The authors acknowledge Dr. Mark Shaaya for his contribution to the development of LightR enzymes and associated protocols. pCAG-iCre was a gift from Wilson Wong (Addgene plasmid #89573), pcDNA3.1 Floxed-STOP mCherry was a gift from Mositoshi Sato (Addgene plasmid #122963), bRaf-Venus construct bearing V600E mutation was a gift from Dr. John O&#39;Bryan (MUSC); ERK2 gene from pCEFL-ERK2 (a gift from Dr. Channing Der&#39;s lab, UNC) was cloned into mCherry-C1 backbone to obtain mCherry-ERK2 plasmid; and pmiRFP670-N1 was a gift from Vladislav Verkhusha (Addgene plasmid # 79987). The work was supported by NIH grants R33CA258012, R35GM145318, and P01HL151327 to AK. This work was further supported by the T32 VBST training fellowship T32HL144459 to NL.

Optogenetic Control of Enzyme Activity Using Light-Regulated Allosteric Switch

The authors have nothing to disclose.

Our study presents an optogenetic approach for the investigation of diverse signaling pathways and demonstrates its wide applicability in addressing different biological questions. The LightR tool system provides several essential advantages: (1) Allosteric regulation of protein activity, (2) Tight temporal control of activity that can be tuned to achieve different kinetics of activation and inactivation, (3) Spatial resolution of activity at the subcellular level, (4) Specificity of signaling modulation and biological a...

The ability of the cell to interpret external signals and convert them into specific responses in physiological or pathological contexts is directed by dedicated groups of proteins. The contribution of any protein to such complex responses is often defined by its subcellular location, level of expression, and the timing of transient, sustained, or oscillatory activation. Dissecting the role of these individual parameters in the regulation of signaling demands methods capable of replicating intricate spatiotemporal control of protein activity at the subcellular level. Traditional techniques such as genetic manipulation and small molecule inhibitors fall short in this regard. In contrast, optogenetic techniques promise the potential for the dissection of biological processes by manipulating or mimicking physiological and pathological processes. However, current tools often lack broad applicability or subcellular control. Several existing strategies achieve tight regulation of protein localization and interactions; but lack direct control over enzymatic activity1,2,3,4. Others enable regulation of enzymatic activity but may lack subcellular control or have limited applicability across different enzyme classes5,6,7,8,9. In this protocol paper, we describe a novel protein engineering method that combines the advantages of optogenetics into one tool: tight temporal regulation, tunable kinetics, subcellular control, and broad enzyme applicability10. We engineered a Light-regulated (LightR) domain that functions as an allosteric switch when inserted into the target protein of interest. This strategy enables tight spatiotemporal control of the activation and deactivation of a protein of interest in living cells.
Here, the design and application strategy for the LightR optogenetic tool across several enzyme classes is discussed. This study offers a step-by-step protocol for the development, characterization, and application of light-regulated tyrosine kinase Src (LightR-Src). The study further demonstrates the tunability of LightR switch inactivation kinetics. The slow-inactivating LightR switch can maintain enzyme activity with reduced frequency of illumination, whereas fast cycling version, FastLightR, requires more frequent illumination for activation but shows fast inactivation when illumination is turned off. Activation/inactivation of FastLightR-Src in living cells induces cycles of cell spreading and retraction. FastLightR-Src-induced cell spreading agrees with the physiological role of Src kinase11,12. Due to fast inactivation kinetics, FastLightR-Src can also be regulated at a subcellular level, resulting in stimulation of local protrusions and cell polarization. To demonstrate the applicability of the LightR tool to other types of enzymes, we briefly walk the readers through similar success with engineered light-regulated kinase bRaf (LightR-bRaf, FastLightR-bRaf) and a site-specific DNA recombinase Cre (LightR-Cre). Overall, this approach has the potential to advance our understanding of complex signaling pathways shaping the pathophysiology of diverse diseases.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<tr>
			<td>#1.5 Glass Coverslips 25 mm Round</td>
			<td>Warner Instruments&#160;</td>
			<td>64-0715&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Tubes&#160;</td>
			<td>USA Scientific&#160;</td>
			<td>cc7682-3394</td>
			<td></td>
		</tr>
		<tr>
			<td>2x Laemmli Buffer</td>
			<td></td>
			<td></td>
			<td>For 500 mL: 5.18 g of&#160; Tris-HCL, 131.5 mL of glycerol, 52.5 mL of 20% SDS, 0.5 g of bromophenol blue, final pH 6.8</td>
		</tr>
		<tr>
			<td>4-20% Mini-PROTEAN TGX Precast Gel</td>
			<td>Biorad</td>
			<td>4561096</td>
			<td></td>
		</tr>
		<tr>
			<td>5x Phusion Plus Buffer&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>F538L</td>
			<td></td>
		</tr>
		<tr>
			<td>60 LED Microscope Ring Light</td>
			<td>Boli Optics</td>
			<td>ML46241324</td>
			<td>Blue LED, 60 mm diameter, 5 W</td>
		</tr>
		<tr>
			<td>Agarose&#160;</td>
			<td>GoldBiotech</td>
			<td>A-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Erk 1/2 Antibody</td>
			<td>Cell Signaling</td>
			<td>9102</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GAPDH Antibody</td>
			<td>invitrogen</td>
			<td>AM4300</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP</td>
			<td>Clontech</td>
			<td>632380</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mCherry Antibody</td>
			<td>invitrogen</td>
			<td>M11217</td>
			<td></td>
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		<tr>
			<td>Anti-MEK</td>
			<td>Cell Signaling</td>
			<td>9122</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-p130Cas</td>
			<td>BD Biosciences</td>
			<td>610271</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-paxillin</td>
			<td>Cell Signaling</td>
			<td>2542</td>
			<td></td>
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			<td>Anti-phospho-Erk 1/2 T202/Y204 Antibody</td>
			<td>Cell Signaling</td>
			<td>9101</td>
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		<tr>
			<td>Anti-phospho-pY249 p130Cas</td>
			<td>Cell Signaling</td>
			<td>4014</td>
			<td></td>
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		<tr>
			<td>Anti-phospho-Y118 Paxillin</td>
			<td>Cell Signaling</td>
			<td>2541</td>
			<td></td>
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			<td>Anto-phospho-S217/221 MEK</td>
			<td>Cell Signaling</td>
			<td>9121</td>
			<td></td>
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		<tr>
			<td>Arduino Compatable Power Supply</td>
			<td>Corporate Computer</td>
			<td>LJH -186</td>
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			<td>Arduino Uno Rev3</td>
			<td>Arduino</td>
			<td>&#8206;A000066</td>
			<td></td>
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		<tr>
			<td>Attofluor Cell Chamber</td>
			<td>invitrogen</td>
			<td>A7816</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchmark Fetal Bovine Serum (FBS)&#160;</td>
			<td>Gemini Bio-products</td>
			<td>100-106</td>
			<td>Heat Inactivated Triple 0.1 &#181;m sterile-filtered&#160;</td>
		</tr>
		<tr>
			<td>bRaf-V600E-Venus</td>
			<td></td>
			<td></td>
			<td>Gift from Dr. John O&#39;Bryan, MUSC</td>
		</tr>
		<tr>
			<td>BSA&#160;</td>
			<td>GoldBiotech</td>
			<td>A-420&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbenicillin (Disodium)</td>
			<td>Gold Biotechnology</td>
			<td>C-103-25</td>
			<td></td>
		</tr>
		<tr>
			<td>CellMask Deep Red plasma membrane dye</td>
			<td>invitrogen</td>
			<td>c10046</td>
			<td></td>
		</tr>
		<tr>
			<td>Colony Screen MasterMix&#160;</td>
			<td>Genesee</td>
			<td>42-138</td>
			<td></td>
		</tr>
		<tr>
			<td>DH5a competent cells&#160;</td>
			<td>NEB&#160;</td>
			<td>C2987H</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM&#160;</td>
			<td>Corning&#160;</td>
			<td>15-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Ladder&#160;</td>
			<td>GoldBio&#160;</td>
			<td>D010-500</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs&#160;</td>
			<td>NEB</td>
			<td>N04475</td>
			<td></td>
		</tr>
		<tr>
			<td>Dpn1 Enzyme&#160;</td>
			<td>NEB</td>
			<td>R01765</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>GoldBio</td>
			<td>DTT10</td>
			<td>DL-Dithiothreitol, Cleland&#39;s Reagents&#160;</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Acros&#160;</td>
			<td>409910250</td>
			<td></td>
		</tr>
		<tr>
			<td>FastLightR-bRaf-mVenus</td>
			<td>Addgene</td>
			<td>#162155</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin from bovine plasma&#160;</td>
			<td>Sigma&#160;</td>
			<td>F1141</td>
			<td></td>
		</tr>
		<tr>
			<td>FuGENE(R) 6 Transfection Reagent&#160;</td>
			<td>Promega</td>
			<td>E2692</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>Gel Green Nucleic Acid Stain&#160;</td>
			<td>GoldBio</td>
			<td>G-740-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Loading Dye Purple 6x&#160;</td>
			<td>NEB</td>
			<td>B7024A</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneJET Gel extraction Kit&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>K0692</td>
			<td>Gel Extraction Kit&#160;</td>
		</tr>
		<tr>
			<td>GeneJET Plasmid Miniprep Kit</td>
			<td>Thermo Scientific&#160;</td>
			<td>K0502</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>GlutaMAX-l (100x) 100 mL&#160;</td>
		</tr>
		<tr>
			<td>HEK 293T Cells&#160;</td>
			<td>ATCC&#160;</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa Cells&#160;</td>
			<td>ATCC&#160;</td>
			<td>CRM-CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fischer&#160;</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Iot Relay</td>
			<td>Digital Loggers</td>
			<td>DLI 705020645490</td>
			<td>AC/DC control relay for illumination</td>
		</tr>
		<tr>
			<td>Kanamycin Monosulfate</td>
			<td>Gold Biotechnology</td>
			<td>K-120-25</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma&#160;</td>
			<td>P-4504</td>
			<td></td>
		</tr>
		<tr>
			<td>L-15 1x</td>
			<td>Corning&#160;</td>
			<td>10-045-CV&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar&#160;</td>
			<td>Fisher</td>
			<td>BP1425-2</td>
			<td></td>
		</tr>
		<tr>
			<td>LED Grow Light System</td>
			<td>HQRP</td>
			<td>884667106091218</td>
			<td>LED panel lamp system&#160;</td>
		</tr>
		<tr>
			<td>LightR-bRaf-mVenus</td>
			<td>Addgene</td>
			<td>#162154</td>
			<td></td>
		</tr>
		<tr>
			<td>LightR-iCre-miRFP670</td>
			<td>Addgene</td>
			<td>#162158</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td>R2024a</td>
			<td>Software for running CellGEO Scripts</td>
		</tr>
		<tr>
			<td>Metamorph</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Imaging Analysis Software</td>
		</tr>
		<tr>
			<td>MgCl2&#160;</td>
			<td>Fisher Chemical</td>
			<td>M33-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Sigma&#160;</td>
			<td>M5310</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniPrep Kit&#160;</td>
			<td>Gene Choice&#160;</td>
			<td>96-308</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN TGX Precast Gels 12 well</td>
			<td>Bio-Rad</td>
			<td>4561085</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Biology Grade Water&#160;</td>
			<td>Corning&#160;</td>
			<td>46-000-CV&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiband Polychroic Mirror</td>
			<td>89903BS</td>
			<td>Chroma</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl&#160;</td>
			<td>Fisher Chemical</td>
			<td>S271-3&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS w/o Ca and Mg&#160;</td>
			<td>Corning&#160;</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>pCAG-iCre</td>
			<td>Addgene</td>
			<td>#89573</td>
			<td></td>
		</tr>
		<tr>
			<td>pcDNA3.1_Floxed-STOP mCherry</td>
			<td>Addgene</td>
			<td>#122963</td>
			<td></td>
		</tr>
		<tr>
			<td>pCEFL-ERK2</td>
			<td></td>
			<td></td>
			<td>Gift from Dr. Channing Der&#39;s Lab, UNC</td>
		</tr>
		<tr>
			<td>PCR Tubes&#160;</td>
			<td>labForce</td>
			<td>1149Z65</td>
			<td>0.2 mL 8-Strip Tubes and Caps, Rigid Strip Individually Attached Dome Caps&#160;</td>
		</tr>
		<tr>
			<td>Phusion Plus DNA Polymerase</td>
			<td>Thermo Scientific&#160;</td>
			<td>F630S</td>
			<td></td>
		</tr>
		<tr>
			<td>pmiRFP670-N1</td>
			<td>Addgene</td>
			<td>#79987</td>
			<td></td>
		</tr>
		<tr>
			<td>Polygon 400 Patterned Illuminator</td>
			<td>Mightex</td>
			<td>DSI-G-00C</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers&#160;</td>
			<td>IDT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF Membranes</td>
			<td>BioRad</td>
			<td>1620219</td>
			<td>Immun-Blot PVDF/Filter Paper Sandwiches&#160;</td>
		</tr>
		<tr>
			<td>T0.25% Trypsin, 2.21 mM, eDTA, 1x [-] sodium</td>
			<td>Corning&#160;</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Acetate-EDTA (TAE) 50x&#160;</td>
			<td>Fischer&#160;</td>
			<td>BP1332-1&#160;</td>
			<td>For electrophoresis&#160;</td>
		</tr>
		<tr>
			<td>UPlanSApo 40x Microscope Objective</td>
			<td>Olympus</td>
			<td>1-U2B828</td>
			<td></td>
		</tr>
		<tr>
			<td>USB TTL Box</td>
			<td>National Instruments</td>
			<td>6501</td>
			<td>For TTL interface</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Fisher Chemical&#160;</td>
			<td>O3446I-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

spatiotemporal control of protein activity through optogenetic allosteric regulation

Optogenetics offers the potential for mimicking complex spatiotemporal control of enzyme activity down to a subcellular resolution. However, most optogenetic approaches often face significant challenges in integrating multiple capabilities in a single tool applicable to a wide range of target proteins. Achieving precise control over ON/OFF kinetics, ensuring minimum leakiness in the dark, and demonstrating efficient performance in mammalian cells with subcellular precision are some of the most common challenges faced in this field. A promising solution lies in the application of rationally designed light-sensitive domains to allosterically control protein activity. Using that strategy, we generated an optogenetic method combining all the desired features. The approach involves the incorporation of the Light-regulated allosteric switch module (LightR) in the target protein to regulate enzyme activity using blue (465 nm) light. The LightR domain is generated by linking two Vivid (VVD) photoreceptor domains, creating a light-sensitive clamp that can be incorporated into a small flexible loop within the catalytic domain of an enzyme. In its dark state, LightR clamp is open, thus distorting the enzyme&#39;s catalytic domain and inactivating it. Upon exposure to blue light, the LightR domain closes and restores the catalytic domain&#39;s structure and enzyme activity. In this manuscript, we discuss design strategies&#160;to generate a light-regulated protein kinase and demonstrate its control by blue light, reversibility, kinetics, and precise regulation at the subcellular level, enabling tight spatiotemporal precision. Utilizing Src tyrosine kinase as a model, we showcase a protocol for effectively regulating LightR-Src kinase activity. We also demonstrate LightR applicability across different enzyme classes, expanding the utility of the tool system in addressing mechanistic questions of signaling pathways in different diseases.

The LightR-Src is designed and generated following the strategy described in Figure 1A,B. Biochemical analysis of LightR-Src accesses the phosphorylation of known endogenous Src substrates, p130Cas (Y249)22 and paxillin (Y118)23 in response to blue light at 60 min of continuous illumination (Figure 2B). Notably, no background activation of Src kinase is observed when LightR-Src is expressed only in...

Spatiotemporal Control of Protein Activity through Optogenetic Allosteric Regulation

This protocol describes the application of an engineered blue-light-activated allosteric switch (LightR) domain for reversible spatiotemporal control of protein activity. Utilizing Src tyrosine kinase as a model, this study offers an elaborate protocol for developing and characterizing light-regulated Src (LightR-Src). It demonstrates the versatility of this approach across enzyme classes.

Optogenetics offers the potential for mimicking complex spatiotemporal control of enzyme activity down to a subcellular resolution. However, most ...

spatiotemporal-control-protein-activity-through-optogenetic

Research

JoVE Journal

Biology

385 Views.  University of Illinois at Chicago. This protocol describes the application of an engineered blue-light-activated allosteric switch (LightR) domain for reversible spatiotemporal control of protein activity. Utilizing Src tyrosine kinase as a model, this study offers an elaborate protocol for developing and characterizing light-regulated Src (LightR-Src). It demonstrates the versatility of this approach across enzyme classes.

Video: Spatiotemporal Control of Protein Activity through Optogenetic Allosteric Regulation

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Quantifying Agonist Activity at G Protein-coupled Receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (&#949;) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1.
Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (&tau;). The estimate of &tau;Kobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (&tau;sys). In this case, the Kb value of an agonist is equivalent to &tau;Kobs/&tau;sys 3.
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this principle, the purification of critical proteins required for the functioning of these pathways along with their native interacting partners has not only allowed the mapping of the protein constituents of these pathways, but has also provided a deeper understanding of how these proteins coordinate to regulate these pathways. Within this context, understanding a protein's spatiotemporal localization and its protein-protein interaction network can aid in defining its role within a pathway, as well as how its misregulation may lead to disease pathogenesis. To address this need, several approaches for protein purification such as tandem affinity purification (TAP) and localization and affinity purification (LAP) have been designed and used successfully. Nevertheless, in order to apply these approaches to pathway-scale proteomic analyses, these strategies must be supplemented with modern technological developments in cloning and mammalian stable cell line generation. Here, we describe a method for generating LAP-tagged human inducible stable cell lines for investigating protein subcellular localization and protein-protein interaction networks. This approach has been successfully applied to the dissection of multiple cellular pathways including cell division and is compatible with high-throughput proteomic analyses.

We describe a method for generating localization and affinity purification (LAP)-tagged inducible stable cell lines for investigating protein function, spatiotemporal subcellular localization and protein-protein interaction networks.

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

Myeloid Innate Signaling Pathway Regulation by MALT1 Paracaspase Activity

Besides its function in lymphoid cells, which has been addressed by numerous studies, the paracaspase MALT1 also plays an important role in innate cells downstream of pattern recognition receptors. Best studied are the Dectin-1 and Dectin-2 members of the C-type lectin-like receptor family that induce a SYK- and CARD9-dependent signaling cascade leading to NF-&#954;B activation, in a MALT1-dependent manner. By contrast, Toll-like receptors (TLR), such as TLR-4, propagate NF-&#954;B activation but signal via an MYD88/IRAK-dependent cascade. Nonetheless, whether MALT1 might contribute to TLR-4 signaling has remained unclear. Recent evidence with MLT-827, a potent and selective inhibitor of MALT1 paracaspase activity, indicates that TNF- production downstream of TLR-4 in human myeloid cells is independent of MALT1, as opposed to TNF- production downstream of Dectin-1, which is MALT1 dependent. Here, we addressed the selective involvement of MALT1 in pattern recognition sensing further, using a variety of human and mouse cellular preparations, and stimulation of Dectin-1, MINCLE or TLR-4 pathways. We also provided additional insights by exploring cytokines beyond TNF-, and by comparing MLT-827 to a SYK inhibitor (Cpd11) and to an IKK inhibitor (AFN700). Collectively, the data provided further evidence for the MALT1-dependency of C-type lectin-like receptor &#8212;signaling by contrast to TLR-signaling.

MALT1 regulates innate immunity but how this occurs remains ill-defined. We used the selective MALT1 paracaspase inhibitor MLT-827 to unravel the contribution of MALT1 to innate signaling downstream of Toll-like or C-type lectin-like receptors, demonstrating that MALT1 regulates the production of myeloid cytokines, and downstream of C-type lectin-like receptors, selectively.

Besides its function in lymphoid cells, which has been addressed by numerous studies, the paracaspase MALT1 also plays an important role in innate cells ...

Probing Structural and Dynamic Properties of Trafficking Subcellular Nanostructures by Spatiotemporal Fluctuation Spectroscopy

Imaging-derived mean square displacement (iMSD) is used to address the structural and dynamic properties of subcellular nanostructures, such as vesicles involved in the endo/exocytotic trafficking of solutes and biomolecules. iMSD relies on standard time-lapse imaging, is compatible with any optical setup, and does not need to dwell on single objects to extract trajectories. From each iMSD trace, a unique triplet of average structural and dynamic parameters (i.e., size, local diffusivity, anomalous coefficient) is calculated and combined to build the "iMSD signature" of the nanostructure under study. 
The potency of this approach is proved here with the exemplary case of macropinosomes. These vesicles evolve in time, changing their average size, number, and dynamic properties passing from early to late stages of intracellular trafficking. As a control, insulin secretory granules (ISGs) are used as a reference for subcellular structures that live in a stationary state in which the average structural and dynamic properties of the whole population of objects are invariant in time. The iMSD analysis highlights these peculiar features quantitatively and paves the way to similar applications at the sub-cellular level, both in the physiological and pathological states.

Imaging-derived mean square displacement (iMSD) analysis is applied to macropinosomes to highlight their intrinsic time-evolving nature in terms of structural and dynamic properties. Macropinosomes are then compared to insulin secretory granules (ISGs) as a reference for subcellular structures with time-invariant average structural/dynamic properties.

Imaging-derived mean square displacement (iMSD) is used to address the structural and dynamic properties of subcellular nanostructures, such as vesicles ...