{"id":368,"date":"2024-01-01T15:17:51","date_gmt":"2024-01-01T15:17:51","guid":{"rendered":"http:\/\/pharmsci.createuky.net\/eromedaniel\/?p=368"},"modified":"2024-01-02T15:09:52","modified_gmt":"2024-01-02T15:09:52","slug":"neisslock-a-method-for-labeling-unmodified-endogenous-proteins-in-mild-conditions","status":"publish","type":"post","link":"https:\/\/pharmsci.createuky.net\/eromedaniel\/e-views\/neisslock-a-method-for-labeling-unmodified-endogenous-proteins-in-mild-conditions\/","title":{"rendered":"NeissLock: a method for labeling unmodified endogenous proteins in mild conditions"},"content":{"rendered":"\n

As chemical biology becomes increasingly translational, its tools and strategies must adhere to higher standards of resolution to ensure success and longevity in the clinic. As a result, labeling approaches often incorporate avenues for spatial and temporal control for in vivo<\/em> scenarios. In this review, I focus on summarizing a labeling strategy designed to do exactly that. NeissLock is a method for targeting endogenous proteins through proximity-induced conjugation. Initially reported by the Howarth lab<\/a> using ornithine decarboxylase (ODC) and ornithine decarboxylase antienzyme (OAZ) complex as a model system, NeissLock enables covalent linkage between binding and target proteins.

1. Self-processing module (SPM) of Neisseria meningitidis<\/em><\/strong><\/em><\/strong> has autoproteolysis capabilities<\/strong>: SPM is a key component of NeissLock. It displays Ca2+<\/sup>-induced autoproteolysis at an Asp-Pro dipeptide site. Autoproteolysis cleaves between the Asp and Pro residues generating an aspartic anhydride in the process. <\/p>\n\n\n\n

2. SPM can be repurposed for tagging endogenous proteins<\/strong>: SPM is fused to the C-terminal of a binding protein. Upon exposure to Ca2+<\/sup> (0.5 mM and above), SPM autopreotolysis releases an SPM-free binding protein with a C-terminal anhydride. When in proximity to the “activated” binding protein, a nucleophilic Lys in the target protein reacts with the C-terminal anhydride of the binding protein to form a covalent bond between the two (NeissLock). In vitro<\/em> reactions with OAZ-SPM and ODC showed calcium-induced ligation of OAZ and ODC. Additionally, that affinity is crucial for NeissLock was demonstrated by showing selective reaction of ODC to OAZ in reactions of OAZ-SPM with cell lysate (Figure source is the article). <\/p>\n\n\n\n

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3. A computational analysis software NeissDist was created to predict NeissLock sites in protein-protein complexes<\/strong>: A software called NeissDist was designed to identify nucleophilic amines close to the anhydride of a binding for protein protein-protein complexes in the PDB. It should be noted, however, that the authors report that the anhydride reacts with nucleophiles other than amines, including thiols, amides, and hydroxyls, implicating Cys, Asn, Gln, and Tyr in NeissLock-mediated conjugation of protein-protein complexes. <\/p>\n\n\n\n

4. NeissLock is efficient at neutral pH and is compatible with different buffers<\/strong>: The authors were concerned that the high pKa of amines might necessitate higher pH conditions for NeissLock. However, reactions were efficient between pH 6.5-8.5. Reactions were also shown to be efficient in HEPES and TBS buffers. Phosphate-based buffers are not recommended due to precipitation upon calcium addition.  
<\/p>\n\n\n\n

5. NeissLock works for cell surface labeling<\/strong>: SPM-tagged TGF-\u03b1 is shown to efficiently label sEGFR in A431 cells. Although the authors observed significant staining with an SPM-tagged TGF-\u03b1 variant that should be non-reactive, this could be abolished with an R42A mutation in TGF-\u03b1 that weakens its affinity to EGFR, demonstrating once-again that NeissLock is a proximity-dependent conjugation process. <\/p>\n\n\n\n

The advantage for spatiotemporal control in this technology is that no reaction occurs until reactivity is initiated by addition of calcium. Additionally, no conjugation occurs without proximity, conferring spatial resolution on the NeissLock approach. This advantages are crucial for applications in imaging and diagnostics. Limitations include possibly having to remove nucleophilic residues near the anhydride to minimize self-reaction, and relative ambiguity regarding the conjugation sites due to anhydride reactivity with different nucleophilic residues. <\/p>\n\n\n\n

<\/p>\n\n\n\n

Read the article here<\/a>!  <\/p>\n","protected":false},"excerpt":{"rendered":"

As chemical biology becomes increasingly translational, its tools and strategies must adhere to higher standards of resolution to ensure success and longevity in the clinic. As a result, labeling approaches often incorporate avenues for spatial and temporal control for in vivo scenarios. In this review, I focus on summarizing a labeling strategy designed to do … <\/p>\n

Continue reading “NeissLock: a method for labeling unmodified endogenous proteins in mild conditions”<\/span><\/a><\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[6,1],"tags":[8,9,10],"class_list":["post-368","post","type-post","status-publish","format-standard","hentry","category-article-reviews","category-e-views","tag-chemical-biology","tag-covalent-conjugation","tag-self-processing-module"],"_links":{"self":[{"href":"https:\/\/pharmsci.createuky.net\/eromedaniel\/wp-json\/wp\/v2\/posts\/368","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/pharmsci.createuky.net\/eromedaniel\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/pharmsci.createuky.net\/eromedaniel\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/pharmsci.createuky.net\/eromedaniel\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/pharmsci.createuky.net\/eromedaniel\/wp-json\/wp\/v2\/comments?post=368"}],"version-history":[{"count":37,"href":"https:\/\/pharmsci.createuky.net\/eromedaniel\/wp-json\/wp\/v2\/posts\/368\/revisions"}],"predecessor-version":[{"id":433,"href":"https:\/\/pharmsci.createuky.net\/eromedaniel\/wp-json\/wp\/v2\/posts\/368\/revisions\/433"}],"wp:attachment":[{"href":"https:\/\/pharmsci.createuky.net\/eromedaniel\/wp-json\/wp\/v2\/media?parent=368"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/pharmsci.createuky.net\/eromedaniel\/wp-json\/wp\/v2\/categories?post=368"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/pharmsci.createuky.net\/eromedaniel\/wp-json\/wp\/v2\/tags?post=368"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}