molecules Review Moonlighting Proteins in the Fuzzy Logic of Cellular Metabolism Haipeng Liu 1 and Constance J. Jeffery 2,* 1 Center for Biomolecular Sciences, College of Pharmacy, University of Illinois at Chicago, 900 South Ashland Avenue, Chicago, IL 60607, USA; [email protected] 2 Department of Biological Sciences, University of Illinois at Chicago, 900 South Ashland Avenue, Chicago, IL 60607, USA * Correspondence: cjeff[email protected]; Tel.: +1-312-996-3168 Academic Editor: Pier Luigi Gentili Received: 30 May 2020; Accepted: 23 July 2020; Published: 29 July 2020 Abstract: The numerous interconnected biochemical pathways that make up the metabolism of a living cell comprise a fuzzy logic system because of its high level of complexity and our inability to fully understand, predict, and model the many activities, how they interact, and their regulation. Each cell contains thousands of proteins with changing levels of expression, levels of activity, and patterns of interactions. Adding more layers of complexity is the number of proteins that have multiple functions. Moonlighting proteins include a wide variety of proteins where two or more functions are performed by one polypeptide chain. In this article, we discuss examples of proteins with variable functions that contribute to the fuzziness of cellular metabolism. Keywords: moonlighting proteins; fuzzy logic; intrinsically disordered proteins; metamorphic proteins; morpheeins 1. Introduction Fuzzy logic systems include variables that can be any real number between 0 and 1 instead of being limited to the Boolean logic variables of only 0 and 1. This enables expression of complexity, uncertainty, and imprecision. In general, the vast interconnected biochemical pathways that make up the metabolism of a living cell can appear fuzzy because they are complex and hard to predict, and we have incomplete and not yet accurate knowledge. A single cell contains thousands of proteins performing a wide variety of activities, and the proteins have complex and constantly changing levels of expression, levels of activity, and patterns of interactions with other proteins and other molecules. Adding even more layers of complexity is the ability of many proteins, called moonlighting proteins, to perform more than one function. Moonlighting proteins are proteins in which one polypeptide chain performs more than one physiologically relevant biochemical or biophysical function [1–3] (Figure1). The MoonProt Database (www.moonlightingproteins.org) contains annotations for over 300 experimentally confirmed moonlighting proteins, of which about 130 proteins are from human [4,5]. Although the mechanisms by which one protein performs two different functions are not always understood, it is clear that the function (or functions) performed at any specific time can be affected by multiple factors, and sometimes combinations of factors, including targeting to different cellular compartments, changes in the intracellular concentration of ligands, and changes in environmental conditions. In this paper, we describe examples of moonlighting proteins and some of the mechanisms by which they change function. These examples help illustrate and complement the ideas in this collection of papers on the topic of “The Fuzziness in Molecular Supramolecular, and Systems Chemistry”, where Gentili presents the “Fuzziness of the Molecular World” and describes natural information systems that involve fuzzy logic in large part due to proteins having multiple features Molecules 2020, 25, 3440; doi:10.3390/molecules25153440 www.mdpi.com/journal/molecules Molecules 2020, 25, 3440 2 of 18 Molecules 2020, 25, x FOR PEER REVIEW 2 of 19 featuresand functions and functions that vary that in vary a context-dependent in a context-dependent manner manner [6]. In addition,[6]. In addition, the paper the bypaper Fuxreiter by Fuxreiterdescribes describes using fuzzy using set theoryfuzzy inset a quantitativetheory in frameworka quantitative for describingframework the for relationships describing between the relationshipschanging protein between structures, changing interactions, protein structures, and functions interactio underns, changing,and functions and under somewhat changing, unknown and or somewhat unknown or unpredictable, cellular conditions [7]. unpredictable, cellular conditions [7]. Figure 1. In a moonlighting protein (purple oval), more than one physiologically relevant biochemical Figure 1. In a moonlighting protein (purple oval), more than one physiologically relevant biochemical or biophysical function is performed by a single polypeptide chain. Note: This figure was “Created or biophysical function is performed by a single polypeptide chain. Note: This figure was “Created with BioRender.com”. with BioRender.com”. 2. Examples of Moonlighting Proteins and Factors that Affect Function 2. Examples of Moonlighting Proteins and Factors that Affect Function 2.1. Cellular Localization 2.1. Cellular Localization The most often observed subclass of moonlighting protein includes proteins that perform different functionsThe most in dioftenfferent observed cellular localizations.subclass of moonlighting Over 100 enzymes protein and includes chaperones proteins that catalyze that perform reactions diffeinrent the cytosolfunctions can in be different secreted cellular and act localizations. as cytokines Over that modify100 enzymes the host’s and immunechaperones system that catalyze or become reactionsbound toin thethe cellcytosol membrane can be secreted where they and serve act as as cytokines cell surface that receptors, modify the and host’s in some immune cases thesesystem second or becomefunctions bound contribute to the cell to virulencemembrane [8 –where11]. they serve as cell surface receptors, and in some cases these secondEnolase functions is one of contribute these intracellular to virulence/surface [8–11]. moonlighting proteins in many species, including eukaryotesEnolase is as one well of as these prokaryotes. intracellular/surface Inside the cell, moonlighting it catalyzes proteins the conversion in many of species, 2-phosphoglycerate including eukaryotesto phosphoenolpyruvate as well as prokaryotes. in glycolysis. 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StreptococcusThe binding pneumonia of plasminogene, Trichomoniasis plays an important vaginalis role and in invasionLactobacillus of host crispatus tissues can because, bind once to boundhost plasminogento the cell surface[12–17]. receptor,The binding the of plasminogen plasminogen becomes plays an convertedimportant role to the in invasion active protease, of host tissues plasmin, because,which canonce aid bound in breaking to the cell down surface host receptor, extracellular the plasminogen matrix and invasion becomes of converted tissues [18 to,19 the]. active In some protease,species, plasmin, surface-located which can enolase aid in and breaking other intracellulardown host extracellular/surface moonlighting matrix and proteins invasion bind of tissues to other [18,19].host proteinsIn some for species, colonization surface or for-located modulating enolase the hostand immuneother intracellular/surface system. Streptococcus moonlighting suis enolase can proteinsalso bind bind to hostto other fibronectin, host proteins and Staphylococcus for colonization aureus orenolase for modulating exhibits laminin the host binding immune activity system. [20,21 ]. StreptococcusGlyceraldehyde suis enolase 3-phosphate can also bind dehydrogenase to host fibronectin, (GAPDH) and is Staphylococcus another commonly aureusfound enolase intracellular exhibits / lamininsurface binding moonlighting activity protein.[20,21]. It catalyzes the conversion of glyceraldehyde 3-phosphate to glycerate 1,3-bisphosphateGlyceraldehyde in glycolysis3-phosphate in the dehydrogenase cytoplasm. Some (GAPDH) commensal bacteriais another that colonizecommonly the humanfound gut intracellular/surfaceuse GAPDH on the moonlighting cell surface protein. to bind It to catalyzes host mucin the conversion and enable of colonizationglyceraldehyde of 3 the-phosphategut [22,23 ]. to Whenglycerate expressed 1,3-bisphosphate on the cell in glycolysis surface, Streptococcusin the cytoplasm. pyogenes SomeGADPH commensal can ba bindcteria to that plasminogen colonize theand human fibronectin, gut use andGAPDH can alsoon the function cell surface as a to ADP-ribosylating bind to host mucin enzyme and enable and assist colonization with neutrophil of the gutevasion [22,23]. [24 When–27]. In expressed addition, onStreptococcus the cell surface, agalactiae StreptococcusGADPH can pyogenes act as a GADPH modulator can of bind the host’s to plasminogenimmune system and fibronectin, [28]. and can also function as a ADP-ribosylating enzyme and assist with neutrophilAs theevasion bacterial [24– HSP7027]. In addition, (heat shock Streptococcus protein 70), agalactiae DnaK is GADPH abundantly can act expressed as a modulator in the cytosol of the as host’sa stress-inducible immune system chaperone. [28]. Mycobacterium tuberculosis DnaK can be displayed on