title: Assessment of proteasome impairment and accumulation/aggregation of ubiquitinated proteins in neuronal cultures.
abstract: The ubiquitin/proteasome pathway (UPP) is the major proteolytic quality control system in cells and involves tightly regulated removal of unwanted proteins and retention of those that are essential. In addition to its function in normal protein degradation, the UPP plays a critical role in the quality control process by degrading mutated or abnormally folded proteins. The proteolytic component of the UPP is a multiprotein complex known as the proteasome. Many factors, including the aging process, can cause proteasome impairment leading to formation of abnormal ubiquitin-protein aggregates that are found in most progressive neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. In this chapter, we describe protocols to measure proteasome activity, evaluate its state of assembly, and assess the accumulation and aggregation of ubiquitinated proteins in two types of neuronal cultures: human neuroblastoma cells and rat primary cortical cultures. These protocols can be used with different types of neuronal cultures to estimate proteasome activity and the levels and aggregation of ubiquitinated proteins. In addition, they can be used to identify compounds potentially capable of preventing a decline in proteasome activity and formation of ubiquitin-protein aggregates associated with neurodegeneration.
authors.affiliation: Department of Biological Sciences, Hunter College of City University of New York, New York, NY, USA.
authors.forename: Natura
authors.initials: N
authors.lastname: Myeku
authors.forename: Maria Jose
authors.initials: MJ
authors.lastname: Metcalfe
authors.forename: Qian
authors.initials: Q
authors.lastname: Huang
authors.forename: Maria
authors.initials: M
authors.lastname: Figueiredo-Pereira
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002457:Cell Extracts; D045744:Cell Line, Tumor; D002469:Cell Separation; D002498:Centrifugation; D002540:Cerebral Cortex / Q000166:cytology; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005455:Fluorescent Antibody Technique; D005990:Glycerol / Q000737:chemistry; D006801:Humans; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D011247:Pregnancy; D046988:Proteasome Endopeptidase Complex / Q000302:isolation & purification / Q000378:metabolism*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D057149:Ubiquitinated Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D002457:Cell Extracts; D021122:Protein Subunits; D057149:Ubiquitinated Proteins; D002918:Chymotrypsin; D046988:Proteasome Endopeptidase Complex; D005990:Glycerol