111d1e8dc2d600657538c92125e7b120 ijms-26-10375.pdf fb500948be3883ee81211240688e8253aa0d4016 ijms-26-10375.pdf 0e1c472450502d69a8c2262c984d712d5bb29e0bc2238f0c98cf099cd897805d ijms-26-10375.pdf Title: Moonlighting Proteins: Some Hypotheses on the Structural Origin of Their Multifunctionality Subject: Moonlighting proteins—single polypeptides performing multiple, often unrelated functions—are increasingly recognized as key players in human disease and microbial pathogenesis, making their identification crucial for understanding disease mechanisms and developing targeted therapies. This study addresses the unresolved question of how such multifunctionality evolves, focusing on two potential structural mechanisms: Non-Orthologous Gene Displacement/Non-Homologous Isofunctional Enzymes (NOGD/NHIE), where evolutionarily unrelated proteins perform the same function, and Fold-Switching Proteins (FSP), which adopt alternative secondary structures to switch functions without sequence changes. We analyzed the overlap between known human moonlighting proteins (from MultitaskProtDB-II) and curated datasets of NOGD/NHIE (Non-Orthologous Gene Displacement/Non-Homologous Isofunctional Enzymes) and fold-switching proteins (FSPs), using Fisher’s exact test for statistical validation. Moonlighting proteins showed extraordinary enrichment for NOGD/NHIE (19.89% vs. 0.39% in non-moonlighting proteins; odds ratio = 63.1, p < 2.2 10-16) and strong enrichment for FSPs (6.99% vs. 0.26%; odds ratio = 28.7, p = 1.13 10-14), corresponding to ~51-fold and ~27-fold higher risks, respectively. These findings establish intrinsic structural plasticity—whether through evolutionary replacement (NOGD/NHIE) or conformational switching (FSP)—as a central mechanism enabling functional moonlighting in the human proteome. The results suggest that such plasticity facilitates functional innovation while preserving sequence integrity, and that both NOGD/NHIE and FSP features may serve as predictive signatures for identifying novel moonlighting proteins, particularly those with implications for disease mechanisms and therapeutic targeting. Keywords: moonlighting proteins; multitasking proteins; moonlighting proteins evolution;non-orthologous gene displacement; non-homologous isofunctional enzymes;fold-switching proteins; protein structure and function; conformational plasticity Author: Juan Cedano, Mario Huerta, Angel Mozo-Villarias and Enrique Querol Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Fri Oct 24 17:19:06 2025 CEST ModDate: Fri Oct 24 17:23:32 2025 CEST Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 11 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 759111 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- PFCGPU+URWPalladioL-Roma Type 1 Custom yes yes yes 98 0 SOSCBV+CMSY10 Type 1 Builtin yes yes yes 99 0 KXCHGN+URWPalladioL-Ital Type 1 Custom yes yes yes 100 0 IAJRSH+URWPalladioL-Bold Type 1 Custom yes yes yes 101 0 JGJJDB+PalatinoLinotype TrueType WinAnsi yes yes no 160 0 JGJJDB+PalatinoLinotype TrueType WinAnsi yes yes no 170 0 QDCWHF+EURM10 Type 1 Builtin yes yes yes 229 0 PLOLAK+VnURWPalladioL Type 1 Custom yes yes yes 275 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-11-26 03:10:51 CET RepresentationInformation: ijms-26-10375.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-11-25 10:23:33 CET Size: 759111 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 345 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Children: Item: Title: Structural and Regulatory Mechanisms of Moonlighting Item: Title: Evolutionary Origins: Key Unanswered Questions Item: Title: Results and Discussion Children: Item: Title: Mechanistic Hypotheses for Moonlighting Acquisition Item: Title: Strategies for Acquiring Moonlighting Functions: Exploiting Intrinsic Properties Item: Title: Redox-Sensitive Functional Switching: Exploiting Catalytic Residues Item: Title: Oligomerization as a Conformational Switch Item: Title: Fold-Switching Proteins: Subtle Conformational Changes Beyond Oligomerization Item: Title: Why Is Not Moonlighting More Widespread? Item: Title: Materials and Methods Item: Title: Conclusions Item: Title: References Info: Title: Moonlighting Proteins: Some Hypotheses on the Structural Origin of Their Multifunctionality Author: Juan Cedano, Mario Huerta, Angel Mozo-Villarias and Enrique Querol Subject: Moonlighting proteins—single polypeptides performing multiple, often unrelated functions—are increasingly recognized as key players in human disease and microbial pathogenesis, making their identification crucial for understanding disease mechanisms and developing targeted therapies. This study addresses the unresolved question of how such multifunctionality evolves, focusing on two potential structural mechanisms: Non-Orthologous Gene Displacement/Non-Homologous Isofunctional Enzymes (NOGD/NHIE), where evolutionarily unrelated proteins perform the same function, and Fold-Switching Proteins (FSP), which adopt alternative secondary structures to switch functions without sequence changes. We analyzed the overlap between known human moonlighting proteins (from MultitaskProtDB-II) and curated datasets of NOGD/NHIE (Non-Orthologous Gene Displacement/Non-Homologous Isofunctional Enzymes) and fold-switching proteins (FSPs), using Fisher’s exact test for statistical validation. Moonlighting proteins showed extraordinary enrichment for NOGD/NHIE (19.89% vs. 0.39% in non-moonlighting proteins; odds ratio = 63.1, p < 2.2 10-16) and strong enrichment for FSPs (6.99% vs. 0.26%; odds ratio = 28.7, p = 1.13 10-14), corresponding to ~51-fold and ~27-fold higher risks, respectively. These findings establish intrinsic structural plasticity—whether through evolutionary replacement (NOGD/NHIE) or conformational switching (FSP)—as a central mechanism enabling functional moonlighting in the human proteome. The results suggest that such plasticity facilitates functional innovation while preserving sequence integrity, and that both NOGD/NHIE and FSP features may serve as predictive signatures for identifying novel moonlighting proteins, particularly those with implications for disease mechanisms and therapeutic targeting. 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