.001DOI Listing
June 2019

Sustained NFκB inhibition improves insulin sensitivity but is detrimental to muscle health.

Aging Cell 2017 08 29;16(4):847-858. Epub 2017 May 29.

Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX, 78245, USA.

Older adults universally suffer from sarcopenia and approximately 60-70% are diabetic or prediabetic. Nonetheless, the mechanisms underlying these aging-related metabolic disorders are unknown. NFκB has been implicated in the pathogenesis of several aging-related pathologies including sarcopenia and type 2 diabetes and has been proposed as a target against them. NFκB also is thought to mediate muscle wasting seen with disuse, denervation, and some systemic diseases (e.g., cancer, sepsis). We tested the hypothesis that lifelong inhibition of the classical NFκB pathway would protect against aging-related sarcopenia and insulin resistance. Aged mice with muscle-specific overexpression of a super-repressor IκBα mutant (MISR) were protected from insulin resistance. However, MISR mice were not protected from sarcopenia; to the contrary, these mice had decreases in muscle mass and strength compared to wild-type mice. In MISR mice, NFκB suppression also led to an increase in proteasome activity and alterations in several genes and pathways involved in muscle growth and atrophy (e.g., myostatin). We conclude that the mechanism behind aging-induced sarcopenia is NFκB independent and differs from muscle wasting due to pathologic conditions. Our findings also indicate that, while suppressing NFκB improves insulin sensitivity in aged mice, this transcription factor is important for normal muscle mass maintenance and its sustained inhibition is detrimental to muscle function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/acel.12613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5506420PMC
August 2017

Cholesterol Regulates Monocyte Rolling through CD44 Distribution.

Biophys J 2017 Apr;112(7):1481-1488

Department of Biomedical Engineering, The University of Texas at San Antonio, San Antonio, Texas; Department of Biomedical, Chemical and Materials Engineering, San José State University, San José, California. Electronic address:

Cholesterol is an important risk factor of atherosclerosis, due to its active uptake by monocytes/macrophages. Monocyte recruitment from flowing blood to atherosclerotic foci is the key first step in the development of atherosclerosis. Cholesterol content alters cell membrane stiffness, and lateral lipid and protein diffusion. We hypothesized that cholesterol content will modulate the recruitment of monocytes to inflamed endothelial surface by altering the dynamics of adhesion receptors. We depleted or enriched the cellular cholesterol levels using methyl-β-cyclodextran in freshly isolated human monocytes. We investigated the effect of these changes on the mechanics of monocyte rolling on E-selectin surfaces at 1 dyn/cm in microchannels. Using imaging flow cytometry and atomic force microscopy, we characterized the distribution of lipid rafts and the E-selectin counterreceptor CD44 on the monocyte surface. We observed that lower levels of cholesterol resulted in the uniform, CD44-mediated rolling of monocytes on the E-selectin-coated surfaces. We also observed that cells depleted of cholesterol had higher membrane fluidity, and more uniform distribution of CD44 counterreceptor, which resulted in smooth motion of the cells compared to cells enriched with cholesterol. This work demonstrates that cholesterol can modulate monocyte adhesion by regulating the receptor mobility, and our results provide insights into the biophysical regulation of inflammation for the better understanding of diseases like atherosclerosis and hypercholesterolemia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2017.02.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389964PMC
April 2017

EGFR-Dependent Regulated Intramembrane Proteolysis of EpCAM-Response.

Cancer Res 2017 04 22;77(7):1777. Epub 2017 Mar 22.

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

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-16-3440DOI Listing
April 2017

Cellular cholesterol regulates monocyte deformation.

J Biomech 2017 02 30;52:83-88. Epub 2016 Dec 30.

Department of Biomedical, Chemical and Materials Engineering, San José State University, San José, CA, United States. Electronic address:

The role of cholesterol content on monocyte biomechanics remains understudied despite the well-established link between cholesterol and monocytes/macrophages in atherosclerosis, and the effect on other cell types. In this work, we have investigated the effect of cholesterol on monocyte deformability and the underlying molecular mechanisms. We altered the baseline cholesterol in human monocytic cell line THP-1, and investigated the changes in monocyte deformability using a custom microfluidic platform and atomic force microscopy. We observed that the cholesterol depletion lowered deformability while enrichment increased deformability compared to untreated cells. As a consequence of altered deformability, cholesterol depleted cells spread more on collagen-coated surfaces with elongated morphology, whereas cholesterol enriched cells had a more rounded morphology. We observed that the decreased deformability in cholesterol depleted cells, despite an increase in the fluidity of the membrane, is due to an increase in phosphorylation of Protein Kinase C (PKC), which translates to a higher degree of actin polymerization. Together, our results highlight the importance of biophysical regulation of monocyte response to cholesterol levels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jbiomech.2016.12.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736503PMC
February 2017

EpCAM-Regulated Transcription Exerts Influences on Nanomechanical Properties of Endometrial Cancer Cells That Promote Epithelial-to-Mesenchymal Transition.

Cancer Res 2016 11 28;76(21):6171-6182. Epub 2016 Aug 28.

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

Overexpression of epithelial cell adhesion molecule (EpCAM) has been implicated in advanced endometrial cancer, but its roles in this progression remain to be elucidated. In addition to its structural role in modulating cell-surface adhesion, here we demonstrate that EpCAM is a regulatory molecule in which its internalization into the nucleus turns on a transcription program. Activation of EGF/EGFR signal transduction triggered cell-surface cleavage of EpCAM, leading to nuclear internalization of its cytoplasmic domain EpICD. ChIP-seq analysis identified target genes that are coregulated by EpICD and its transcription partner, LEF-1. Network enrichment analysis further uncovered a group of 105 genes encoding functions for tight junction, adherent, and cell migration. Furthermore, nanomechanical analysis by atomic force microscopy revealed increased softness and decreased adhesiveness of EGF-stimulated cancer cells, implicating acquisition of an epithelial-mesenchymal transition (EMT) phenotype. Thus, genome editing of EpCAM could be associated with altering these nanomechanical properties towards a less aggressive phenotype. Using this integrative genomic-biophysical approach, we demonstrate for the first time an intricate relationship between EpCAM-regulated transcription and altered biophysical properties of cells that promote EMT in advanced endometrial cancer. Cancer Res; 76(21); 6171-82. ©2016 AACR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-16-0752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5405773PMC
November 2016

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/1568026615666150519103206DOI Listing
April 2016

Nanomechanical biomarkers of single circulating tumor cells for detection of castration resistant prostate cancer.

Prostate 2014 Sep 25;74(13):1297-307. Epub 2014 Jul 25.

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

Background: Emerging evidence shows that nanomechanical phenotypes of circulating tumor cells (CTC) could become potential biomarkers for metastatic castration resistant prostate cancer (mCRPC).

Methods: To determine the nanomechanical phenotypes of CTCs we applied atomic force microscopy (AFM) employing the PeakForce quantitative nanomechanical (QNM) imaging. We assessed biophysical parameters (elasticity, deformation, and adhesion) of 130 CTCs isolated from blood samples from five castration sensitive (CS) and 12 castration resistant prostate cancer (CRPCa) patients.

Results: We found that CTCs from CRPCa patients are three times softer, three times more deformable, and seven times more adhesive than counterparts from CSPCa patients. Both nonsupervised hierarchical clustering and principle component analysis show that three combined nanomechanical parameters could constitute a valuable set to distinguish between CSPCa and CRPCa.

Conclusions: [corrected] Our study indicates that nanomechanical phenotypes of CTCs may serve as novel and effective biomarkers for mCRPC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pros.22846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4142568PMC
September 2014

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neurobiolaging.2013.03.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4496028PMC
October 2013

Single-cell analysis of circulating tumor cells identifies cumulative expression patterns of EMT-related genes in metastatic prostate cancer.

Prostate 2013 Jun 31;73(8):813-26. Epub 2012 Dec 31.

Department of Molecular Medicine, Cancer Therapy and Research Center, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.

Background: Prostate tumors shed circulating tumor cells (CTCs) into the blood stream. Increased evidence shows that CTCs are often present in metastatic prostate cancer and can be alternative sources for disease profiling and prognostication. Here we postulate that CTCs expressing genes related to epithelial-mesenchymal transition (EMT) are strong predictors of metastatic prostate cancer.

Methods: A microfiltration system was used to trap CTCs from peripheral blood based on size selection of large epithelial-like cells without CD45 leukocyte marker. These cells individually retrieved with a micromanipulator device were assessed for cell membrane physical properties using atomic force microscopy. Additionally, 38 CTCs from eight prostate cancer patients were used to determine expression profiles of 84 EMT-related and reference genes using a microfluidics-based PCR system.

Results: Increased cell elasticity and membrane smoothness were found in CTCs compared to noncancerous cells, highlighting their potential invasiveness and mobility in the peripheral circulation. Despite heterogeneous expression patterns of individual CTCs, genes that promote mesenchymal transitioning into a more malignant state, including IGF1, IGF2, EGFR, FOXP3, and TGFB3, were commonly observed in these cells. An additional subset of EMT-related genes (e.g., PTPRN2, ALDH1, ESR2, and WNT5A) were expressed in CTCs of castration-resistant cancer, but less frequently in castration-sensitive cancer.

Conclusions: The study suggests that an incremental expression of EMT-related genes in CTCs is associated with metastatic castration-resistant cancer. Although CTCs represent a group of highly heterogeneous cells, their unique EMT-related gene signatures provide a new opportunity for personalized treatments with targeted inhibitors in advanced prostate cancer patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pros.22625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882087PMC
June 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
July 2013

Altered composition of liver proteasome assemblies contributes to enhanced proteasome activity in the exceptionally long-lived naked mole-rat.

PLoS One 2012 2;7(5):e35890. Epub 2012 May 2.

Sam and Ann Barshop Institute for Aging and Longevity Studies, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.

The longest-lived rodent, the naked mole-rat (Bathyergidae; Heterocephalus glaber), maintains robust health for at least 75% of its 32 year lifespan, suggesting that the decline in genomic integrity or protein homeostasis routinely observed during aging, is either attenuated or delayed in this extraordinarily long-lived species. The ubiquitin proteasome system (UPS) plays an integral role in protein homeostasis by degrading oxidatively-damaged and misfolded proteins. In this study, we examined proteasome activity in naked mole-rats and mice in whole liver lysates as well as three subcellular fractions to probe the mechanisms behind the apparently enhanced effectiveness of UPS. We found that when compared with mouse samples, naked mole-rats had significantly higher chymotrypsin-like (ChT-L) activity and a two-fold increase in trypsin-like (T-L) in both whole lysates as well as cytosolic fractions. Native gel electrophoresis of the whole tissue lysates showed that the 20S proteasome was more active in the longer-lived species and that 26S proteasome was both more active and more populous. Western blot analyses revealed that both 19S subunits and immunoproteasome catalytic subunits are present in greater amounts in the naked mole-rat suggesting that the observed higher specific activity may be due to the greater proportion of immunoproteasomes in livers of healthy young adults. It thus appears that proteasomes in this species are primed for the efficient removal of stress-damaged proteins. Further characterization of the naked mole-rat proteasome and its regulation could lead to important insights on how the cells in these animals handle increased stress and protein damage to maintain a longer health in their tissues and ultimately a longer life.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035890PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3342291PMC
September 2012

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-61779-105-5_9DOI Listing
October 2011

Phosphorylation by Nek1 regulates opening and closing of voltage dependent anion channel 1.

Biochem Biophys Res Commun 2010 Apr 15;394(3):798-803. Epub 2010 Mar 15.

Department of Medicine, Division of Endocrinology, University of California, Irvine, USA.

VDAC1 is a key component of the mitochondrial permeability transition pore. To initiate apoptosis and certain other forms of cell death, mitochondria become permeable such that cytochrome c and other pre-apoptotic molecules resident inside the mitochondria enter the cytosol and activate apoptotic cascades. We have shown recently that VDAC1 interacts directly with never-in-mitosis A related kinase 1 (Nek1), and that Nek1 phosphorylates VDAC1 on Ser193 to prevent excessive cell death after injury. How this phosphorylation regulates the activity of VDAC1, however, has not yet been reported. Here, we use atomic force microscopy (AFM) and cytochrome c conductance studies to examine the configuration of VDAC1 before and after phosphorylation by Nek1. Wild-type VDAC1 assumes an open configuration, but closes and prevents cytochrome c efflux when phosphorylated by Nek1. A VDAC1-Ser193Ala mutant, which cannot be phosphorylated by Nek1 under identical conditions, remains open and constitutively allows cytochrome c efflux. Conversely, a VDAC1-Ser193Glu mutant, which mimics constitutive phosphorylation by Nek1, remains closed by AFM and prevents cytochrome c leakage in the same liposome assays. Our data provide a mechanism to explain how Nek1 regulates cell death by affecting the opening and closing of VDAC1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2010.03.077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2859727PMC
April 2010

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.mad.2010.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2849732PMC
February 2010