This work was funded primarily by grants from the CHDI foundation and the Swiss National Science Foundation. We thank Dr. Sophie Vieweg for useful discussions during the development of this new expression system and other members of the Lashuel group for sharing their experience with this expression system and for their input and valuable feedback. We also thank Prof. Oliver Hantschel for providing the ULP1 plasmid. The authors thank Dr. John B. Warner and Dr. Senthil K. Thangaraj for critical review of the manuscript

The authors declare that they have no conflicts of interest with the contents of this article.

In this protocol, we have outlined an efficient procedure for obtaining milligram quantities of native, untagged Httex1 containing 23 or 43 glutamine residues. This was achieved by expressing Httex1 as a C-terminal fusion to a His6-SUMO tag, which is used to isolate the fusion protein from the cell lysate by IMAC and is cleaved prior to HPLC purification of Httex1. While the SUMO strategy has been used in the production of several other proteins, our method shows that the unique properties SUMO could also be used to generate intrinsically disordered, aggregation-prone, amyloidogenic protein that have previously proved to be extremely difficult to handle and produce43,44. We present a protocol that is straightforward, easy to use and comparable to a protocol for the generation of a &#34;well-behaved&#34; protein. The SUMO fusion solubilizes and stabilizes Httex1 during expression and the IMAC purification step. Premature cleavage of the tag, as observed with the intein strategy10 and aggregation were no longer an issue.
Intrinsically disordered proteins are especially vulnerable to degradation. While N-terminal degradation in the N17 region is not an issue using this protocol, truncations in the PRD of Httex1 can occur. As the truncated proteins are very similar to Httex1 in hydrophobicity, charge and size, removing them by chromatographic means is challenging, thus it is best to prevent their formation in the first place. Sticking closely to the protocol, always working on ice and using a sufficient amount of protease inhibitor should help keep the level of observed truncation very low. Applying a fusion tag at the C-terminus of Httex1 could remove truncations in the PRD easily as the truncated protein would lose the affinity tag as well. However, if the native sequence needs to be maintained this option cannot be applied as Httex1 ends with proline and to the best of our knowledge there are no C-terminal fusion tags that are known to induce traceless and efficient cleavage after proline.
The most critical part of the protocol is the handling of the Httex1 liberated after cleavage of the SUMO tag by ULP1. The protein should be purified immediately by RP-HPLC. Fortunately, this is an efficient and fast reaction that is usually completed in 10-20 min at 4 &#176;C. In contrast, the intein strategy required several hours for complete cleavage of the intein, thus requiring a trade-off between incomplete cleavage and beginning aggregation in order to maximize the yield. A fast workup is required for mutant Httex1, as it will start to aggregate at the comparatively high concentration present in the cleavage reaction, whereas the 23Q variant is stable for a longer time. During the RP-HPLC purification, another advantage of SUMO becomes apparent: While the Ssp DnaB intein is hydrophobic and sticks strongly to the column, SUMO is more hydrophilic and elutes completely from the C4 reversed-phase column. Although commercial ULP1 is quite costly, the protein can be easily produced in high yield following previously published protocols29.
The critical importance of applying a disaggregation protocol prior to using Httex1 cannot be stressed enough. Lyophilized polyQ proteins such as Httex1 are stable and can be stored long periods, but are not completely soluble in water and buffers. The presence of preformed oligomers or fibrils could have a significant impact on aggregation kinetics and biophysical properties of the protein45. The disaggregation protocol described here allows the disaggregation of the protein, removal of preformed aggregates and generation of a solution of monomeric Httex1 from a lyophilized sample. We observed similar aggregation kinetics and fibril morphology for Httex1 obtained with the SUMO and the intein strategy.
Compared to previous methods for producing Httex1, the SUMO strategy described here offers several advantages and expands the range of possible studies to investigate the structure and functional properties of this protein in health and disease. The SUMO-Httex1 fusion protein is easy to handle, it can be frozen and stored or kept in solution for 24 h at ambient temperature, while the free mHttex1 would aggregate quickly. The stability and high solubility of the SUMO-Httex1 fusion proteins provide greater flexibility to manipulate the protein and/or introduce enzymatic and chemical modifications into mHttex1 that would otherwise not be possible after cleavage. This includes the introduction of post-translational modifications, fluorophores, spin labels, biotin tags, etc. The advances presented here should 1) facilitate future studies to elucidate structure-function relationships of Httex1; 2) generate new tools to investigate Htt aggregation and pathology spreading; 3) enable the development of new assays to identify molecules that stabilize mutant Httex1 and prevent its aggregation; and 4) encourage scientists from other fields to bring to work on this protein and join our quest to find cures for Huntington&#39;s disease.

Htt is a 348 kDa protein and has been implicated in several physiological functions1. When Htt contains an expanded polyQ region of more than 36 residues in its N-terminus, it causes HD2,3. HD pathology is characterized by cellular inclusions in the striatum and cortex, which leads to neuronal death and atrophy of the affected tissues4,5. Several N-terminal Htt fragments that contain the polyQ repeat tract have been detected in post-mortem brains from HD patients and are thought to be generated by proteolytic processing of the huntingtin protein6. Recent studies suggest that Httex1 could also be formed due to aberrant mRNA splicing. Httex1 contains the pathological polyQ mutation and its overexpression in animals can recapitulate many of the key features of HD7, thus highlighting a possible central role of this fragment in HD pathology and disease progression6,8,9.
Due to the high aggregation propensity of mutant Httex1 (mHttex1) with expanded polyQ tract, the majority of existing expression systems are based on the transient fusion of Httex1 to proteins (such as glutathione-S-transferase (GST), thioredoxin (TRX) or maltose-binding-protein (MBP) and/or peptides (poly-histidine) that differentially improve its expression, stability, purification and/or solubility10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28. The fusion partner is linked to Httex1 with a short sequence containing a cleavage site for proteases such as trypsin, tabacco etch virus (TEV) protease or PreScission to allow for the cleavage and release of Httex1 prior to the initiation of aggregation or purification. Shortcomings of these methods include the possibility of leaving additional residues due to non-traceless cleavage and the creation of truncated fragments due to miscleavage within the sequence of Httex1, in addition to heterogeneity due to incomplete cleavage (see Vieweg et al. for more in-depth discussion on the advantages and limitations of this approach)10. To address these limitations, we recently developed an expression strategy enabling the generation of tag-free native Httex1 for the first time by utilizing a transient N-terminal fusion of the Synechocystis sp. (Ssp) DnaB intein to Httex110. While the intein cleavage is traceless and specific and yields mg quantity of proteins, it still suffers two drawbacks that could reduce the yield: namely, premature cleavage of the intein which can occur during the expression, and the fact that cleavage occurs over several hours, which could lead to loss of protein due to aggregation, especially for Httex1 with expanded polyQ repeats.
To address these limitations and to refine our strategy for the production of native, tag-free Httex1, we developed a new expression system based on the transient fusion of SUMO, more exactly the yeast homolog Smt3 to Httex1. The application of the SUMO system for the production of recombinant proteins was first published in 200429, where an increased rate of expression and solubility of SUMO fusion protein was demonstrated. The SUMO tag can be cleaved by the ubiquitin like protein-specific protease 1 (ULP1), which does not require a recognition site, but recognizes the tertiary structure of SUMO and practically eliminates the possibility of miscleavage30. Furthermore, the ULP1-mediated cleavage is fast and traceless and does not leave additional residues behind. The premature cleavage of the fusion tag, as observed with the autocatalytic intein10, is completely avoided by the requirement of an external protease. While the SUMO strategy is nowadays widely used for recombinant protein production31,32,33, we demonstrate in this paper that it is especially useful for the generation of an intrinsically disordered, aggregation-prone, amyloidogenic protein such as Httex1. We believe that the simplicity, efficiency and robustness of our SUMO-fusion-based method will make native, tag-free Httex1 more accessible to researchers from different disciplines and eliminate the need to use non-native sequences of Httex1 in vitro. This is an important advance that will facilitate future studies to elucidate the structure-function relationship of Httex1.
The protocol describes the purification of Httex1 from 12 L of bacterial culture, but the protocol could be easily adapted for smaller or larger scale productions. The protocol describes the production of wild type Httex1 (wtHttex1) with a polyQ repeat length below (23Q) and mutant Httex1 (mHttex1) with a polyQ repeat length above (43Q) the pathogenic threshold (36Q).

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			<td height="20" style="height:20px;">High Precision Cell made of Quartz SUPRASIL 1 mm light path from Hellma Analytics</td>
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			<td height="20" style="height:20px;">Anti-Huntingtin Antibody, a.a. 1-82</td>
			<td>Merck Millipore Corporation</td>
			<td>MAB5492</td>
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			<td height="20" style="height:20px;">IRDye 680RD Goat anti-Mouse IgG (H + L)</td>
			<td>Licor</td>
			<td>925-68070</td>
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			<td height="20" style="height:20px;">PMSF</td>
			<td>AxonLab</td>
			<td>A0999.0005</td>
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			<td height="20" style="height:20px;">HisPrep 16/10 column&#160;</td>
			<td>GE Healthcare</td>
			<td>28936551</td>
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			<td height="20" style="height:20px;">C4 HPLC column</td>
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			<td>00G-4168.P0</td>
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			<td height="20" style="height:20px;">Filtre seringue Filtropur S 0,45 ul sans prefiltre sterile</td>
			<td>Sarstedt AG</td>
			<td>83.1826</td>
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			<td height="20" style="height:20px;">Spectrophotometer semi-micro cuvette</td>
			<td style="width:193px;">Reactolab S.A.</td>
			<td>2534</td>
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			<td height="20" style="height:20px;">Superloop, 1/16&#34; fittings (&#196;KTAdesign), 50 ml</td>
			<td>GE Healthcare</td>
			<td>18111382</td>
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			<td height="20" style="height:20px;">Trifluoroacetic acid</td>
			<td>Sigma</td>
			<td>302031</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">GREINER Tubes fo FPLC 16 x 100 mm, cap. 12.0 ml</td>
			<td style="width:193px;">Greiner Bio-One</td>
			<td>7.160 102</td>
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			<td height="20" style="height:20px;">&#160;100 kD Microcon&#160; fast flow filters</td>
			<td style="width:193px;">Merck Millipore Corporation</td>
			<td>MRCF0R100</td>
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			<td height="20" style="height:20px;">Vibra-cell VCX130 ultrasonic liquid processor</td>
			<td>Sonics</td>
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			<td height="20" style="height:20px;">&#196;kta 900 equipped with a fraction collector&#160;</td>
			<td>GE Healthcare</td>
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			<td></td>
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			<td height="20" style="height:20px;">Jasco J-815 Circular Dichroism</td>
			<td>Jasco</td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;">Waters UPLC system&#160;</td>
			<td>Waters</td>
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			<td>C8 BEH acquity 2.1x100 mm 1.7 micron column&#160; , preheated column (40 &#176;C), flow rate of 0.6 mL/min, injection volume of 4 &#956;L</td>
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			<td height="20" style="height:20px;">waters HPLC system</td>
			<td>Waters</td>
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			<td>2489 UV detector and 2535 quaternary gradient module, 20 mL loop in a FlexInject housing</td>
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			<td height="20" style="height:20px;">ESI-MS: Finnigan LTQ</td>
			<td>Thermo Fisher Scientific</td>
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			<td height="20" style="height:20px;">lyophylizer instrument</td>
			<td>FreeZone 2.5 Plus</td>
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			<td height="20" style="height:20px;">Tecnai Spirit BioTWIN&#160;</td>
			<td>FEI</td>
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			<td>electron microscope equipped with a LaB6 gun and a 4K x 4K FEI Eagle CCD camera (FEI) and operated at 80 kV</td>
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			<td height="20" style="height:20px;">37 &#176;C shaking&#160; incubator</td>
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			<td height="20" style="height:20px;">Biophotometer plus</td>
			<td>Eppendorf</td>
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			<td></td>
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generation of native, untagged huntingtin exon1 monomer and fibrils using a sumo fusion strategy

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, resulting in the expression of the Huntingtin protein (Htt) or N-terminal fragments thereof with an expanded polyglutamine (polyQ) stretch.
The exon1 of the Huntingtin protein (Httex1) is the smallest Htt fragment that recapitulates many of the features of HD in cellular and animal models and is one of the most widely studied fragments of Htt. The small size of Httex1 makes it experimentally more amenable to biophysical characterization using standard and high-resolution techniques in comparison to longer fragments or full-length Htt. However, the high aggregation propensity of mutant Httex1 (mHttex1) with increased polyQ content (&#8805;42) has made it difficult to develop efficient expression and purification systems to produce these proteins in sufficient quantities and make them accessible to scientists from different disciplines without the use of fusion proteins or other strategies that alter the native sequence of the protein. We present here a robust and optimized method for the production of milligram quantities of native, tag-free Httex1 based on the transient fusion of small ubiquitin related modifier (SUMO). The simplicity and efficiency of the strategy will eliminate the need to use non-native sequences of Httex1, thus making this protein more accessible to researchers and improving the reproducibility of experiments across different laboratories. We believe that these advances will also facilitate future studies aimed at elucidating the structure-function relationship of Htt as well as developing novel diagnostic tools and therapies to treat or slow the progression of HD.

Httex1 is expressed in E. coli with an N-terminal His6-SUMO tag. The representative results of the expression and purification of the fusion protein are summarized in Figure 1. The sequence of Httex1 consists of the residues 2-90 of Htt and starts with Ala2, because Met1 is fully cleaved in vivo42. The numbering of the amino acids refers to the 23Q variant, the complete sequence of the expressed fusion protein is shown in Figure 1A. The plasmids will be deposited at Addgene in the near future to be shared with the community. A schematic of the plasmid and the expressed fusion protein is shown in Figure 1B. His6-SUMO Httex1 expresses at a medium level (Figure 1C) and most of the fusion protein is present in the soluble fraction after lysis, both for the 23Q and the 43Q variant. The fusion protein migrates higher than expected, based on the molecular weight. This is partly due to the strong fold of SUMO but mostly due to the unusual sequence composition of Httex1, containing mainly glutamine and proline residues. Both the wildtype (23Q) and the mutant (43Q) fusion protein can be enriched to ~80% purity by IMAC (Figure 1D) The presence of co-purifying host protein can be explained by the comparatively low expression level of Httex1 and the big sample volume applied to the column.
The cleavage of the His6-SUMO tag and the purification of Httex1 is shown in Figure 2A. UPLC is an efficient tool to monitor the cleavage of the His6-SUMO tag (Figure 2B). The original peak of the fusion protein is consumed and two new and well-separated peaks corresponding the His6-SUMO tag and Httex1 appear. The cleavage reaction is finished in 10-20 min. The Western Blot (WB) is too slow to monitor the cleavage reaction efficiently, but it has been included in the figure for reference and to demonstrate the completeness of the SUMO cleavage. Both Httex1 23Q and 43Q can be separated well from the His6-SUMO tag by RP-HPLC (Figure 2C) and were obtained in high purity as shown by UPLC, MS and SDS-PAGE analysis (Figure 2D).
To illustrate that the Httex1 proteins prepared by this method retain the expected aggregation properties of Httex1, we assessed the fibrillization kinetics of mutant Httex1 at 37 &#176;C by a sedimentation assay, monitored the changes in secondary structure by CD spectroscopy, and characterized the morphology of the aggregates by TEM. A representative data set of the aggregation kinetics of mHttex1 fibril formation as determined by a sedimentation assay is shown inFigure 3A. The loss of soluble Httex1 43Q over time, due to fibril formation was quantified by UPLC. We observe a complete depletion of soluble protein after 48 hours of incubation. Additionally, we determined the secondary structure of the protein by CD spectroscopy (Figure 3B). Httex1 43Q shifts from unstructured (&#955;min 205 nm) to mainly &#946;-sheet rich conformation (&#955;min 215 nm) after 48 hours of incubation. This structural change is accompanied by the formation of long fibrillar aggregates as observable by TEM at 48 hours (Figure 3C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57506/57506fig1.jpg" /> 
Figure 1. Expression and purification of the His6-SUMO Httex1 fusion protein.&#160; 
(A) The amino acid sequence of the His6-SUMO-Httex1-QN fusion constructs (His6-SUMO in green and Httex1-QN in orange); (B) Schematic overview of the expression and purification of the fusion protein; (C) SDS-PAGE analysis of the expression and the solubility of the fusion protein after lysis; (D) Chromatogram of the IMAC purification of the fusion protein and analysis of the fractions by SDS-PAGE (red bar: unbound fraction, blue bar: wash fraction, black bar: fractions containing the elution peak);&#160;<a href="/files/ftp_upload/57506/57506fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57506/57506fig2.jpg" /> 
Figure 2. Cleavage of the His6 SUMO tag and purification of tag-free Httex1-QN proteins. 
(A) schematic overview; (B) analysis of the cleavage of the SUMO tag with ULP1 by UPLC (blue: before addition of ULP1; black: 20 min (23Q), respectively 10 min (43Q) after addition of ULP1) and WB (MAB5492 1:2000, secondary goat anti mouse antibody 1:5000); (C) Chromatogram of the preparative RP-HPLC purification of Httex1; D: analysis of the purified Httex1 by UPLC, SDS-PAGE and ESI-MS; the expected molecular weight is 9943 Da (23Q) and 12506 Da (43Q) respectively.&#160;<a href="/files/ftp_upload/57506/57506fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57506/57506fig3.jpg" /> 
Figure 3. Aggregation of Httex1-43Q: (A) Sedimentation assay based on UPLC. (B) CD spectra of the secondary structure at 0 h and 48 h. (C) TEM micrographs of the aggregates at 48 h (scale bars are 200 nm). <a href="/files/ftp_upload/57506/57506fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy at JoVE.com

Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy

Here, we present a robust and optimized protocol for the production of milligram quantities of native, tag-free monomers and fibrils of the exon1 of the Huntingtin protein (Httex1) based on the transient fusion of small ubiquitin related modifier (SUMO).

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, ...

generation-native-untagged-huntingtin-exon1-monomer-fibrils-using

Nanyang Technological University

Research

JoVE Journal

Biochemistry

7.9K Views.  Ecole Polytechnique Fédérale de Lausanne (EPFL). Here, we present a robust and optimized protocol for the production of milligram quantities of native, tag-free monomers and fibrils of the exon1 of the Huntingtin protein (Httex1) based on the transient fusion of small ubiquitin related modifier (SUMO).

Video: Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a key pathological feature of HD is the aberrant accumulation of mutant HTT (mHTT) protein into high molecular weight complexes and intracellular inclusion bodies composed of fragments and other proteins. Conventional methods to measure and understand the contributions of various forms of mHTT-containing aggregates include fluorescence microscopy, western blot analysis, and filter trap assays. 
However, most of these methods are conformation specific, and therefore may not resolve the full state of mHTT protein flux due to the complex nature of aggregate solubility and resolution.
For the identification of aggregated mHTT and various modified forms and complexes, separation and solubilization of the cellular aggregates and fragments is mandatory. Here we describe a method to isolate and visualize soluble mHTT, monomers, oligomers, fragments, and an insoluble high molecular weight (HMW) accumulated mHTT species. HMW mHTT tracks with disease progression, corresponds with mouse behavior readouts, and has been beneficially modulated by certain therapeutic interventions1. This approach can be used with mouse brain, peripheral tissues, and cell culture but may be adapted to other model systems or disease contexts.

A method is described for fractionation of insoluble and soluble mutant huntingtin species from mouse brain and cell culture. The method described is useful for characterization and quantification of huntingtin protein flux and aids in analyzing protein homeostasis in disease pathogenesis and in the presence of perturbations

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes.
In this assay system, the applied substrates contain N-terminal hexahistidine (His6) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.

Here, we present the detailed procedure of a recently developed protease assay platform utilizing N-terminal hexahistidine/maltose-binding protein and fluorescent protein-fused recombinant substrates attached to the surface of nickel-nitrilotriacetic acid magnetic agarose beads. A subsequent in-gel analysis of the assay samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is also presented.

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

A Strategy to Identify Compounds that Affect Cell Growth and Survival in Cultured Mammalian Cells at Low-to-Moderate Throughput

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug screening assays utilize cytotoxicity as one of the critical characteristics to be profiled for individual compounds. Cells in culture are a useful model to assess cytotoxicity before proceeding to follow up on promising lead compounds in more costly and labor-intensive animal models. We describe a strategy to identify compounds that affect cell growth in a tdTomato expressing human neural stem cells (NSC) line. The strategy uses two complementary assays to assess cell number. One assay works via the reduction of 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan as a proxy for cell number and the other directly counts the tdTomato expressing NSCs. The two assays can be performed simultaneously in a single experiment and are not labor intensive, rapid, and inexpensive. The strategy described in this demonstration tested 57 compounds in an exploratory primary screen for toxicity in a 96-well plate format. Three of the hits were characterized further in a six-point dose response using the same assay set-up as the primary screen. In addition to providing excellent corroboration for toxicity, comparison of results from the two assays may be effective in identifying compounds affecting other aspects of cell growth.

It is often necessary to assess the potential cytotoxicity of a set of compounds on cultured cells. Here, we describe a strategy to reliably screen for toxic compounds in a 96-well format.

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug ...

Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. This method utilizes a recombinant modified SUMO-trapping protein fragment, kmUTAG, derived from the Ulp1 SUMO protease of the stress-tolerant budding yeast Kluyveromyces marxianus. We have adapted the properties of the kmUTAG for the purpose of studying sumoylation in a variety of model systems without the use of antibodies. For the study of SUMO, KmUTAG has several advantages when compared to antibody-based approaches. This stress-tolerant SUMO-trapping reagent is produced recombinantly, it recognizes native SUMO isoforms from many species, and unlike commercially available antibodies it shows reduced affinity for free, unconjugated SUMO. Representative results shown here include the localization of SUMO conjugates in mammalian tissue culture cells and nematode oocytes.

SUMO is an essential and highly conserved, small ubiquitin-like modifier protein. In this protocol we are describing the use of a stress-tolerant recombinant SUMO-trapping protein (kmUTAG) to visualize native, untagged SUMO conjugates and their localization in a variety of cell types.

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in liver cancer, the modification by SUMO (Small Ubiquitin MOdifier) has been given the most attention. Isolation of endogenous SUMOylated proteins in vivo is challenging due to the presence of active SUMO-specific proteases. Initial studies of SUMOylation in vivo were based on the molecular detection of specific SUMOylated proteins (e.g., by western blot). However, in many cases, antibodies, generally made with non-modified recombinant protein, did not immunoprecipitate SUMOylated forms of the protein of interest.&#160;Nickel chromatography has been the other approach to study SUMOylation by capturing histidine-tagged versions of SUMO molecules. This approach is mainly used in cells stably expressing or transiently transfected with His-SUMO molecules. To overcome these limitations, SUMO-binding entities (SUBEs) were developed to isolate endogenous SUMOylated proteins. Herein, we describe all the steps required for the enrichment, isolation, and identification of SUMOylated substrates from human hepatoma cells and hepatic tissues from a liver cancer mouse model by using SUBEs. Firstly, we describe methods involved in the preparation and lysis of the human hepatoma cells and liver tumor tissue samples. Then, a thorough explanation of the preparation of SUBEs and controls is detailed along with the protocol for the protein pull-down assays. Finally, some examples are provided regarding the options available for the identification and characterization of the SUMOylated proteome, namely the use of western-blot analysis for the detection of a specific SUMOylated substrate from liver tumors or the use of proteomics by mass spectrometry for high-throughput characterization of the SUMOylated proteome and interactome in hepatoma cells.

SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Here, we present a protocol to enrich, isolate, identify, and characterize proteins modified by SUMO in vivo both from human hepatoma cells and liver tumors obtained from mouse models of hepatocellular carcinoma by using SUMO-binding entities (SUBEs).

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in ...