We thank S. Raychaudhuri for providing the destabilized firefly luciferase mutant plasmid, and A. Glavis-bloom and N. Charan for technical assistance. This work was supported, in part, by grants from NIH (CA088868, GM060911, and CA182675).

<ol>
	<li>Dobson, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+folding+and+misfolding.">Protein folding and misfolding.</a> Nature. 426, 884-890 (2003).</li><li>Goldberg, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+degradation+and+protection+against+misfolded+or+damaged+proteins.">Protein degradation and protection against misfolded or damaged proteins.</a> Nature. 426, 895-899 (2003).</li><li>Hartl, F. U., Bracher, A., Hayer-Hartl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+chaperones+in+protein+folding+and+proteostasis.">Molecular chaperones in protein folding and proteostasis.</a> Nature. 475, 324-332 (2011).</li><li>Taylor, J. P., Hardy, J., Fischbeck, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxic+proteins+in+neurodegenerative+disease.">Toxic proteins in neurodegenerative disease.</a> Science. 296, 1991-1995 (2002).</li><li>Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+proteins+in+fatal+ways.">Folding proteins in fatal ways.</a> Nature. 426, 900-904 (2003).</li><li>Vousden, K. H., Prives, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blinded+by+the+Light:+The+Growing+Complexity+of+p53.">Blinded by the Light: The Growing Complexity of p53.</a> Cell. 137, 413-431 (2009).</li><li>Xu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain+of+function+of+mutant+p53+by+coaggregation+with+multiple+tumor+suppressors.">Gain of function of mutant p53 by coaggregation with multiple tumor suppressors.</a> Nat Chem Biol. 7, 285-295 (2011).</li><li>Orr, H. T., Zoghbi, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trinucleotide+repeat+disorders.">Trinucleotide repeat disorders.</a> Annu Rev Neurosci. 30, 575-621 (2007).</li><li>Guo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cellular+system+that+degrades+misfolded+proteins+and+protects+against+neurodegeneration.">A cellular system that degrades misfolded proteins and protects against neurodegeneration.</a> Mol Cell. 55, 15-30 (2014).</li><li>Chu, Y., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO+E3+ligase+activity+of+TRIM+proteins.">SUMO E3 ligase activity of TRIM proteins.</a> Oncogene. 30, 1108-1116 (2011).</li><li>Sun, H., Leverson, J. D., Hunter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+function+of+RNF4+family+proteins+in+eukaryotes:+targeting+a+ubiquitin+ligase+to+SUMOylated+proteins.">Conserved function of RNF4 family proteins in eukaryotes: targeting a ubiquitin ligase to SUMOylated proteins.</a> EMBO J. 26, 4102-4112 (2007).</li><li>Janer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PML+clastosomes+prevent+nuclear+accumulation+of+mutant+ataxin-7+and+other+polyglutamine+proteins.">PML clastosomes prevent nuclear accumulation of mutant ataxin-7 and other polyglutamine proteins.</a> J Cell Biol. 174, 65-76 (2006).</li><li>Bauer, P. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+chaperone-mediated+autophagy+for+the+selective+degradation+of+mutant+huntingtin+protein.">Harnessing chaperone-mediated autophagy for the selective degradation of mutant huntingtin protein.</a> Nat Biotechnol. 28, 256-263 (2010).</li><li>Austin, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease+causing+mutants+of+TDP-43+nucleic+acid+binding+domains+are+resistant+to+aggregation+and+have+increased+stability+and+half-life.">Disease causing mutants of TDP-43 nucleic acid binding domains are resistant to aggregation and have increased stability and half-life.</a> Proc Natl Acad Sci U S A. 111, 4309-4314 (2014).</li><li>Barmada, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+induction+enhances+TDP43+turnover+and+survival+in+neuronal+ALS+models.">Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models.</a> Nat Chem Biol. 10, 677-685 (2014).</li><li>Paxinou, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+alpha-synuclein+aggregation+by+intracellular+nitrative+insult.">Induction of alpha-synuclein aggregation by intracellular nitrative insult.</a> J Neurosci. 21, 8053-8061 (2001).</li><li>Gupta, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Firefly+luciferase+mutants+as+sensors+of+proteome+stress.">Firefly luciferase mutants as sensors of proteome stress.</a> Nat Methods. 8, 879-884 (2011).</li><li>Cooper, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-synuclein+blocks+ER-Golgi+traffic+and+Rab1+rescues+neuron+loss+in+Parkinson's+models.">Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models.</a> Science. 313, 324-328 (2006).</li><li>Elden, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ataxin-2+intermediate-length+polyglutamine+expansions+are+associated+with+increased+risk+for+ALS.">Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS.</a> Nature. 466, 1069-1075 (2010).</li><li>Fortini, M. E., Bonini, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+human+neurodegenerative+diseases+in+Drosophila:+on+a+wing+and+a+prayer.">Modeling human neurodegenerative diseases in Drosophila: on a wing and a prayer.</a> Trends Genet. 16, 161-167 (2000).</li><li>Bogdanov, A. M., Kudryavtseva, E. I., Lukyanov, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-fading+media+for+live+cell+GFP+imaging.">Anti-fading media for live cell GFP imaging.</a> PLoS One. 7, (2012).</li><li>Wanker, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+filter+assay+for+detection+of+amyloid-like+polyglutamine-containing+protein+aggregates.">Membrane filter assay for detection of amyloid-like polyglutamine-containing protein aggregates.</a> Methods Enzymol. 309, 375-386 (1999).</li><li>McDonald, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Biological Statistics. 3, 173-179 (2014).</li><li>Holmberg, C. I., Staniszewski, K. E., Mensah, K. N., Matouschek, A., Morimoto, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inefficient+degradation+of+truncated+polyglutamine+proteins+by+the+proteasome.">Inefficient degradation of truncated polyglutamine proteins by the proteasome.</a> EMBO J. 23, 4307-4318 (2004).</li><li>Verhoef, L. G., Lindsten, K., Masucci, M. G., Dantuma, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggregate+formation+inhibits+proteasomal+degradation+of+polyglutamine+proteins.">Aggregate formation inhibits proteasomal degradation of polyglutamine proteins.</a> Human molecular genetics. 11, 2689-2700 (2002).</li><li>Jana, N. R., Zemskov, E. A., Wang, G., Nukina, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+proteasomal+function+due+to+the+expression+of+polyglutamine-expanded+truncated+N-terminal+huntingtin+induces+apoptosis+by+caspase+activation+through+mitochondrial+cytochrome+c+release.">Altered proteasomal function due to the expression of polyglutamine-expanded truncated N-terminal huntingtin induces apoptosis by caspase activation through mitochondrial cytochrome c release.</a> Human molecular genetics. 10, 1049-1059 (2001).</li><li>Fribley, A., Zeng, Q., Wang, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteasome+inhibitor+PS-341+induces+apoptosis+through+induction+of+endoplasmic+reticulum+stress-reactive+oxygen+species+in+head+and+neck+squamous+cell+carcinoma+cells.">Proteasome inhibitor PS-341 induces apoptosis through induction of endoplasmic reticulum stress-reactive oxygen species in head and neck squamous cell carcinoma cells.</a> Molecular and cellular biology. 24, 9695-9704 (2004).</li><li>Bence, N. F., Sampat, R. M., Kopito, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairment+of+the+ubiquitin-proteasome+system+by+protein+aggregation.">Impairment of the ubiquitin-proteasome system by protein aggregation.</a> Science. 292, 1552-1555 (2001).</li><li>Chen, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrasarcoplasmic+amyloidosis+impairs+proteolytic+function+of+proteasomes+in+cardiomyocytes+by+compromising+substrate+uptake.">Intrasarcoplasmic amyloidosis impairs proteolytic function of proteasomes in cardiomyocytes by compromising substrate uptake.</a> Circulation research. 97, 1018-1026 (2005).</li><li>Link, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+green+fluorescent+protein+into+a+toxic,+aggregation-prone+protein+by+C-terminal+addition+of+a+short+peptide.">Conversion of green fluorescent protein into a toxic, aggregation-prone protein by C-terminal addition of a short peptide.</a> The Journal of biological chemistry. 281, 1808-1816 (2006).</li></ol>

The authors declare that they have no competing financial interests.

الآليات التي تنظم تدهور البروتينات تتجمع ضرورية للحفاظ على توازن البروتينات الخلوية، وعلى الأرجح أنها تمثل أهدافا المخدرات قيمة لعلاج الاضطرابات العصبية وغيرها من الأمراض misfolding البروتين. هنا، يتم وصف المقايسات التي تدرس تدهور البروتينات تتجمع، وذلك باستخدام بروت...

البروتينات هي الجزيئات الأكثر وفرة في الخلايا، وأنها تلعب دورا أساسيا في جميع العمليات الحيوية تقريبا. النشاط البيولوجي لمعظم البروتينات يتطلب قابلة للطي وبياناتهم إلى والمحافظة عليها، والأم هياكل ثلاثية الأبعاد. البروتينات مع التشكل الشاذة ليس فقط على فقدان وظائفهم العادية، ولكن أيضا في كثير من الأحيان تشكل الأنواع بلازميدة قليلة القسيمات القابلة للذوبان أو المجاميع التي تعوق وظائف بروتينات أخرى، وهي مواد سامة للخلايا 1،2. لمواجهة misfolding البروتين وخلايا توظف كل من المحرمين الجزيئية، التي تساعد البروتينية المطوية أو مطوية جزئيا للوصول إلى التشكل وطنهم، ومسارات التحلل، والتي تزيل البروتينات تتجمع 3. نظرا لتعقيد وطبيعة العشوائية لعملية قابلة للطي، والبروتين misfolding أمر لا مفر منه، وأنه قد لا يكون عكس في حالة من الطفرات، وأخطاء السكروز، والأضرار بعد متعدية 1. وبالتالي، فإن الخلايا تعتمد في نهاية المطاف على degradatمسارات أيون للحفاظ على نوعية البروتين. وشدد على أهمية مراقبة الجودة البروتين الخلوي (PQC) نظم من قبل انتشار الأمراض misfolding البروتين، بما في ذلك السرطان والسكري والعديد من الأمراض العصبية مثل مرض الزهايمر ومرض باركنسون، التصلب الجانبي الضموري، مرض هنتنغتون (HD)، و الرنح النخاعي المخيخي (SCA) 4،5. على سبيل المثال، والطفرات في البروتين p53 الكابتة للورم هي الآفة الأكثر الوراثية في كثير من الأحيان واحدة في الأورام، ويرتبط مع ~ 50-70٪ من جميع حالات 6. وجزء كبير من الطفرات البروتين p53 والطفرات مغلطة التي تغير التشكل من البروتين p53، مما يؤدي إلى تشكيل المجاميع 7. علاوة على ذلك، وراثيا ومرضي البروتينات المرتبطة مع مساحات polyQ موسع مع HD وهيئة السلع التموينية. هذه الأمراض التقدمية وغالبا مميتة واضح عندما يتجاوز طول امتداد polyQ في البروتينات المتضررة معينة thresعقد، وتصبح شديدة على نحو متزايد على طول امتداد polyQ يطيل 8. مقاربة جذابة لعلاج هذه الأمراض هو تعزيز علاجيا أنظمة PQC الخلوية، وخصوصا مسارات التحلل. ومع ذلك، فإن الممرات المشتركة في تدهور البروتينات التالفة، ولا سيما في خلايا الثدييات، لا تزال غير مفهومة. وعلى الرغم من الاعتراف بأن proteasome ومهم للغاية لتحلل البروتينات تتجمع، تبقى مسألة حيوية غير محددة: كيف البروتينات تتجمع معترف بها على وجه التحديد والمستهدفة للتدهور. وعلاوة على ذلك، على الرغم من أن أنظمة PQC تم تحديدها في مقصورات الخلوية بما في ذلك السيتوبلازم، الشبكة الإندوبلازمية، والحبيبات الخيطية، لا تزال أنظمة PQC في نواة واضحة 2. وقد حددت الدراسات الأخيرة التي مختبرنا نظام يعترف ويحط مجموعة متنوعة من البروتينات تتجمع في النواةخلايا الثدييات من 9. ويتألف هذا النظام من البروتين البرولمفوسيت (الرابطة الاسلامية الباكستانية)، وهو بروتين النووي وعضوا في أسرة البروتين الثلاثية التي تحتوي على عزر (TRIM)، وRNF4، حلقة المجال تحتوي على البروتين. حزب الرابطة الاسلامية، والعديد من البروتينات TRIM أخرى، تمتلك سومو (صغير مثل اليوبيكويتين معدل) E3 النشاط يغاز، مما يسهل خصوصية وكفاءة البروتين SUMOylation 10. RNF4 ينتمي إلى مجموعة صغيرة من ligases اليوبيكويتين المستهدفة سومو (STUbL)، التي تحتوي على واحد أو أكثر من الزخارف-التفاعل السومو (بالمشترك) بالإضافة إلى المجال RING التي تتيح لهم النشاط يغاز اليوبيكويتين 11. وجدنا أن حزب الرابطة الاسلامية تعترف تحديدا البروتينات تتجمع من خلال مواقع التعرف على الركيزة المنفصلة التي يمكن أن نتبين سمات مميزة على البروتينات تتجمع. على الربط، والعلامات حزب الرابطة الاسلامية تتجمع البروتينات مع بولي سلاسل من SUMO2 / 3، اثنين من البروتينات SUMO الثدييات متطابقة تقريبا التي يمكن أن تشكل بولي سلاسل بسبب وجود موقع SUMOylation الداخلي. Sثم يتم التعرف على البروتينات تتجمع UMOylated التي كتبها RNF4، الأمر الذي يؤدي إلى ubiquitination وproteasomal تدهورها. أثبتنا أيضا أن نظام حزب الرابطة الاسلامية-RNF4 مهم للحماية من التنكس العصبي، ونقص في حزب الرابطة الاسلامية يفاقم من العيوب السلوكية وعصبية مرضية من نموذج الفأر من نوع SCA 1 (SCA1) 9. للتمييز تدهور البروتين من الآليات الخلوية الأخرى التي يمكن أن تنظم مستويات البروتين، تم قياس معدلات دوران البروتين 9. من بين الأساليب الأكثر استخداما لتحديد دوران البروتين هي مطاردة النبض وسيكلوهيكسيميد (فروج) مطاردة. هاتين الطريقتين تدرس على مر الزمن، على التوالي، والبروتينات وصفت النظائر المشعة من الاهتمام في خلايا الترجمة يتقن ومجموع البروتينات الموجودة مسبقا من الفائدة في الخلايا تحول دون ترجمة. ومع ذلك، تحديا كبيرا لدراسة البروتينات المسببة للأمراض وعرضة للmisfolding هو أن عمر النصف لهذه البروتينات يمكن أن يكون لو للغايةنانوغرام. على سبيل المثال، Ataxin-1، Ataxin-7، هنتنغتين، ألفا-synuclein، وTDP-43 جميعا حياة نصف من أكثر من 12 إلى 24 ساعة 9،12-16. معدلات دوران بطيئة من هذه البروتينات تمنع استخدام تحليل فروج مطاردة لأن الخلايا إيواء هذه البروتينات قد لا البقاء على قيد الحياة لفترة طويلة تثبيط الترجمة، خصوصا لأن البروتينات تتجمع أنفسهم يمكن أن تكون شديدة السمية للخلايا. تحليل نبض مطاردة مع وضع العلامات النظائر قد يكون أيضا تحديا للبروتينات التي تكون عرضة تجميع-للغاية. معظم فحوصات نبض مطاردة تعتمد على مناعي لفصل البروتين من الاهتمام من جميع البروتينات الأخرى التي هي أيضا المسمى بالإشعاع. ويشمل هذا الإجراء عادة مناعي وغسل الإجراءات المطولة خلالها المجاميع SDS-غير قابلة للذوبان يمكن أن تشكل، مما يجعل تحليل مع SDS-PAGE الكهربائي غير دقيقة. هنا بروتوكول لتحليل البروتينات تتجمع النووية مع يوصف معدل دوران بطيء 9. ويستخدم شكلهما من Ataxin-1 (Atxn1) الذي يحتوي على امتداد 82 glutamines (Atxn1 82Q) لهذا الغرض (8). عندما أعرب في الخلايا، وتعزيز الخضراء بروتين فلوري (GFP) انصهار أشكال Atxn1 82Q مجهريا شوائب مرئية في نواة (الشكل 1A). ويكشف تحليل نبض مطاردة أن نصف عمر Ataxn1 82Q هي أكثر من 18 ساعة 9. يتكون Atxn1 82Q الأنواع مع التشكل تتجمع من خصائص مختلفة، فضلا عن الأنواع مع التشكل الأصلي. ومن المرجح أن هذه الأنواع تدهور بمعدلات مختلفة، وبالتالي يجب أن يتم تحليلها بشكل منفصل. لست] من Atxn1 82Q، معربا عن GFP ومجزأة الخلايا في NP-40 للذوبان (قابل للذوبان أو NS، من المرجح أن تمثل البروتينات الأم أو البروتينات أحادى / بلازميدة قليلة القسيمات تتجمع) و (NI، مجمعة / تتجمع) أجزاء NP-40-غير قابلة للذوبان. هذا الأخير يمكن تقسيمها الى مزيد من SDS للذوبان (SS، ومن المحتمل المجاميع المختلين) أو SDS مقاومة (SR، الألياف اميلويد المرجح) كسور (الشكل 1B). ويمكن تحليل NS و SS الكسور SDS-PAGE تليها النشاف الغربي، في حين يمكن أن يتم الكشف عن جزء ريال عن طريق فحوصات مرشح التخلف تليها immunoblotting. يتم الجمع بين فروج مطاردة مع أسلوب تجزئة المنظفات، واكتشفوا أن نصف عمر SS Atxn1 82Q أقصر بكثير من NS Atxn1 82Q ومجموعه Atxn1 82Q (الشكل 2A)، مشيرا إلى أن جزء SS يمكن التعرف بسهولة وتتحلل في الخلايا 9. وهكذا، وهذه الطريقة توفر أداة قوية لدراسة ديناميات البروتينات تتجمع ومقارنة نمط تدهورها. وصفنا أيضا الأسلوب الذي هو مناسبة لفحص إنتاجية عالية لتحديد الجزيئات أو المركبات الصغيرة التي يمكن أن تعدل تدهور البروتينات تتجمع. ويستند هذا الأسلوب على متحولة مزعزع conformationally من وسيفيراز يراعة (LucDM) 17، ركيزة نموذج كوصي. لدينا الصماماتد LucDM إلى إشارة توطين النووية (NLS) للتحقيق في أنظمة PQC في هذا الحيز الخلوية، وGFP (NLS-LucDM-GFP) لتسهيل الكشف. أشكال NLS-LucDM-GFP مجهريا المجاميع النووية واضحة في نسبة صغيرة من الخلايا (الشكل 3A). على غرار Atxn1 82Q، NLS-LucDM-GFP، ولكن ليس في البرية من نوع نظيره NLS-LucWT-GFP-يتم تعديل من قبل SUMO2 / 3 والتي تخضع لرقابة مسار حزب الرابطة الاسلامية-RNF4 9. كما يشكل NLS-LucDM-GFP SS والأنواع ريال، على الرغم من أن كميات النسبية لقوات الأمن الخاصة والأنواع ريال ضئيلة مقارنة NS، وذلك باستخدام الشروط الواردة في بروتوكول 3 أدناه. لتبسيط الفحص، نقوم بتحليل فقط SDS LucDM للذوبان (بما في ذلك كل من NS وكسور SS) بواسطة SDS-PAGE وصمة عار الغربية. الأهم من ذلك، أظهر فروج مطاردة الفحص أن نصف عمر SDS للذوبان NLS-LucDM-GFP أقصر بكثير من أن لها من النوع البري نظيره (الشكل 3B)، مما يوحي بأن LucDM هو الركيزة محددة للنظام يقر ويحطالبروتينات تتجمع. تدهور LucDM-GFP يؤدي إلى انخفاض كبير في إشارة مضان الشاملة. لذلك، وقد وضعنا أيضا على بروتوكول للكشف في الوقت الحقيقي الخلوية LucDM-GFP باستخدام فحص أساس مضان صفيحة. يتم تطوير العديد من الأنظمة شاشة عالية الإنتاجية (HTS) للعقاقير أو الجينات تعديل المجاميع الخلوية والجدوى الخلوية التي تسببها البروتينات الشاذة 18-20. ومع ذلك، فقد تم تصميم عدد قليل جدا HTSs خصيصا لاستهداف التدهور في خلايا الثدييات. يخدم هذا البروتوكول على أنه نظام قوي للتحليل واسع النطاق السريع ومن آثار تعبير البروتين، ضربة قاضية، والعلاج من تعاطي المخدرات على الخلوي تدهور البروتين تتجمع. باستخدام خلايا هيلا كمثال أدناه ونحن تصف بروتوكولات لتحليل هذه البروتينات تتجمع اثنين. يمكن أن تطبق أيضا على فحوصات للخطوط الخلايا الأخرى، على الرغم من الظروف ترنسفكأيشن وبالطبع الوقت قد يحتاج إلى أن يكون الأمثل لخطوط الخلايا الفردية.

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			<td height="21" style="height:21px;width:308px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">11995-092</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Fetal Bovine Serum</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">10082147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Lipofectamine 2000</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">11668019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">NP-40</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">NP40S-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">SDS</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">L3771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">MG132</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8699</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Cycloheximide</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">C7698</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Dithiothreitol (DTT)</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:119px;">45000232</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:308px;">Complete Protease Inhibitor Cocktail Tablets</td>
			<td style="width:175px;">Roche Boehringer Mannheim</td>
			<td style="width:119px;">4693159001</td>
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			<td height="21" style="height:21px;width:308px;">Bio-Dot Apparatus&#160;</td>
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			<td style="width:119px;">1706545</td>
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			<td height="21" style="height:21px;width:308px;">Living Colors GFP Monoclonal Antibody</td>
			<td style="width:175px;">Clonetech</td>
			<td style="width:119px;">632375</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Anti-Actin mAb Rabbit, IgG</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">A45060-200UL</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Amino acids</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td>Amino acids are used for making low fluorecence culturing medium</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:308px;">Olympus IX-81&#160; Inverted Fluorescence Microscope</td>
			<td style="width:175px;">Olympus</td>
			<td style="width:119px;">IX71/IX81</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:308px;">96 Well Black TC Plate w/ Transluscent Clear Bottom</td>
			<td style="width:175px;">Sigma-Greiner</td>
			<td style="width:119px;">89135-048</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:308px;">Fluorescence Bottom Plate Reader Infinite 200&#174; PRO</td>
			<td style="width:175px;">TECAN</td>
			<td style="width:119px;">Infinite 200&#174; PRO</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Cellulose acetate membrane 0.2 &#181;m</td>
			<td style="width:175px;">Sterlitech</td>
			<td style="width:119px;">CA023001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Prism 5</td>
			<td style="width:175px;">GraphPad</td>
			<td style="width:119px;"></td>
			<td>Statistical analysis software</td>
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assays for the degradation of misfolded proteins in cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

في تحليل حالة مستقرة، وحدات النووية مرئية المجهر Atxn1 82Q-GFP يمكن ملاحظتها في 30 - 50٪ من خلايا هيلا 20 ساعة بعد ترنسفكأيشن (الشكل 1A). تحليل لطخة الغربي من كسور NS و SS باستخدام الأجسام المضادة لمكافحة GFP يظهر الفرقة متميزة من Atxn1 82Q-GFP بين 100 كيلو دالتون...

Watch this Scientific Journal Video about Assays for the Degradation of Misfolded Proteins in Cells at JoVE.com

Assays for the Degradation of Misfolded Proteins in Cells

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

فحوصات لتدهور تتجمع البروتينات في الخلايا

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

assays-for-the-degradation-of-misfolded-proteins-in-cells

Research

JoVE Journal

Biology

11.9K Views.  University of Pennsylvania Perelman School of Medicine. This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening. 

Video: Assays for the Degradation of Misfolded Proteins in Cells

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

بلورة البروتينات في الغشاء Mesophases Lipidic

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

Photobleaching فحوصات (FRAP و FLIP) لقياس حيوية الكروماتين البروتين في الخلايا الحية الجذعية الجنينية

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

فحوصات قابلية للخلايا في الثقافة

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

زراعة خلايا الإنسان الظهارية الأنف في واجهة الهواء السائل

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

في دراسة لالإلتقام وإعادة غشاء البلازما من البروتينات القائم على خلية L-الجلوتاثيون حماية فحوصات

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

إعادة تشكيل β-catenin تدهور في<em&gt; القيطم</em&gt; مستخلصات البيض

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

الفحص النمو القائم على مراسل لتحليل منهجي من تدهور البروتين

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

Comparing the Affinity of GTPase-binding Proteins using Competition Assays

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the binding capabilities of GTPases for two reasons: The fact that all interactions involve the same face of the GTPases means that binding events must be considered in the context of competitors, and the fact that the bound nucleotide must also be controlled means that conventional approaches such as immunoprecipitation are unsuitable for GTPase biochemistry. The assay relies on the use of purified proteins. Purified Rac1 immobilized on beads is used as the bait protein, and can be loaded with GDP, a non-hydrolyzable version of GTP or left nucleotide free, so that the signaling stage to be investigated can be controlled. The binding proteins to be investigated are purified from mammalian cells, to allow correct folding, by means of a GFP tag. Use of the same tag on both proteins is important because not only does it allow rapid purification and elution, but also allows detection of both competitors with the same antibody during elution. This means that the relative amounts of the two bound proteins can be determined accurately.

This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate.

مقارنة الانجذاب من البروتينات ملزم GTPase باستخدام فحوصات المنافسة

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

سم كلوي حقن مكروي في الزرد لنموذج قصور كلوي حاد

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

واجهة المستخدم الرسومية لتتبع بمساعدة برنامج تركيز البروتين في النتوءات الخلوية الديناميكية

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...