Difference between revisions of "Team:Slovenia/Protease signaling/Orthogonality"

 
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<h1 class = "ui left dividing header"><span class="section">nbsp;</span>Protease-based signaling and logic</h1>
 
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<p><b><ul>
 
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<li>New antiparallel and destabilized coiled coil pairs were designed and functionally characterized in mammalian cells.
 
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<li>Coiled coils were combined with split luciferase fragments to design functions with logical negation.
 
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<li>Light and chemically inducible proteases were used as mediators in a functional proof of concept for fast regulated logic gates.
 
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<p>As the main challenge of our project was to create fast responsive synthetic circuits in cells, we sought to implement logic operations based on protein posttranslational 
 
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modification, rather than slower transcriptional activation. The developed set of <a href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Orthogonality">orthogonal
 
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proteases</a> that could additionally be split, provided the modules to implement logic functions, for which we had to design the appropriate framework. An inspiration
 
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was provided by the study by Shekhawat et al. in which they presented an <i>in vitro</i> protease sensor using autoinhibited coiled-coil <x-ref>Shekhawat2009</x-ref>.
 
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The principle of their approach was that the two segments of a split reporter are linked to the coiled coil dimer forming peptides. Dimerization of the two chains is
 
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prevented by the presence of an antiparallel coiled coil segment that inhibits the binding of its partner to other CC peptides. Reconstitution is enabled by the proteolytic
 
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cleavage of the linker between the coiled coil fused to the split reporter and the autoinhibitory segment, which dissociates and can therefore be replaced by a second
 
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coiled-coil forming peptide with the second segment of the split reporter <ref>4.12.0</ref>.</p>
 
  
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                        <b>Reporters</b>
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                        <b>Orthogonality</b>
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                        <b>Achievements</b>
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                        <b>Introduction</b>
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                        <b>TEVp variants</b>
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                        <b>TEVp homologs</b>
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                    <a class="item" href="//2016.igem.org/Team:Slovenia/Protease_signaling/Split_proteases">
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                        <b>Split proteases</b>
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                    </a>
  
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  <figcaption><b> Principle of the protease sensor based on autoinhibited coiled-coil interactions </b><br/> Coiled coil segments can reconstitute the active split
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  reporter after cleavage of the autoinhibitory segment.</figcaption>
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<p>We realized that the same design could be adapted for our orthogonal proteases by replacing the cleavage sites with appropriate protease target motif, such as
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for the orthogonal proteases PPVp and TEVp.</p>
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<p>The constructs B:nLuc, cLuc:A, A’:TEVs:B:nLuc and cLuc:A:PPVs:B’2A, which do not possess autoinhibitory segments, were tested for CC binding by measuring luciferase
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reconstitution. Constructs without protease cleavage sites (B:nLuc, cLuc:A ) were used as a control (<ref>4.12.1.</ref>). A’:TEVs:B:nLuc and cLuc:A:PPVs:B’2A were
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tested in the presence of TEVp and PPVp, which cleave off the autoinhibitory CC, resulting in split luciferase reconstitution. Additionally, different ratios of
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constructs were tested in order to obtain the best luciferase activity ((<ref>4.12.1.</ref>).</p>
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                            <h1 class="ui left dividing header"><span id="ach" class="section colorize">&nbsp;</span>Protease
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  <figcaption><b> Interactions and protease activated AB coiled-coil formation.</b><br/> HEK293T cells were transfected with appropriate plasmids, 24 h after
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                                orthogonality</h1>
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  transfection cells were lysed and double luciferase assay was performed. (A) B:nLuc and cLuc:A coiled-coils constructs fused with split firefly luciferase
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                            <div class="ui segment" style="background-color: #ebc7c7; ">
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  system spontaneously interact and reconstitute firefly luciferase. (B) A’:TEVs:B:nLuc and cLuc:A:PPVs:B’2A autoinhibitory CCs reconstitute activity of firefly
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                                <p><b>
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  luciferase upon cleavage by TEVp and PPV. Successive luciferase reconstitution is observed only when high amounts of both A’:TEVs:B:nLuc and cLuc:A:PPVs:B’2A.
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                                    <ul>
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  </figcaption>
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                                        <li>Two TEVp variants (TEVpE and TEVpH) were tested and proved to be mutually
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</figure>
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                                            orthogonal.
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<p>Results showed that very high amounts of the constructs based on same coiled-coil sequences used by Shekhawat et al <x-ref>Shekhawat2009</x-ref> (i.e. 50 ng of each)
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                                        <li>Upon overexpression none of the tested proteases were toxic to mammalian
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were needed to detect the firefly luciferase signal in mammalian cells (<ref>4.12.0</ref>). Therefore, we decided to engineer designed coiled-coils from a toolbox, used
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                                            cells, demonstrating that they do not interfere with essential cellular
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by the (<a href=” https://2009.igem.org/Team:Slovenia/Orthogonal_coiled-coils.html > 2009 Slovenian iGEM team </a>) <x-ref>Gradisar2011a</x-ref>. In order to design an
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                                            processes.
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antiparallel coiled coil-based system applicable for logic operation in living cells we took into consideration the rules that establish the orientation and strength
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of the affinity of the CCs and designed new coiled coils, expanding on the available collection of orthogonal CC and
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(<a href="https://2016.igem.org/Team:Slovenia/CoiledCoilInteraction"> modeled </a>) the ratio of the affinities that are required to obtain the optimal response at low
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leakage.</p>
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Further explanation ...
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<p><h3>Coiled coils<h/3>
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<p>Alpha-helical segment interaction is a common feature in protein tertiary and quaternary structures, where helices form complexes of two or more coils<ref>4.12.1.2</ref>. The most frequent interaction is between two alpha-helices, which form a dimeric coiled-coil. Interactions can occur both in the two parallel or antiparallel orientation of the coil pairs <x-ref>Hadley2006</x-ref>. The interaction strength of different coiled-coil pairs depends on their amino acid sequence and their structure, which determine the underlying noncovalent forces of attraction and repulsion the helices exert on each other. Understanding the rules that govern the interactions between coiled-coils is thus inherently linked to understanding their amino acid sequences <x-ref>Woolfson2005</x-ref>. </p>
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<p>Sequences of coiled-coils that form interactions have a characteristic seven amino acid repeat, called heptad repeat. The position of each amino acid within a heptad is presented in a unified nomenclature (<i>a,b,c,d,e,f,g</i>). Interaction between two coils occurs on a continuous patch along the side of each alpha-helix with each patch facing the core of the dimer’s interface <ref>4.12.1.2 </ref>B. The amino acid residues which occupy this strip correspond to the <i>a</i> and <i>d</i> positions of the heptad; they are generally hydrophobic and represent the driving force behind dimerization <x-ref>Woolfson2005</x-ref>. Coiled coils are additionally stabilized by ionic interactions between polar amino acids (Asp, Glu, His, Lys, Asn, Gln, Arg, Ser or Thr) in positions e and g <x-ref>Woolfson2005, Gradisar2011a</x-ref>; while amino acids in positions <i>b, c</i> and <i>f</i>, which are less important to interactions between the helices; contribute to helix stability and solubility. </p>
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  <figcaption><b>
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<figcaption><b> Coiled coil structure and schematic representation of heptad repeats</b><br/>(A) Structure of coiled-coil. Specific coiled-coil interactions in (B) parallel and (C) antiparallel orientation        </figcaption>
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<p>Two alpha-helices that form a coiled-coil can interact either in a parallel or in an antiparallel orientation <x-ref>Oakley1998</x-ref> (<ref>4.12.1.2</ref> B and C). The orientation of coiled coils is largely determined through interactions between amino acid residues in positions <i>e</i> and <i>g</i> <x-ref>Woolfson2005, Oakley1998</x-ref>. In coiled-coils with a parallel orientation, electrostatic interactions form between position g on the first and position e on the second alpha-helix. In coiled-coils with an antiparallel orientation, electrostatic interactions occur between <i>g:g’</i> and <i>e:e’</i> positions of the two helices <x-ref>Litowski2001</x-ref>. The repeating and predicable nature of these interactions can be used for the rational design of coiled coils <x-ref>Gradisar2011a</x-ref>. Antiparallel CC orientation allows for fusion of C-termini of N-part of split protein to N-termini of CC via a shorter linker, thereby likely resulting in more efficient reconstitution upon binding with appropriate CC partner.  As represented in the wheel helical projection in <ref>4.12.1.3</ref parallel CC are stabilized by electrostatic interactions <i>g:e’<i> and <i>e:g’</i>, while interactions between <i>g:g’</i> and <i>e:e’</i> positions stabilize antiparallel CC. While CC orientation is mainly influenced by electrostatic interactions specific amino acid residues such as Asn inside CC core can contribute to the orientation as well. Due to polarity of the Asn residue two asparagines prefer interaction with each other rather than with other hydrophobic residues in vicinity such as Leu and Ile. These interactions stabilize the core of intended CC orientation and destabilize the core of CC in the opposite orientation.</p>
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<p>In order to compare the reconstitution efficiency of split protein dictated by parallel or antiparallel coiled coil interaction, we prepared fusion proteins with split firefly luciferase where we designed a new antiparallel peptide (AP4) and tested their activity in cells. Antiparallel coiled coils (AP4:P3) worked significantly better than parallel coiled coils (P4:P3) (<ref>4.12.4.</ref>), thus demonstrating that a shorter linker between reporters and dimerizing units helps in the reconstitution of the split protein.</p>
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                                        <li>Additionally, three TEVp homologues (PPVp, SbMVp and SuMMVp) were tested and
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<p>In order to compare the reconstitution efficiency of split protein dictated by parallel or antiparallel coiled coil interaction, we prepared fusion proteins with split firefly luciferase where we designed a new antiparallel peptide (AP4) and tested their activity in cells. Antiparallel coiled coils (AP4:P3) worked significantly better than parallel coiled coils (P4:P3) (<ref>4.12.4.</ref>), thus demonstrating that a shorter linker between reporters and dimerizing units helps in the reconstitution of the split protein.</p>
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                                            proved to be fully orthogonal.
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  <figcaption><b> Comparison of the efficiency of the split luciferase reconstitution by parallel and antiparallel coiled coils. </b><br/> Reconstituted activity of the luciferase dictated by the parallel (left) and antiparallel coiled coil formation (right). HEK293-T cells were transfected with genetic fusions of coiled coil forming peptides and split luciferase. 24 h after transfection luciferase activity was measured. Coiled coil orientation is represented by coloring of each helix form blue (N-terminus) to red (C-terminus). N and C termini of split luciferase are represented by N or C, respectively.</figcaption>
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                            <h4><span id="mot" class="section colorize">&nbsp;</span></h4>
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                            <p>The first challenge in construction of a new protease-based signaling cascade was the
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                                selection of appropriate proteases. The candidate proteases should:
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                                i) recognize defined target cleavage sequences, preferably as long as possible; ii) be
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                                active in mammalian cells, but not toxic to them and iii) be inducible,
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                                ideally through the reconstitution of split protein fragments. Most importantly, a large
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                                number of proteases with similar properties but different
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                                substrates should be available to allow for modular construction of signaling pathways
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                                and logic functions. These proteases should be orthogonal to each
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                                other, meaning that their specific cleavage sites should not be recognized by other
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                                proteases in the system.
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                            </p>
  
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<p>To investigate whether the newly designed antiparallel CC is suited for implementation as logic unit into our system, the constructs nLuc:AP4 and P3:cLuc were compared to the coiled coil cLuc:A and B:nLuc from Shekhawat et al <x-ref>Shekhawat2009</x-ref>. Measurement of the reconstituted firefly luciferase activity showed that our designed coiled coils provided far higher (~50 fold) signal (<ref>4.12.5.</ref>), thus proposing the use of this new coiled coils for more sensitive logic gates that functions well in the cellular milieu.</p>
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                            <p>We found that the tobacco etch virus protease (TEVp) was the only protease described in
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                                the literature to match our criteria.</p>
  
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                                    Further explanation ...
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                                <div class="content">
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                                    <p>TEV protease is a highly specific, 242 amino acids long, 27 kDa cysteine
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                                        protease, that originates from the tobacco etch virus (TEV) of the Potyvirus
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                                        genus.
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                                        It has a target recognition sequence of seven amino acids, ENLYFQ-S/G, where
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                                        cleavage occurs after the glutamine residue (denoted by the – symbol),
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                                        and the final residue of the recognition sequence can be either S or G (denoted
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                                        by the / symbol). This substrate sequence is scarcely represented in the
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                                        proteome. TEV protease is therefore relatively non-toxic
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                                        <x-ref>Parks1994</x-ref>
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                                        and can be safely expressed in host cells. Due to this non-toxicity and
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                                        its high cleavage specificity, TEVp is an attractive protease for use in several
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                                        biotechnological applications, such as removal of affinity tags
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                                        from recombinant proteins.
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                                    </p>
 +
                                    <p>Despite its widespread use in biotechnology, TEVp also displays some
 +
                                        shortcomings, the most prominent of them being self-cleavage. Substitution
 +
                                        of Ser-219 with Val or Pro
 +
                                        <x-ref>Cesaratto2015</x-ref>
 +
                                        or a replacement of the C-terminal sequence MSELVYSQ with the sequence MNEGGGLE
 +
                                        <x-ref>Cesaratto2015</x-ref>
 +
                                        decreased the self-cleavage of TEVp and thereby increased its activity.
 +
                                    </p>
 +
                                </div>
 +
                            </div>
 +
                            <br/>
 +
                            <p>To overcome the lack of inducible orthogonal proteases, we looked for the characterized
 +
                                TEVp mutants and naturally occurring proteases, closely related to
 +
                                TEVp that might also be used to function as split proteases.</p>
 +
                        </div>
 +
                        <h1><span class="section colorize">&nbsp;</span>Results</h1>
 +
                        <div class="ui segment">
 +
                            <div>
 +
                                <h3><span id="var" class="section colorize">&nbsp;</span>TEVp variants</h3>
 +
                                <p>Based on the sequence alterations described by Yi et al.
 +
                                    <x-ref>Yi2013</x-ref>
 +
                                    , our team decided to test two mutated variants of TEVp - TEVpE and TEVpH.
 +
                                </p>
  
−
  <figcaption><b> Comparison of the split protein reconstitution based on two different sets of CCs. </b><br/> HEK293T cells were transfected with different amounts of constructs. (A) Previously reported CCs were tested in different B:nLuc to cLuc:A ratios. Luciferase reconstitution can be observed at higher plasmid amounts. (B) nLuc:AP4 to P3:cLuc CCs were tested in different ratios. Luciferase activity was detected even with lower plasmid amounts used. Overall, the comparison between pairs of CCs B:nLuc and cLuc:A to nLuc:AP4 and P3:cLuc showed that our own CCs give a much higher signal, so lower amounts can be used for integration into our whole system.
+
                                <div class="ui styled fluid accordion">
−
</figcaption>
+
                                    <div class="title">
−
</figure>
+
                                        <i class="dropdown icon"></i>
−
</div>
+
                                        Further explanation ...
 +
                                    </div>
 +
                                    <div class="content">
 +
                                        <p>Yi et al.
 +
                                            <x-ref>Yi2013</x-ref>
 +
                                            tackled the problem of acquiring novel orthogonal proteases by screening a
 +
                                            library of TEVp mutants for orthogonal
 +
                                            specificity. They designed a novel yeast ER sequestration screening assay
 +
                                            that allowed them to identify variants of TEVp that recognize
 +
                                            alternative substrates. The two most prominent mutants were TEVpE, which
 +
                                            cleaves the substrate ENLYFE-S (TEVsE) and TEVpH, cleaving the substrate
 +
                                            ENLYFH-S (TEVsH). Although the two variants were also able to cleave the
 +
                                            wild type TEVp substrate
 +
                                            ENLYFQ-S (TEVs), they displayed a high preference for their own variation of
 +
                                            the substrate and were mutually orthogonal.
 +
                                        </p>
 +
                                    </div>
 +
                                </div>
 +
                                <br/>
  
−
<p>The system presented by Shekhawat is able to process AND or OR logic functions but not those including negation (such as NOR, NAND etc.) We realized that this type of logic functions could be accomplished by introducing an additional cleavage site between the split reporter and coiled coil segment (<ref>4.12.6.1.</ref>).</p>
+
                                <p>To test these two proteases we used a <a
 +
                                        href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Reporters#cle">cleavable
 +
                                    firefly luciferase
 +
                                    (fLuc) reporter</a> with an appropriate cleavage sequence inserted in a permissible
 +
                                    site. We observed a significant decrease in luciferase activity upon
 +
                                    co-expression of the reporters and their corresponding proteases, whereas
 +
                                    co-expression of an orthogonal protease did not decrease the luciferase activity (<ref>1</ref>). These results were additionally confirmed by results from western blotting, where
 +
                                    cleavage of the luciferase reporter was
 +
                                    detected only in the presence of the corresponding protease, but not in the presence
 +
                                    of orthogonal proteases (<ref>2</ref>).
 +
                                </p>
 +
                                <div style="float:left; width:100%">
 +
                                    <figure data-ref="1">
 +
                                        <img src="https://static.igem.org/mediawiki/2016/8/87/T--Slovenia--4.4.1.png">
 +
                                        <figcaption><b>Activity and orthogonality of TEVp variants.</b><br/>
 +
                                            <p style="text-align:justify">(A) Activity of cleavable fLuc with the wild type TEVs cleavage sequence is reduced only in the presence of wild type TEVp. (B) Activity of cleavable fLuc with the TEVsE cleavage sequence is reduced only in the presence of TEVpE. (C) Activity of cleavable fLuc with the  TEVsH cleavage sequence is reduced only in the presence of TEVpH. HEK293T cells were transfected with the
 +
                                                indicated fLuc:TEVs and TEVp variant constructs. Luciferase activity was
 +
                                                determined 24 h after transfection. (D) Table of cleavage sequences for the wild type TEVp and its variants. The cleavage occurs before the final Ser and is indicated by -.
 +
                                            </p>
 +
                                        </figcaption>
 +
                                    </figure>
 +
                                </div>
  
−
<div align = "left">  
+
                                <p style="clear:left;">No data has previously been reported on TEVpE and TEVpH toxicity,
−
<figure data-ref="4.12.6.1 ">
+
                                    therefore we expressed each TEVp variant in
−
<img class="ui medium image" src="https://static.igem.org/mediawiki/2016/5/56/T--Slovenia--4.12.6.1.png">
+
                                    HEK293T cells and performed a viability assay. Even after transfection with a high
 +
                                    amount of the plasmid for each respective protease, the cells showed high viability,
 +
                                    with practically
 +
                                    no difference when compared to cells transfected with a mock plasmid (<ref>3</ref>).
 +
                                </p>
  
−
  <figcaption><b> Introduction of protease cleavage site between the reporter (effector) and coiled-coil segment(s) </b><br/> Cleavage sites in between CCs and reporter protein introduces logical negation. </figcaption>
+
                                <div style="width:60%; float:left;">
−
</figure>
+
                                    <figure data-ref="2">
−
</div>
+
                                        <img src="https://static.igem.org/mediawiki/2016/f/ff/T--Slovenia--4.4.2.png">
 +
                                        <figcaption><b>Orthogonality and activity of TEVpE and TEVpH.</b><br/>
 +
                                            <p style="text-align:justify">HEK293T cells were transfected with 2000 ng of
 +
                                                the indicated protease and 500 ng of the indicated reporter. Cells
 +
                                                were lysed and analyzed by western blotting with anti-AU1 antibodies.
 +
                                                The cleaved reporter (55 kDa) was detected only in the presence of the
 +
                                                corresponding
 +
                                                TEVp variant.
 +
                                            </p>
 +
                                        </figcaption>
 +
                                    </figure>
 +
                                </div>
  
−
<p>Constructs were therefore modified by the addition of TEVp cleavage site (TEVs) between nLuc and AP4 and PPVp cleavage site (PPVs) between P3 and cLuc. This represents a logic NOR gate based on the input signals, represented by TEVp and PPVp.</p>
+
                                <div style="width:40%; float:left;">
−
 
+
                                    <figure data-ref="3">
−
 
+
                                        <img src="https://static.igem.org/mediawiki/2016/8/8f/T--Slovenia--4.4.3.png">
−
 
+
                                        <figcaption><b>Different TEVp variants are not toxic to HEK293T cells.</b><br/>
−
 
+
                                            <p style="text-align:justify">HEK293T cells were transfected with plasmids
−
 
+
                                                coding for different TEVp variants. 48 hours after transfection, cells
−
 
+
                                                were stained with the trypan blue dye, which stains only dead cells. The
−
 
+
                                                ratio of live and dead cells was analysed with the Countess automated
−
<div align = "left">  
+
                                                cell counter.
−
<figure data-ref="4.12.6.">
+
                                            </p>
−
<img class="ui medium image" src="https://static.igem.org/mediawiki/2016/6/61/T--Slovenia--4.12.6.png" >
+
                                        </figcaption>
−
 
+
                                    </figure>
−
  <figcaption><b> Optimization of protease and substrate plasmid amounts.</b><br/> HEK293T cells were transfected with plasmids for constructs with introduced TEVs and PPVs (substrates) and different plasmid amounts of either PPVp (left) or TEVp (right) protease. Results show that 1:5 ratio of substrate and protease, respectively, was needed to achieve adequate cleavage followed by the decrease in protease activity. /figcaption>
+
                                </div><p style="clear:both"></p>
−
</figure>
+
                           
 
</div>
 
</div>
  
 +
                            <div>
 +
                                <h3 style="clear:both"><span id="hom" class="section colorize">&nbsp;</span>TEVp homologs</h3>
 +
                                <p>Introduction of two new TEVp variants expanded our repertoire of tools, demonstrating
 +
                                    that we can use the results of mutational screenings for
 +
                                    protease variants. However, a larger number of strictly orthogonal proteases would
 +
                                    be required for modular design of complex logic circuits. We therefore decided to
 +
                                    investigate the activity of de novo created split proteases from the potyviridae
 +
                                    family.
 +
                                </p>
  
−
<p>Indeed the system performed nicely (<ref>4.12.6.</ref>). Using this type of cleavage sites enabled us to design protease-based logic gates NOR, NOT A and NOT B (<ref>4.12.7.</ref>).  
+
                                <div class="ui styled fluid accordion">
 +
                                    <div class="title">
 +
                                        <i class="dropdown icon"></i>
 +
                                        Further explanation ...
 +
                                    </div>
 +
                                    <div class="content">
 +
                                        <p>The NIa proteases from the potyviridae group of plant viruses in general
 +
                                            recognize a seven amino acid sequence motif as their substrate. They are
 +
                                            are classified as cysteine proteases with an active site closely related to
 +
                                            eukaryotic serine proteases. The NIa proteases adopt a characteristic
 +
                                            two-domain antiparallel β-barrel fold. The active site of the protease
 +
                                            comprises a catalytic triad: His-46, Asp-81, Cys-151 (amino acids numbered
 +
                                            according to the TEVp sequence) with a Gly-x-Cys-Gly motif around the active
 +
                                            cysteine residue
 +
                                            <x-ref>Yoon2000, Nunn2005</x-ref>
 +
                                            .
 +
                                        </p>
 +
                                    </div>
 +
                                </div>
 +
                                <br/>
  
−
<div align = "left">  
+
                                <p>We searched for different potyviruses available on UniProt (<ref>4</ref>), paying particular
−
<figure data-ref="4.12.7">
+
                                    attention to any evidence of orthogonality among their target substrates.
−
<img class="ui medium image" src="https://static.igem.org/mediawiki/2016/a/af/T--Slovenia--4.12.7.png" >
+
                                    We decided to test the plum pox virus protease (PPVp), the soybean mosaic virus
 +
                                    protease (SbMVp), and the sunflower mild mosaic virus protease (SuMMVp).
 +
                                </p>
  
−
  <figcaption><b> Design of protease based logic operations NOT, NOT A and NOT B in HEK203 cells.</b><br/> HEK293 cells were transfected plasmids for nLuc:TEVs:AP4, P3:PPVs:cLuc, nLuc:AP4, P3:cLuc, TEVp and/or PPV as indicated in graphs. 24 h after transfection cells were lysed and double luciferase assay was performed. (A) Logic gate NOR, where the output signal is active only when none of the input signals are present. (B) Logic gate NOT A, in which output signal is active when none or just B input signals (TEVp) is present.(C) Logic gate NOT B, in which the output signal is active when none or just A input signal (PPV) is present.</figcaption>
 
−
</figure>
 
−
</div>
 
  
−
 
+
                                <div style="clear:right; width:70%">
−
<p>For implementation of the system with additional logic operations further modifications on our own CCs collection were needed. Analysis of the equilibrium model reveals that the affinity of the autoinhibitory segment should not be too strong, otherwise the inhibition will remain; but should also not be too weak, otherwise the system would be leaky and active already without cleavage. Stability of the coiled-coil interaction can be tuned by introduction of non-favorable interactions e.g. by introducing Ala residues at a and d positions <x-ref>Acharya2002</x-ref>.  We designed four different destabilized P3 coils by substituting b and c position with polar amino acids and <i>a</i> and <i>d</i> positions of different heptads with alanine residues (<ref>4.12.8.</ref>).</p>
+
                                    <figure data-ref="4">
−
 
+
                                        <img src="https://static.igem.org/mediawiki/2016/1/19/T--Slovenia--4.3.5.png">
−
<div align = "left">  
+
                                        <figcaption><b>Orthogonal proteases from the potyviridae family.</b><br>
−
<figure data-ref="4.12.8.">
+
                                            <p style="text-align:justify">Homology models obtained from <a style="padding-right: 0"
−
<img class="ui medium image" src="https://static.igem.org/mediawiki/2016/5/5f/T--Slovenia--4.12.8.png" >
+
                                                    href="https://swissmodel.expasy.org/">SWISS-MODEL</a> using the
−
 
+
                                                crystal structure of TEVp (red) (<a style="padding-right: 0"
−
  <figcaption><b> Sequence alignment of different coils used to tune the affinity of antiparallel coiled-coils.</b><br/> We designed different destabilized coils from our coiled coil pair AP4 and P3; Ile and Leu were substituted with Ala at the a and/or d position of the first and/or second heptad of P3mS (a more soluble variant of the original P3).</figcaption>
+
                                                    href="http://www.rcsb.org/pdb/explore.do?structureId=1LVB">PDB 1LVB</a>) as template. The
−
</figure>
+
                                                homology models of the potyviridae family proteases PPVp (blue), SbMVp
−
</div>
+
                                                (cyan) and
−
 
+
                                                SuMMVp (yellow) are shown in ribbons.
−
<p>Those variably destabilized peptides were used as autoinhibitory coiled-coil forming segments to test the difference in activity between the uncleaved and TEVp cleaved forms.</p>
+
                                            </p>
−
 
+
                                        </figcaption>
−
<div align = "left">  
+
                                    </figure>
−
<figure data-ref="4.12.9.">
+
                                </div>
−
<img class="ui medium image" src="https://static.igem.org/mediawiki/2016/b/bc/T--Slovenia--4.12.9.png" >
+
−
 
+
−
  <figcaption><b> P3mS-2A and P3mS were the best autoinhibitory coiled-coil constructs.</b><br/>A) In the presence of TEVp the auto inhibitory coil is cleaved off, allowing P3 to dimerize with AP4 and reconstitute the split luciferase. B) Normalized luciferase activity was compared between samples with and without added TEVp to calculate the fold change of luciferase activity. Out of the four different constructs, the constructs which contained the inhibitory coils P3mS and P3mS-2A worked best, where we observed up to 15 times fold increase with the addition of TEVp</figcaption>
+
−
</figure>
+
−
</div>
+
−
 
+
−
<p>To test which one of our four destabilized CCs worked best, all constructs were tested in vivo with and without the presence of TEVp (<ref>4.12.9.</ref>). We concluded that P3mS and P3mS-2A demonstrated the highest fold increase in the luciferase activity upon the addition of TEVp. The other two constructs showed little to no increase in luciferase activity upon the addition of TEVp, suggesting that the peptides were destabilized too much leading to the leakage in the uninduced form.</p>
+
−
 
+
−
<p>The final test was to investigate if the system could indeed be controlled by two signals at the same time. In order to test this we constructed NOR gate with logic processing (nLuc:TEVs:AP4 and P3:PPVs:cLuc) and inducible components (split PPVp and TEVp inducible by the rapamycin and light, respectively) (<ref>4.12.13.</ref>).</p>
+
−
 
+
−
<div align = "left">
+
−
<figure data-ref="4.12.13.">
+
−
<img class="ui medium image" src="https://static.igem.org/mediawiki/2016/0/06/T--Slovenia--4.12.13.png">
+
−
 
+
−
  <figcaption><b> Protease-based NOR logic gate regulated by light and rapamycin. </b><br/> HEK293 cells were transfected with appropriate plasmids as indicated in the graph. 24 hours after the transfection the cells were induced with light and rapamycin for 15min and after 4 hours, lysed and double luciferase assay was performed.</figcaption>
+
−
</figure>
+
−
</div>
+
−
 
+
−
 
+
−
<p>The types building blocks that we developed made possible to construct all of the possible 16 two-input logic function based on the proteolysis. To demonstrate this we tested additional logic operations (A and A nimply B), where the response was measured directly after 15 minutes of induction to demonstrate the increased speed of protein-based processing, as shown on <ref>4.12.11.</ref>.</p>
+
−
 
+
−
<div align = "left">
+
−
<figure data-ref="4.12.11.">
+
−
<img class="ui medium image" src="https://static.igem.org/mediawiki/2016/a/ab/T--Slovenia--4.12.11.png">
+
−
 
+
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  <figcaption><b> Logic gates A and A nimply B regulated by light (input A) and rapalycin (input B) based on protease processing produce correct and measurable output after 15 minutes. </b><br/> HEK293 cells were transfected with appropriate plasmids as indicated in graph. 24 hours after the transfection the cells were induced with light and rapamycin for 15 minutes, lysed and double luciferase assay was performed. (A) Logic gate A, in which output signal has a value of 1 when A input signal (TEVp induced with light) is present. (B) Logic gate A, in which output signal has a value of 1 when only A input signal (TEVp induced with light) is present.</figcaption>
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<p>In both cases the system performed very well, producing clear difference between the active and inactive output states within 15 min after the stimulation by combinations of two signals. Logic function A nimply B is relatively difficult to implement but on the other hand it can be quite useful as for example the signal B may identify the cell type and trigger  activation by an external signal only in a selected cell types or cells in a selected state.</p>
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                                <div class="ui styled fluid accordion">
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<p>The availability of a set of <a href=”https://2016.igem.org/Team:Slovenia/Protease_signaling/Split_proteases>orthogonal proteases</a> as well as an <a href=” https://2016.igem.org/Team:Slovenia/CoiledCoilInteraction> orthogonal coiled-coil dimers  toolset</a> enabled the construction of a fast complex logic processing circuits. The Previous CC toolbox has been further expanded with a strategy of generating antiparallel and destabilized CC. Furthermore, designed system based on <a href=” https://2016.igem.org/Team:Slovenia/Mechanosensing/CaDependent_mediator> split proteases can also be linked to many other input signals such as e.g. intracellular calcium increase</a>. An important advance is the adaptation of the system to function in vivo in mammalian cells. Further, reporter as the output signal could be substituted by a split protease, enabling multi-layered processing, or used as an trigger for other cellular processes, such as the <a href=” https://2016.igem.org/Team:Slovenia/Implementation/ProteaseInducible_secretion
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> release of therapeutics </a>. Therefore we believe that those results represent a valuable foundational advance in synthetic biology.</p>
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                                        <i class="dropdown icon"></i>
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                                        Further explanation ...
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                                    <div class="content">
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                                        <p>
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                                            PPVp is one of the most studied potyviral proteases after the TEV protease.
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                                            Its substrate (PPVs) has an amino acid sequence NVVVHQ-A.
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                                            In contrast to TEVp, it has been reported that PPVp is resistant to
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                                            self-cleavage at the C-terminus
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                                            <x-ref>Zheng2008, Garcia1991</x-ref>
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                                            .<br/>SbMVp has been
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                                            recently studied by Seo et al. as a tool for protein-protein interaction
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                                            studies in the soybean plant. The substrate (SbMVs) has been determined to
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                                            be the
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                                            sequence ESVSLQ-S
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                                            <x-ref>Seo2016, Yoon2000</x-ref>
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                                            .<br>Similarly, SuMMVp has been used by Fernandez-Rodriguez et al.
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                                            <x-ref>Fernandez-Rodriguez2016</x-ref>
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                                            . in a study of genetic circuits using potyviral proteases. The substrate
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                                            (SuMMVs) has been defined as the sequence
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                                            EEIHLQ-S/G
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                                            <x-ref>Fernandez-Rodriguez2016</x-ref>
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                                            .
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                                <p>All selected potyviral proteases were designed as synthetic genes and their activity
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                                    was tested in mammalian cells using
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                                    <a href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Reporters#cyc">cyclic
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                                        luciferase reporters</a>, which propagate luciferase activity only upon
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                                    cleavage.
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                                    We detected an increase of luciferase activity only in the corresponding
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                                    protease-reporter pairs, confirming exquisite orthogonality of the selected
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                                    proteases and
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                                    their activity in the human cell chassis (<ref>5</ref>).
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                                    <figure data-ref="5">
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                                        <figcaption><b>Protease orthogonality.</b><br/>
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                                            <p style="text-align:justify">(A) Table of cleavage sequences for TEVp and its homologues. The cleavage occurs before the final Ser or Ala and is indicated by -. (B) Heat map showing orthogonality of the TEVp homologues. Dark red corresponds to higher luciferase activity. (C) Orthogonality of TEVp homologues shown as bar graphs. HEK293T cells were transfected with the
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                                                indicated cycLuc reporters and proteases. Luciferase activity was
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                                                detected only in the presence
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                                                of a protease and cycLuc reporter containing appropriate protease
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                                                cleavage site.
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                                            </p>
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                                        </figcaption>
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                        <h3 class="ui left dividing header"><span id="ref-title" class="section colorize">&nbsp;</span>References
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Latest revision as of 13:56, 19 October 2016

Orthogonality

Reporters Orthogonality Achievements Introduction TEVp variants TEVp homologs Split proteases

 Protease orthogonality

  • Two TEVp variants (TEVpE and TEVpH) were tested and proved to be mutually orthogonal.
  • Upon overexpression none of the tested proteases were toxic to mammalian cells, demonstrating that they do not interfere with essential cellular processes.
  • Additionally, three TEVp homologues (PPVp, SbMVp and SuMMVp) were tested and proved to be fully orthogonal.

 

The first challenge in construction of a new protease-based signaling cascade was the selection of appropriate proteases. The candidate proteases should: i) recognize defined target cleavage sequences, preferably as long as possible; ii) be active in mammalian cells, but not toxic to them and iii) be inducible, ideally through the reconstitution of split protein fragments. Most importantly, a large number of proteases with similar properties but different substrates should be available to allow for modular construction of signaling pathways and logic functions. These proteases should be orthogonal to each other, meaning that their specific cleavage sites should not be recognized by other proteases in the system.

We found that the tobacco etch virus protease (TEVp) was the only protease described in the literature to match our criteria.

Further explanation ...

TEV protease is a highly specific, 242 amino acids long, 27 kDa cysteine protease, that originates from the tobacco etch virus (TEV) of the Potyvirus genus. It has a target recognition sequence of seven amino acids, ENLYFQ-S/G, where cleavage occurs after the glutamine residue (denoted by the – symbol), and the final residue of the recognition sequence can be either S or G (denoted by the / symbol). This substrate sequence is scarcely represented in the proteome. TEV protease is therefore relatively non-toxic Parks1994 and can be safely expressed in host cells. Due to this non-toxicity and its high cleavage specificity, TEVp is an attractive protease for use in several biotechnological applications, such as removal of affinity tags from recombinant proteins.

Despite its widespread use in biotechnology, TEVp also displays some shortcomings, the most prominent of them being self-cleavage. Substitution of Ser-219 with Val or Pro Cesaratto2015 or a replacement of the C-terminal sequence MSELVYSQ with the sequence MNEGGGLE Cesaratto2015 decreased the self-cleavage of TEVp and thereby increased its activity.


To overcome the lack of inducible orthogonal proteases, we looked for the characterized TEVp mutants and naturally occurring proteases, closely related to TEVp that might also be used to function as split proteases.

 Results

 TEVp variants

Based on the sequence alterations described by Yi et al. Yi2013 , our team decided to test two mutated variants of TEVp - TEVpE and TEVpH.

Further explanation ...

Yi et al. Yi2013 tackled the problem of acquiring novel orthogonal proteases by screening a library of TEVp mutants for orthogonal specificity. They designed a novel yeast ER sequestration screening assay that allowed them to identify variants of TEVp that recognize alternative substrates. The two most prominent mutants were TEVpE, which cleaves the substrate ENLYFE-S (TEVsE) and TEVpH, cleaving the substrate ENLYFH-S (TEVsH). Although the two variants were also able to cleave the wild type TEVp substrate ENLYFQ-S (TEVs), they displayed a high preference for their own variation of the substrate and were mutually orthogonal.


To test these two proteases we used a cleavable firefly luciferase (fLuc) reporter with an appropriate cleavage sequence inserted in a permissible site. We observed a significant decrease in luciferase activity upon co-expression of the reporters and their corresponding proteases, whereas co-expression of an orthogonal protease did not decrease the luciferase activity (1). These results were additionally confirmed by results from western blotting, where cleavage of the luciferase reporter was detected only in the presence of the corresponding protease, but not in the presence of orthogonal proteases (2).

Activity and orthogonality of TEVp variants.

(A) Activity of cleavable fLuc with the wild type TEVs cleavage sequence is reduced only in the presence of wild type TEVp. (B) Activity of cleavable fLuc with the TEVsE cleavage sequence is reduced only in the presence of TEVpE. (C) Activity of cleavable fLuc with the TEVsH cleavage sequence is reduced only in the presence of TEVpH. HEK293T cells were transfected with the indicated fLuc:TEVs and TEVp variant constructs. Luciferase activity was determined 24 h after transfection. (D) Table of cleavage sequences for the wild type TEVp and its variants. The cleavage occurs before the final Ser and is indicated by -.

No data has previously been reported on TEVpE and TEVpH toxicity, therefore we expressed each TEVp variant in HEK293T cells and performed a viability assay. Even after transfection with a high amount of the plasmid for each respective protease, the cells showed high viability, with practically no difference when compared to cells transfected with a mock plasmid (3).

Orthogonality and activity of TEVpE and TEVpH.

HEK293T cells were transfected with 2000 ng of the indicated protease and 500 ng of the indicated reporter. Cells were lysed and analyzed by western blotting with anti-AU1 antibodies. The cleaved reporter (55 kDa) was detected only in the presence of the corresponding TEVp variant.

Different TEVp variants are not toxic to HEK293T cells.

HEK293T cells were transfected with plasmids coding for different TEVp variants. 48 hours after transfection, cells were stained with the trypan blue dye, which stains only dead cells. The ratio of live and dead cells was analysed with the Countess automated cell counter.

 TEVp homologs

Introduction of two new TEVp variants expanded our repertoire of tools, demonstrating that we can use the results of mutational screenings for protease variants. However, a larger number of strictly orthogonal proteases would be required for modular design of complex logic circuits. We therefore decided to investigate the activity of de novo created split proteases from the potyviridae family.

Further explanation ...

The NIa proteases from the potyviridae group of plant viruses in general recognize a seven amino acid sequence motif as their substrate. They are are classified as cysteine proteases with an active site closely related to eukaryotic serine proteases. The NIa proteases adopt a characteristic two-domain antiparallel β-barrel fold. The active site of the protease comprises a catalytic triad: His-46, Asp-81, Cys-151 (amino acids numbered according to the TEVp sequence) with a Gly-x-Cys-Gly motif around the active cysteine residue Yoon2000, Nunn2005 .


We searched for different potyviruses available on UniProt (4), paying particular attention to any evidence of orthogonality among their target substrates. We decided to test the plum pox virus protease (PPVp), the soybean mosaic virus protease (SbMVp), and the sunflower mild mosaic virus protease (SuMMVp).

Orthogonal proteases from the potyviridae family.

Homology models obtained from SWISS-MODEL using the crystal structure of TEVp (red) (PDB 1LVB) as template. The homology models of the potyviridae family proteases PPVp (blue), SbMVp (cyan) and SuMMVp (yellow) are shown in ribbons.

Further explanation ...

PPVp is one of the most studied potyviral proteases after the TEV protease. Its substrate (PPVs) has an amino acid sequence NVVVHQ-A. In contrast to TEVp, it has been reported that PPVp is resistant to self-cleavage at the C-terminus Zheng2008, Garcia1991 .
SbMVp has been recently studied by Seo et al. as a tool for protein-protein interaction studies in the soybean plant. The substrate (SbMVs) has been determined to be the sequence ESVSLQ-S Seo2016, Yoon2000 .
Similarly, SuMMVp has been used by Fernandez-Rodriguez et al. Fernandez-Rodriguez2016 . in a study of genetic circuits using potyviral proteases. The substrate (SuMMVs) has been defined as the sequence EEIHLQ-S/G Fernandez-Rodriguez2016 .


All selected potyviral proteases were designed as synthetic genes and their activity was tested in mammalian cells using cyclic luciferase reporters, which propagate luciferase activity only upon cleavage. We detected an increase of luciferase activity only in the corresponding protease-reporter pairs, confirming exquisite orthogonality of the selected proteases and their activity in the human cell chassis (5).

Protease orthogonality.

(A) Table of cleavage sequences for TEVp and its homologues. The cleavage occurs before the final Ser or Ala and is indicated by -. (B) Heat map showing orthogonality of the TEVp homologues. Dark red corresponds to higher luciferase activity. (C) Orthogonality of TEVp homologues shown as bar graphs. HEK293T cells were transfected with the indicated cycLuc reporters and proteases. Luciferase activity was detected only in the presence of a protease and cycLuc reporter containing appropriate protease cleavage site.

 References