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==Combinatorial Engineering of Proteolytically Resistant APPI Variants that Selectively Inhibit Human Kallikrein 6 for Cancer Therapy==
<StructureSection load='5nx1' size='340' side='right'caption='[[5nx1]], [[Resolution|resolution]] 1.85&Aring;' scene=''>
== Structural highlights ==
<table><tr><td colspan='2'>[[5nx1]] is a 4 chain structure with sequence from [http://en.wikipedia.org/wiki/Human Human]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5NX1 OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=5NX1 FirstGlance]. <br>
</td></tr><tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">KLK6, PRSS18, PRSS9 ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=9606 HUMAN]), APP, A4, AD1 ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=9606 HUMAN])</td></tr>
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=5nx1 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5nx1 OCA], [http://pdbe.org/5nx1 PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=5nx1 RCSB], [http://www.ebi.ac.uk/pdbsum/5nx1 PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=5nx1 ProSAT]</span></td></tr>
</table>
== Disease ==
[[http://www.uniprot.org/uniprot/A4_HUMAN A4_HUMAN]] Defects in APP are the cause of Alzheimer disease type 1 (AD1) [MIM:[http://omim.org/entry/104300 104300]]. AD1 is a familial early-onset form of Alzheimer disease. It can be associated with cerebral amyloid angiopathy. Alzheimer disease is a neurodegenerative disorder characterized by progressive dementia, loss of cognitive abilities, and deposition of fibrillar amyloid proteins as intraneuronal neurofibrillary tangles, extracellular amyloid plaques and vascular amyloid deposits. The major constituent of these plaques is the neurotoxic amyloid-beta-APP 40-42 peptide (s), derived proteolytically from the transmembrane precursor protein APP by sequential secretase processing. The cytotoxic C-terminal fragments (CTFs) and the caspase-cleaved products such as C31 derived from APP, are also implicated in neuronal death.<ref>PMID:8476439</ref> <ref>PMID:15201367</ref> <ref>PMID:1671712</ref> <ref>PMID:1908231</ref> <ref>PMID:1678058</ref> <ref>PMID:1944558</ref> <ref>PMID:1925564</ref> <ref>PMID:1415269</ref> <ref>PMID:1303239</ref> <ref>PMID:1302033</ref> <ref>PMID:1303275</ref> <ref>PMID:8267572</ref> <ref>PMID:8290042</ref> <ref>PMID:8577393</ref> <ref>PMID:9328472</ref> <ref>PMID:9754958</ref> <ref>PMID:10097173</ref> <ref>PMID:10631141</ref> <ref>PMID:10665499</ref> <ref>PMID:10867787</ref> <ref>PMID:11063718</ref> <ref>PMID:11311152</ref> <ref>PMID:11528419</ref> <ref>PMID:12034808</ref> <ref>PMID:15365148</ref> <ref>PMID:15668448</ref>   Defects in APP are the cause of cerebral amyloid angiopathy APP-related (CAA-APP) [MIM:[http://omim.org/entry/605714 605714]]. A hereditary localized amyloidosis due to amyloid-beta A4 peptide(s) deposition in the cerebral vessels. The principal clinical characteristics are recurrent cerebral and cerebellar hemorrhages, recurrent strokes, cerebral ischemia, cerebral infarction, and progressive mental deterioration. Patients develop cerebral hemorrhage because of the severe cerebral amyloid angiopathy. Parenchymal amyloid deposits are rare and largely in the form of pre-amyloid lesions or diffuse plaque-like structures. They are Congo red negative and lack the dense amyloid cores commonly present in Alzheimer disease. Some affected individuals manifest progressive aphasic dementia, leukoencephalopathy, and occipital calcifications.<ref>PMID:10821838</ref> <ref>PMID:2111584</ref> <ref>PMID:11409420</ref> <ref>PMID:12654973</ref> <ref>PMID:16178030</ref>  
== Function ==
[[http://www.uniprot.org/uniprot/A4_HUMAN A4_HUMAN]] Functions as a cell surface receptor and performs physiological functions on the surface of neurons relevant to neurite growth, neuronal adhesion and axonogenesis. Involved in cell mobility and transcription regulation through protein-protein interactions. Can promote transcription activation through binding to APBB1-KAT5 and inhibits Notch signaling through interaction with Numb. Couples to apoptosis-inducing pathways such as those mediated by G(O) and JIP. Inhibits G(o) alpha ATPase activity (By similarity). Acts as a kinesin I membrane receptor, mediating the axonal transport of beta-secretase and presenilin 1. Involved in copper homeostasis/oxidative stress through copper ion reduction. In vitro, copper-metallated APP induces neuronal death directly or is potentiated through Cu(2+)-mediated low-density lipoprotein oxidation. Can regulate neurite outgrowth through binding to components of the extracellular matrix such as heparin and collagen I and IV. The splice isoforms that contain the BPTI domain possess protease inhibitor activity. Induces a AGER-dependent pathway that involves activation of p38 MAPK, resulting in internalization of amyloid-beta peptide and leading to mitochondrial dysfunction in cultured cortical neurons. Provides Cu(2+) ions for GPC1 which are required for release of nitric oxide (NO) and subsequent degradation of the heparan sulfate chains on GPC1.<ref>PMID:9168929</ref> <ref>PMID:11544248</ref> <ref>PMID:11943163</ref> <ref>PMID:19225519</ref> <ref>PMID:19901339</ref>   Beta-amyloid peptides are lipophilic metal chelators with metal-reducing activity. Bind transient metals such as copper, zinc and iron. In vitro, can reduce Cu(2+) and Fe(3+) to Cu(+) and Fe(2+), respectively. Beta-amyloid 42 is a more effective reductant than beta-amyloid 40. Beta-amyloid peptides bind to lipoproteins and apolipoproteins E and J in the CSF and to HDL particles in plasma, inhibiting metal-catalyzed oxidation of lipoproteins. Beta-APP42 may activate mononuclear phagocytes in the brain and elicit inflammatory responses. Promotes both tau aggregation and TPK II-mediated phosphorylation. Interaction with Also bind GPC1 in lipid rafts.<ref>PMID:9168929</ref> <ref>PMID:11544248</ref> <ref>PMID:11943163</ref> <ref>PMID:19225519</ref> <ref>PMID:19901339</ref>   Appicans elicit adhesion of neural cells to the extracellular matrix and may regulate neurite outgrowth in the brain (By similarity).<ref>PMID:9168929</ref> <ref>PMID:11544248</ref> <ref>PMID:11943163</ref> <ref>PMID:19225519</ref> <ref>PMID:19901339</ref>   The gamma-CTF peptides as well as the caspase-cleaved peptides, including C31, are potent enhancers of neuronal apoptosis.<ref>PMID:9168929</ref> <ref>PMID:11544248</ref> <ref>PMID:11943163</ref> <ref>PMID:19225519</ref> <ref>PMID:19901339</ref>   N-APP binds TNFRSF21 triggering caspase activation and degeneration of both neuronal cell bodies (via caspase-3) and axons (via caspase-6).<ref>PMID:9168929</ref> <ref>PMID:11544248</ref> <ref>PMID:11943163</ref> <ref>PMID:19225519</ref> <ref>PMID:19901339</ref>  [[http://www.uniprot.org/uniprot/KLK6_HUMAN KLK6_HUMAN]] Serine protease which exhibits a preference for Arg over Lys in the substrate P1 position and for Ser or Pro in the P2 position. Shows activity against amyloid precursor protein, myelin basic protein, gelatin, casein and extracellular matrix proteins such as fibronectin, laminin, vitronectin and collagen. Degrades alpha-synuclein and prevents its polymerization, indicating that it may be involved in the pathogenesis of Parkinson disease and other synucleinopathies. May be involved in regulation of axon outgrowth following spinal cord injury. Tumor cells treated with a neutralizing KLK6 antibody migrate less than control cells, suggesting a role in invasion and metastasis.<ref>PMID:12878203</ref> <ref>PMID:12928483</ref> <ref>PMID:15557757</ref> <ref>PMID:16987227</ref> <ref>PMID:16321973</ref> <ref>PMID:11983703</ref>  
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== Publication Abstract from PubMed ==
Human tissue kallikrein (KLK) proteases are hormone-like signaling molecules with important functions in cancer pathophysiology. KLK-related peptidase 6 (KLK6), specifically, is highly up-regulated in several types of cancer, where its increased activity promotes cancer invasion and metastasis. This characteristic suggests KLK6 as an attractive target for therapeutic interventions. However, inhibitors that specifically target KLK6 have not yet been reported, possibly because KLK6 shares a high sequence homology and structural similarity with other serine proteases and resists inhibition by many polypeptide inhibitors. Here, we present an innovative combinatorial approach to engineering KLK6 inhibitors via flow cytometry-based screening of a yeast-displayed mutant library of the human amyloid precursor protein Kunitz protease inhibitor domain (APPI), an inhibitor of other serine proteases, such as anionic and cationic trypsins. On the basis of this screening, we generated APPIM17L,I18F,S19F,F34V (APPI-4M), an APPI variant with a KLK6 inhibition constant (Ki ) of 160 pm and a turnover time of 10 days. To the best of our knowledge, APPI-4M is the most potent KLK6 inhibitor reported to date, displaying 146-fold improved affinity and 13-fold improved proteolytic stability compared with WT APPI (APPIWT). We further demonstrate that APPI-4M acts as a functional inhibitor in a cell-based model of KLK6-dependent breast cancer invasion. Finally, the crystal structures of the APPIWT/KLK6 and APPI-4M/KLK6 complexes revealed the structural and mechanistic bases for the improved KLK6 binding and proteolytic resistance of APPI-4M. We anticipate that APPI-4M will have substantial translational potential as both imaging agent and therapeutic.

A potent, proteolysis-resistant inhibitor of kallikrein-related peptidase 6 (KLK6) for cancer therapy, developed by combinatorial engineering.,Sananes A, Cohen I, Shahar A, Hockla A, De Vita E, Miller AK, Radisky ES, Papo N J Biol Chem. 2018 Aug 17;293(33):12663-12680. doi: 10.1074/jbc.RA117.000871. Epub, 2018 Jun 22. PMID:29934309<ref>PMID:29934309</ref>

From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
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<div class="pdbe-citations 5nx1" style="background-color:#fffaf0;"></div>

==See Also==
*[[Kallikrein|Kallikrein]]
== References ==
<references/>
__TOC__
</StructureSection>
[[Category: Human]]
[[Category: Large Structures]]
[[Category: Papo, N]]
[[Category: Radisky, E S]]
[[Category: Sananes, A]]
[[Category: Shahar, A]]
[[Category: Complex]]
[[Category: Hydrolase]]
[[Category: Inhibitor]]
[[Category: Serine_protease]]