Protein degradation analysis by affinity microfluidics

Protein degradation mediated by the ubiquitin-proteasome pathway regulates signaling events in all eukaryotic cells, with implications in pathological conditions such as cancer and neurodegenerative diseases. Detection of protein degradation is an elementary need in basic and translational research. In vitro degradation assays, in particular, have been instrumental in the understanding of how cell proliferation and other fundamental cellular processes are regulated. These assays are direct, quantitative and highly informative but also laborious, typically relying on low-throughput polyacrylamide gel-electrophoresis followed by autoradiography or immunoblotting. We present protein degradation on chip (pDOC), a MITOMI-based integrated microfluidic device for discovery and analysis of ubiquitin mediated proteolysis. The platform accommodates microchambers on which protein degradation is assayed quickly and simultaneously in physiologically relevant environments, using minute amount of reagents. Essentially, pDOC provides a multiplexed, sensitive and colorimetric alternative to the conventional degradation assays, with relevance to biomedical and translational research.

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Abstract 37: Centrosome Destabilization Through the Ubiquitin-Proteasome Pathway Regulates Proplatelet Production

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.37 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Kellie R Machlus ◽

Prakrith Vijey ◽

Thomas Soussou ◽

Joseph E Italiano

Keyword(s):

Protein Degradation ◽

Proteasome Inhibitors ◽

Aurora Kinase ◽

Proteasome Activity ◽

Major Pathway ◽

Ubiquitin Proteasome Pathway ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Proplatelet Formation

Background: Proteasome inhibitors such as bortezomib, a chemotherapeutic used to treat multiple myeloma, induce thrombocytopenia within days of initiation. The mechanism for this thrombocytopenia has been tied to data revealing that proteasome activity is essential for platelet formation. The major pathway of selective protein degradation uses ubiquitin as a marker that targets proteins for proteolysis by the proteasome. This pathway is previously unexplored in megakaryocytes (MKs). Objectives: We aim to define the mechanism by which the ubiquitin-proteasome pathway affects MK maturation and platelet production. Results: Pharmacologic inhibition of proteasome activity blocks proplatelet formation in megakaryocytes. To further characterize how this degradation was occurring, we probed distinct ubiquitin pathways. Inhibition of the ubiquitin-activating enzyme E1 significantly inhibited proplatelet formation up to 73%. In addition, inhibition of the deubiquitinase proteins UCHL5 and USP14 significantly inhibited proplatelet formation up to 83%. These data suggest that an intact ubiquitin pathway is necessary for proplatelet formation. Proteomic and polysome analyses of MKs undergoing proplatelet formation revealed a subset of proteins decreased in proplatelet-producing megakaryocytes, consistent with data showing that protein degradation is necessary for proplatelet formation. Specifically, the centrosome stabilizing proteins Aurora kinase (Aurk) A/B, Tpx2, Cdk1, and Plk1 were decreased in proplatelet-producing MKs. Furthermore, inhibition of AurkA and Plk1, but not Cdk1, significantly inhibited proplatelet formation in vitro over 83%. Conclusions: We hypothesize that proplatelet formation is triggered by centrosome destabilization and disassembly, and that the ubiquitin-proteasome pathway plays a crucial role in this transformation. Specifically, regulation of the AurkA/Plk1/Tpx2 pathway may be key in centrosome integrity and initiation of proplatelet formation. Determination of the mechanism by which the ubiquitin-proteasome pathway regulates the centrosome and facilitates proplatelet formation will allow us to design better strategies to target and reverse thrombocytopenia.

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Identification and Mechanistic Studies of a Novel Ubiquitin E1 Inhibitor

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057111433843 ◽

2012 ◽

Vol 17 (4) ◽

pp. 421-434 ◽

Cited By ~ 34

Author(s):

Dana Ungermannova ◽

Seth J. Parker ◽

Christopher G. Nasveschuk ◽

Douglas A. Chapnick ◽

Andrew J. Phillips ◽

...

Keyword(s):

Small Molecule ◽

Small Molecule Inhibitors ◽

Nucleotide Exchange ◽

Cellular Processes ◽

Small Molecule Compound ◽

Ubiquitin Proteasome ◽

A Cell ◽

Proteasome Pathway

Protein degradation via the ubiquitin-proteasome pathway is important for a diverse number of cellular processes ranging from cell signaling to development. Disruption of the ubiquitin pathway occurs in a variety of human diseases, including several cancers and neurological disorders. Excessive proteolysis of tumor suppressor proteins, such as p27, occurs in numerous aggressive human tumors. To discover small-molecule inhibitors that potentially prevent p27 degradation, we developed a series of screening assays, including a cell-based screen of a small-molecule compound library and two novel nucleotide exchange assays. Several small-molecule inhibitors, including NSC624206, were identified and subsequently verified to prevent p27 ubiquitination in vitro. The mechanism of NSC624206 inhibition of p27 ubiquitination was further unraveled using the nucleotide exchange assays and shown to be due to antagonizing ubiquitin activating enzyme (E1). We determined that NSC624206 and PYR-41, a recently reported inhibitor of ubiquitin E1, specifically block ubiquitin-thioester formation but have no effect on ubiquitin adenylation. These studies reveal a novel E1 inhibitor that targets a specific step of the E1 activation reaction. NSC624206 could, therefore, be potentially useful for the control of excessive ubiquitin-mediated proteolysis in vivo.

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Effects of Postmortem Storage on Muscle Protein Degradation: Analysis by SDS-Polyacrylamide Gel Electrophoresis

Journal of Food Science ◽

10.1111/j.1365-2621.1984.tb13732.x ◽

1984 ◽

Vol 49 (1) ◽

pp. 292-293 ◽

Cited By ~ 49

Author(s):

M. KOOHMARAIE ◽

W. H. KENNICK ◽

E. A. ELGASIM ◽

A. F. ANGLEMIER

Keyword(s):

Protein Degradation ◽

Gel Electrophoresis ◽

Muscle Protein ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

Degradation Analysis ◽

Muscle Protein Degradation

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Derailing the UPS of Protein Turnover in Cancer and other Human Diseases

Journal of Cancer Research ◽

10.1155/2013/167576 ◽

2013 ◽

Vol 2013 ◽

pp. 1-11 ◽

Cited By ~ 2

Author(s):

Jit Kong Cheong ◽

Stephen I-Hong Hsu

Keyword(s):

Protein Turnover ◽

Cellular Differentiation ◽

Cell Cycle Control ◽

Therapeutic Strategies ◽

Organelle Biogenesis ◽

Covalent Linkage ◽

Ubiquitin Proteasome Pathway ◽

Cellular Processes ◽

Ubiquitin Proteasome ◽

Proteasome Pathway

Protein modifications by the covalent linkage of ubiquitin have significant involvement in many cellular processes, including stress response, oncogenesis, viral infection, transcription, protein turnover, organelle biogenesis, DNA repair, cellular differentiation, and cell cycle control. We provide a brief overview of the fundamentals of the regulation of protein turnover by the ubiquitin-proteasome pathway and discuss new therapeutic strategies that aim to mitigate the deleterious effects of its dysregulation in cancer and other human disease pathophysiology.

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Assay Systems for Profiling Deubiquitinating Activity

International Journal of Molecular Sciences ◽

10.3390/ijms21165638 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5638

Author(s):

Jinhong Cho ◽

Jinyoung Park ◽

Eunice EunKyeong Kim ◽

Eun Joo Song

Keyword(s):

Protein Degradation ◽

Protein Interactions ◽

Live Cells ◽

Deubiquitinating Enzyme ◽

Protein Protein Interactions ◽

Deubiquitinating Enzymes ◽

Cell Lysates ◽

Cellular Processes

Deubiquitinating enzymes regulate various cellular processes, particularly protein degradation, localization, and protein–protein interactions. The dysregulation of deubiquitinating enzyme (DUB) activity has been linked to several diseases; however, the function of many DUBs has not been identified. Therefore, the development of methods to assess DUB activity is important to identify novel DUBs, characterize DUB selectivity, and profile dynamic DUB substrates. Here, we review various methods of evaluating DUB activity using cell lysates or purified DUBs, as well as the types of probes used in these methods. In addition, we introduce some techniques that can deliver DUB probes into the cells and cell-permeable activity-based probes to directly visualize and quantify DUB activity in live cells. This review could contribute to the development of DUB inhibitors by providing important information on the characteristics and applications of various probes used to evaluate and detect DUB activity in vitro and in vivo.

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S-Nitrosylation of IRP2 Regulates Its Stability via the Ubiquitin-Proteasome Pathway

Molecular and Cellular Biology ◽

10.1128/mcb.24.1.330-337.2004 ◽

2004 ◽

Vol 24 (1) ◽

pp. 330-337 ◽

Cited By ~ 64

Author(s):

Sangwon Kim ◽

Simon S. Wing ◽

Prem Ponka

Keyword(s):

Regulatory Protein ◽

Untranslated Regions ◽

Raw 264.7 Cells ◽

Ubiquitin Proteasome Pathway ◽

Functional Implications ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Iron Responsive Elements

ABSTRACT Nitric oxide (NO) is an important signaling molecule that interacts with different targets depending on its redox state. NO can interact with thiol groups resulting in S-nitrosylation of proteins, but the functional implications of this modification are not yet fully understood. We have reported that treatment of RAW 264.7 cells with NO caused a decrease in levels of iron regulatory protein 2 (IRP2), which binds to iron-responsive elements present in untranslated regions of mRNAs for several proteins involved in iron metabolism. In this study, we show that NO causes S-nitrosylation of IRP2, both in vitro and in vivo, and this modification leads to IRP2 ubiquitination followed by its degradation in the proteasome. Moreover, mutation of one cysteine (C178S) prevents NO-mediated degradation of IRP2. Hence, S-nitrosylation is a novel signal for IRP2 degradation via the ubiquitin-proteasome pathway.

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Mechanisms of Cancer Cachexia

Physiological Reviews ◽

10.1152/physrev.00016.2008 ◽

2009 ◽

Vol 89 (2) ◽

pp. 381-410 ◽

Cited By ~ 672

Author(s):

Michael J. Tisdale

Keyword(s):

Skeletal Muscle ◽

Protein Synthesis ◽

Adipose Tissue ◽

Protein Degradation ◽

Initiation Factor ◽

Tissue Loss ◽

Α Subunit ◽

Ubiquitin Proteasome Pathway ◽

Ubiquitin Proteasome ◽

Proteasome Pathway

Up to 50% of cancer patients suffer from a progressive atrophy of adipose tissue and skeletal muscle, called cachexia, resulting in weight loss, a reduced quality of life, and a shortened survival time. Anorexia often accompanies cachexia, but appears not to be responsible for the tissue loss, particularly lean body mass. An increased resting energy expenditure is seen, possibly arising from an increased thermogenesis in skeletal muscle due to an increased expression of uncoupling protein, and increased operation of the Cori cycle. Loss of adipose tissue is due to an increased lipolysis by tumor or host products. Loss of skeletal muscle in cachexia results from a depression in protein synthesis combined with an increase in protein degradation. The increase in protein degradation may include both increased activity of the ubiquitin-proteasome pathway and lysosomes. The decrease in protein synthesis is due to a reduced level of the initiation factor 4F, decreased elongation, and decreased binding of methionyl-tRNA to the 40S ribosomal subunit through increased phosphorylation of eIF2 on the α-subunit by activation of the dsRNA-dependent protein kinase, which also increases expression of the ubiquitin-proteasome pathway through activation of NFκB. Tumor factors such as proteolysis-inducing factor and host factors such as tumor necrosis factor-α, angiotensin II, and glucocorticoids can all induce muscle atrophy. Knowledge of the mechanisms of tissue destruction in cachexia should improve methods of treatment.

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The effect of denervation on protein synthesis and degradation in adult rat diaphragm muscle

Journal of Applied Physiology ◽

10.1152/japplphysiol.91247.2008 ◽

2009 ◽

Vol 107 (2) ◽

pp. 438-444 ◽

Cited By ~ 41

Author(s):

Heather M. Argadine ◽

Nathan J. Hellyer ◽

Carlos B. Mantilla ◽

Wen-Zhi Zhan ◽

Gary C. Sieck

Keyword(s):

Protein Synthesis ◽

Protein Degradation ◽

Caspase 3 ◽

Diaphragm Muscle ◽

Rat Diaphragm ◽

Ubiquitin Proteasome Pathway ◽

Protein Ubiquitination ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Net Protein

Previous studies showed that unilateral denervation (DNV) of the rat diaphragm muscle (DIAm) results in loss of myosin heavy chain protein by 1 day after DNV. We hypothesize that DNV decreases net protein balance as a result of activation of the ubiquitin-proteasome pathway. In DIAm strips, protein synthesis was measured by incorporation of 3H-Tyr, and protein degradation was measured by Tyr release at 1, 3, 5, 7, and 14 days after DNV. Total protein ubiquitination, caspase-3 expression/activity, and actin fragmentation were analyzed by Western analysis. We found that, at 3 days after DNV, protein synthesis increased by 77% relative to sham controls. Protein synthesis remained elevated at 5 (85%), 7 (53%), and 14 days (123%) after DNV. At 5 days after DNV, protein degradation increased by 43% relative to sham controls and remained elevated at 7 (49%) and 14 days (74%) after DNV. Thus, by 5 days after DNV, net protein balance decreased by 43% compared with sham controls and was decreased compared with sham at 7 (49%) and 14 days (72%) after DNV. Protein ubiquitination increased at 5 days after DNV and remained elevated. DNV had no effect on caspase-3 activity or actin fragmentation, suggesting that the ubiquitin-proteasome pathway rather than caspase-3 activation is important in the DIAm response to DNV. Early loss of contractile proteins, such as myosin heavy chain, is likely the result of selective protein degradation rather than generalized protein breakdown. Future studies should evaluate this selective effect of DNV.

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Oestrogen causes ATBF1 protein degradation through the oestrogen-responsive E3 ubiquitin ligase EFP

Biochemical Journal ◽

10.1042/bj20111890 ◽

2012 ◽

Vol 444 (3) ◽

pp. 581-590 ◽

Cited By ~ 12

Author(s):

Xue-Yuan Dong ◽

Xiaoying Fu ◽

Songqing Fan ◽

Peng Guo ◽

Dan Su ◽

...

Keyword(s):

Cell Proliferation ◽

Protein Degradation ◽

Ubiquitin Ligase ◽

Feedback Loop ◽

E3 Ubiquitin Ligase ◽

Breast Development ◽

Protein Levels ◽

Ubiquitin Proteasome ◽

Dependent Cell ◽

Proteasome Pathway

We reported previously that the tumour suppressor ATBF1 (AT motif-binding factor 1) formed an autoregulatory feedback loop with oestrogen–ERα (oestrogen receptor α) signalling to regulate oestrogen-dependent cell proliferation in breast cancer cells. In this loop ATBF1 inhibits the function of oestrogen–ERα signalling, whereas ATBF1 protein levels are fine-tuned by oestrogen-induced transcriptional up-regulation as well as UPP (ubiquitin–proteasome pathway)-mediated protein degradation. In the present study we show that EFP (oestrogen-responsive finger protein) is an E3 ubiquitin ligase mediating oestrogen-induced ATBF1 protein degradation. Knockdown of EFP increases ATBF1 protein levels, whereas overexpression of EFP decreases ATBF1 protein levels. EFP interacts with and ubiquitinates ATBF1 protein. Furthermore, we show that EFP is an important factor in oestrogen-induced ATBF1 protein degradation in which some other factors are also involved. In human primary breast tumours the levels of ATBF1 protein are positively correlated with the levels of EFP protein, as both are directly up-regulated ERα target gene products. However, the ratio of ATBF1 protein to EFP protein is negatively correlated with EFP protein levels. Functionally, ATBF1 antagonizes EFP-mediated cell proliferation. These findings not only establish EFP as the E3 ubiquitin ligase for oestrogen-induced ATBF1 protein degradation, but further support the autoregulatory feedback loop between ATBF1 and oestrogen–ERα signalling and thus implicate ATBF1 in oestrogen-dependent breast development and carcinogenesis.

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