Name: studying protein function and the role of altered protein expression by antibody interference and three-dimensional reconstructions
Uploaded: 2016-04-21T13:00:00
Description: Watch this Scientific Journal Video about Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions at JoVE.com

The presented work was supported in parts by funding from the Canadian Institutes of Health Research / Fragile X Research Foundation of Canada partnership program to RD, the Jerome Lejeune Foundation to RD, the Interdisziplin&#228;res Zentrum f&#252;r klinische Forschung of the University Erlangen-Nuremberg to RD and from the Deutsche Forschungsgemeinschaft to RD and RE. The authors wish to thank Ingrid Zenger for technical assistance with cell culture maintenance as well as Prof. M. Wegner for making a pCMV5-FLAG vector available. The authors further wish to particularly thank Nadja Schroeder for the helpful support at the video shooting.

1. Stock Solutions
<ol>
	<li>Resuspend the lyophilized active motif powder in sterile H2O to a final concentration of 2 &#181;g/&#181;l. Tap carefully for mixing.</li>
	<li>Prepare small aliquots (e.g., 12 &#181;l each, 2 &#181;l are required per reaction) and store them at -20 &#176;C.</li>
</ol>
2. Preparation of Cells
<ol>
	<li>Seed mammalian cells such as HEK293 cells or primary neurons on a 24 well plate in 500 &#181;l growth medium including antibiotics. 
	NOTE: ...

<ol>
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Here, we present a protocol to study the significance of protein expression levels in controlling cellular functions in a dose driven manner. The protocol described allows for a fine-tuned manipulation of protein expression in various mammalian cell types, including hippocampal neurons, hence facilitating detailed studies of protein function on the cellular level.
Although RNA interference represents a well known strategy to down-regulate POIs, it has its disadvantages (cp. 14). Not...

A tight control of protein expression is essential for every living organism to command its own development as well as to react to environmental signals. Hence, a multitude of mechanisms has been invented during evolution to precisely regulate the expression level of each protein encoded by the app. 20,000 genes existing in any eukaryotic cell at any given time of its life. Taking place at different stages of protein production, regulatory mechanisms range from the management of chromatin structure, transcription and RNA handling to the direction of posttranslational protein modifications, transport and degradation.
It is therefore not surprising that malfunctions in the underlying molecular machineries and altered protein expression levels have been associated with diverse diseases such as cancer or intellectual disabilities. Indeed, looking at the outstanding complexity of neuronal development and mammalian brain function, the sensitivity of these sophisticated systems to alterations in protein expression manifests itself in several well-known intellectual deficits including Alzheimer and Parkinson disease (AD and PD) as well as autism spectrum disorders (ASD) like the Fragile X Syndrome (FXS). The latter disease is characterized by an extensive misexpression of a variety of proteins, which is due to the loss of a single translation regulating protein, FMRP (Fragile X Mental Retardation Protein)1-4. Furthermore, chromosomal rearrangements affecting the Variable charge x linked protein A (VCXA), a protein, that manages mRNA stability and translation by modifying mRNA capping5, have recently been associated with intellectual deficits, while point mutations were not identified in patients with cognitive disabilities as of now6,7, suggesting that the observed mental impairments originate from altered VCXA expression and dysregulated expression of its target proteins. In line with these findings, a study investigating whether de novo copy number variations of genes are associated with ASD established that novel gene duplications and deletions are a significant risk factor for ASD8, thus supporting the idea that elevated or diminished protein expression levels may cause intellectual deficits.
Remarkably, recent research further provided evidence that the expression level of a given protein is precisely adjusted to prevent its aggregation as a consequence of high protein amounts with almost no safety margins9. It has therefore been proposed that even small increments are sufficient to induce diseases such as AD and PD9. Although the variety of molecular machineries contributing to protein expression control suggests a complex regulation scheme in the light of these findings, a study investigating the expression level of over 5,000 mammalian genes10 demonstrated that nature preferred a more parsimonious scheme: The cellular abundance of proteins was shown to be predominantly regulated at the level of translation10, thus illustrating that the management of RNA availability mainly serves to fine-tune protein expression.
Studying the dose of proteins of interest (POIs) is therefore not only important to the understanding of the endogenous functions of a protein, but also to the investigation of many diseases and the development of therapies. Thus, last decades have seen the advance of several strategies using RNA interference to manipulate POI dosage. Though RNA interference is widely used to study protein function and is even being applied in clinical trials to treat cancer or ocular diseases as well as to pursue antiviral therapies in patients11-13, some difficulties may arise which might render the strategy impossible. For example, the seed sequence, which drives the knockdown by homology is comparable short, hence promoting off-target effects. Since highly efficient sequences are rare and need to be found among thousands of options (reviewed in 14), identifying the right sequence can be time-consuming and costly, but results may still be disappointing.
An alternative strategy is to directly target the POI by antibodies. Here, we illustrate the use of the protein carrier Chariot&#160;(manufactured by Active Motif) to reduce cellular protein availability, and the employment of three-dimensional reconstructions to study protein function following knock-down.
The active motif of Chariot, itself a 2.8 kDa peptide, is used to shuttle peptides, proteins and antibodies across membranes of mammalian cells15. The peptide associates with POIs by forming non-covalently coupled macromolecular complexes utilizing hydrophobic interactions, whereupon Chariot-POI complexes are internalized into cells in an endosome-independent manner. Importantly, Chariot was indicated to neither affect the intracellular localization of shuttled proteins, nor to exert cytotoxic effects or to affect the biological activity of its cargo15.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Chariot</td>
			<td>Active Motif</td>
			<td>30025</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Life technologies</td>
			<td>21103-049</td>
			<td>warm up to 37&#176;C before using</td>
		</tr>
		<tr>
			<td>1xB27</td>
			<td>Life technologies</td>
			<td>17504044</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Life technologies</td>
			<td>25030-149</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td>&#160;Penicilline and Streptomycine</td>
			<td>Life technologies</td>
			<td>15140-122</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Bitplane</td>
			<td>n.a.</td>
			<td>expensive, but unmatched</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Microscope</td>
			<td>Zeiss</td>
			<td>n.a.</td>
			<td></td>
		</tr>
	</tbody>
</table>

studying protein function and the role of altered protein expression by antibody interference and three-dimensional reconstructions

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. Therefore, recent research invented different tools to target protein expression in mammalian cell lines or even animal models, including RNA and antibody interference. While the first strategy has gathered much attention during the past two decades, peptides &#160;mediating a translocation of antibody cargos across cellular membranes and into cells, obtained much less interest. In this publication, we provide a detailed protocol how to utilize a peptide carrier named Chariot in human embryonic kidney cells as well as in primary hippocampal neurons to perform antibody interference experiments and further illustrate the application of three-dimensional reconstructions in analyzing protein function. Our findings suggest that Chariot is, probably due to its nuclear localization signal, particularly well-suited to target proteins residing in the soma and the nucleus. Remarkably, when applying Chariot to primary hippocampal cultures, the reagent turned out to be surprisingly well accepted by dissociated neurons.

In the following paragraphs, exemplary results illustrating a functional knockdown of a POI (Simiate, for further details please see 16,17) using Chariot reagent and antibody interference are presented. The findings demonstrate that diminished expression of Simiate impairs transcriptional activity, and, in a dosage dependent manner, induces apoptosis, culminating in mortality rates of over 99% if higher antibody amounts (&#62; 1 &#181;g antibody) are applied (these discoveries were publis...

Watch this Scientific Journal Video about Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions at JoVE.com

Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

Controlling protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. The protocol presented shows the application of antibody interference in mammalian cells including primary hippocampal neurons and demonstrates the use of three-dimensional reconstructions in studying protein function.

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions ...

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