Do protein-protein interaction databases identify moonlighting proteins?

One of the most striking results of the human (and mammalian) genomes is the low number of protein-coding genes. To-date, the main molecular mechanism to increase the number of different protein isoforms and functions is alternative splicing. However, a less-known way to increase the number of protein functions is the existence of multifunctional, multitask, or "moonlighting", proteins. By and large, moonlighting proteins are experimentally disclosed by serendipity. Proteomics is becoming one of the very active areas of biomedical research, which permits researchers to identify previously unseen connections among proteins and pathways. In principle, protein-protein interaction (PPI) databases should contain information on moonlighting proteins and could provide suggestions to further analysis in order to prove the multifunctionality. As far as we know, nobody has verified whether PPI databases actually disclose moonlighting proteins. In the present work we check whether well-established moonlighting proteins present in PPI databases connect with their known partners and, therefore, a careful inspection of these databases could help to suggest their different functions. The results of our research suggest that PPI databases could be a valuable tool to suggest multifunctionality.     

Physical Features of Intracellular Proteins that Moonlight on the Cell Surface

Moonlighting proteins comprise a subset of multifunctional proteins that perform two or more biochemical functions that are not due to gene fusions, multiple splice variants, proteolytic fragments, or promiscuous enzyme activities. The project described herein focuses on a sub-set of moonlighting proteins that have a canonical biochemical function inside the cell and perform a second biochemical function on the cell surface in at least one species. The goal of this project is to consider the biophysical features of these moonlighting proteins to determine whether they have shared characteristics or defining features that might suggest why these particular proteins were adopted for a second function on the cell surface, or if these proteins resemble typical intracellular proteins. The latter might suggest that many other normally intracellular proteins found on the cell surface might also be moonlighting in this fashion. We have identified 30 types of proteins that have different functions inside the cell and on the cell surface. Some of these proteins are found to moonlight on the surface of multiple species, sometimes with different extracellular functions in different species, so there are a total of 98 proteins in the study set. Although a variety of intracellular proteins (enzymes, chaperones, etc.) are observed to be re-used on the cell surface, for the most part, these proteins were found to have physical characteristics typical of intracellular proteins. Many other intracellular proteins have also been found on the surface of bacterial pathogens and other organisms in proteomics experiments. It is quite possible that many of those proteins also have a moonlighting function on the cell surface. The increasing number and variety of known moonlighting proteins suggest that there may be more moonlighting proteins than previously thought, and moonlighting might be a common feature of many more proteins.     

Moonlighting is mainstream: paradigm adjustment required

Moonlighting--the performance of more than one function by a single protein--is becoming recognized as a common phenomenon with important implications for systems biology and human health. The different functions of a moonlighting protein may use different regions of the protein structure, or alternative structures that occur due to post-translational modifications and/or differences in binding partners. Often the different functions of moonlighting proteins are used at different times or in different places. The existence of moonlighting functions complicates efforts to understand metabolic and regulatory networks, as well as physiological and pathological processes in organisms. Because moonlighting functions can play important roles in disease processes, an improved understanding of moonlighting proteins will provide new opportunities for pharmacological manipulations that specifically target a function involved in pathology while sparing physiologically important functions.     

Bacterial bug-out bags: outer membrane vesicles and their proteins and functions

Among the major bacterial secretions, outer membrane vesicles (OMVs) are significant and highly functional. The proteins and other biomolecules identified within OMVs provide new insights into the possible functions of OMVs in bacteria. OMVs are rich in proteins, nucleic acids, toxins and virulence factors that play a critical role in bacteria-host interactions. In this review, we discuss some proteins with multifunctional features from bacterial OMVs and their role involving the mechanisms of bacterial survival and defence. Proteins with moonlighting activities in OMVs are discussed based on their functions in bacteria. OMVs harbour many other proteins that are important, such as proteins involved in virulence, defence, and competition. Overall, OMVs are a power-packed aid for bacteria, harbouring many defensive and moonlighting proteins and acting as a survival kit in case of an emergency or as a defence weapon. In summary, OMVs can be defined as bug-out bags for bacterial defence and, therefore, survival.     

The multifunctional or moonlighting protein CD26/DPPIV

CD26/DPPIV can be considered a moonlighting protein because it is a multifunctional protein that exerts its different functions depending on cell type and intra- or extracellular conditions in which it is expressed. In the present review, we summarize all its known functions in relation to physiological and pathophysiological conditions. The protein is a proteolytic enzyme, receptor, costimulatory protein, and is involved in adhesion and apoptosis. The CD26/DPPIV protein plays a major role in immune response. Abnormal expression is found in the case of autoimmune diseases, HIV-related diseases and cancer. Natural substrates for CD26/DPPIV are involved in immunomodulation, psycho/neuronal modulation and physiological processes in general. Therefore, targeting of CD26/ DPPIV and especially its proteolytic activity has many therapeutic potentials. On the other hand, there are homologous proteins with overlapping proteolytic activity, which thus may prevent specific modulation of CD26/DPPIV. In conclusion, CD26/DPPIV is a protein present both in various cellular compartments and extracellularly where it exerts different functions and thus is a true moonlighting protein.     

Moonlighting Biochemistry of Cysteine Synthase: A Species-specific Global Regulator

Cysteine Synthase (CS), the enzyme that synthesizes cysteine, performs non-canonical regulatory roles by binding and modulating functions of disparate proteins. Beyond its role in catalysis and regulation in the cysteine biosynthesis pathway, it exerts its moonlighting effect by binding to few other proteins which possess a C-terminal “CS-binding motif”, ending with a terminal ILE. Therefore, we hypothesized that CS might regulate many other disparate proteins with the “CS-binding motif”. In this study, we developed an iterative sequence matching method for mapping moonlighting biochemistry of CS and validated our prediction by analytical and structural approaches. Using a minimal protein-peptide interaction system, we show that five previously unknown CS-binder proteins that participate in diverse metabolic processes interact with CS in a species-specific manner. Furthermore, results show that signatures of protein–protein interactions, including thermodynamic, competitive-inhibition, and structural features, highly match the known CS-Binder, serine acetyltransferase (SAT). Together, the results presented in this study allow us to map the extreme multifunctional space (EMS) of CS and reveal the biochemistry of moonlighting space, a subset of EMS. We believe that the integrated computational and experimental workflow developed here could be further modified and extended to study protein-specific moonlighting properties of multifunctional proteins.     

Single-gene disorders: what role could moonlighting enzymes play?

Single-gene disorders with "simple" Mendelian inheritance do not always imply that there will be an easy prediction of the phenotype from the genotype, which has been shown for a number of metabolic disorders. We propose that moonlighting enzymes (i.e., metabolic enzymes with additional functional activities) could contribute to the complexity of such disorders. The lack of knowledge about the additional functional activities of proteins could result in a lack of correlation between genotype and phenotype. In this review, we highlight some notable and recent examples of moonlighting enzymes and their possible contributions to human disease. Because knowledge and cataloging of the moonlighting activities of proteins are essential for the study of cellular function and human physiology, we also review recently reported and recommended methods for the discovery of moonlighting activities.     

Therapeutic potential of β-arrestin- and G protein-biased agonists

Members of the seven-transmembrane receptor (7TMR), or G protein-coupled receptor (GPCR), superfamily represent some of the most successful targets of modern drug therapy, with proven efficacy in the treatment of a broad range of human conditions and disease processes. It is now appreciated that β-arrestins, once viewed simply as negative regulators of traditional 7TMR-stimulated G protein signaling, act as multifunctional adapter proteins that regulate 7TMR desensitization and trafficking and promote distinct intracellular signals in their own right. Moreover, several 7TMR biased agonists, which selectively activate these divergent signaling pathways, have been identified. Here we highlight the diversity of G protein- and β-arrestin-mediated functions and the therapeutic potential of selective targeting of these in disease states.     

General approach to identifying potential targets for cancer imaging by integrated bioinformatics analysis of publicly available genomic profiles

Molecular imaging has moved to the forefront of drug development and biomedical research. The identification of appropriate imaging targets has become the touchstone for the accurate diagnosis and prognosis of human cancer. Particularly, cell surface- or membrane-bound proteins are attractive imaging targets for their aberrant expression, easily accessible location, and unique biochemical functions in tumor cells. Previously, we published a literature mining of potential targets for our in-house enzyme-mediated cancer imaging and therapy technology. Here we present a simple and integrated bioinformatics analysis approach that assembles a public cancer microarray database with a pathway knowledge base for ascertaining and prioritizing upregulated genes encoding cell surface- or membrane-bound proteins, which could serve imaging targets. As examples, we obtained lists of potential hits for six common and lethal human tumors in the prostate, breast, lung, colon, ovary, and pancreas. As control tests, a number of well-known cancer imaging targets were detected and confirmed by our study. Further, by consulting gene-disease and protein-disease databases, we suggest a number of significantly upregulated genes as promising imaging targets, including cell surface-associated mucin-1, prostate-specific membrane antigen, hepsin, urokinase plasminogen activator receptor, and folate receptors. By integrating pathway analysis, we are able to organize and map "focused" interaction networks derived from significantly dysregulated entity pairs to reflect important cellular functions in disease processes. We provide herein an example of identifying a tumor cell growth and proliferation subnetwork for prostate cancer. This systematic mining approach can be broadly applied to identify imaging or therapeutic targets for other human diseases.     

Non-conventional protein secretion in yeast

Many proteins are transported to the cell surface of Saccharomyces cerevisiae and Candida albicans to be either integrated into the cell-wall structure or exported to the external medium. Secretion of many of these proteins through the classical endoplasmic reticulum-Golgi pathway is driven by a canonical N-terminal signal peptide. However, several surface proteins lacking this motif can also access the cell surface and remain loosely bound to the wall. The previous identification of these secretion-signal-less proteins in the cytoplasm as proteins that function as glycolytic enzymes, chaperones, translation factors and others suggests that they could be "moonlighting" (multifunctional) proteins. The accumulated evidence indicates that mechanisms of secretion other than the endoplasmic reticulum-Golgi pathway drive these proteins outside the plasma membrane. The relevance of these secretion-signal-less proteins in virulence and cell-wall dynamics warrants further characterization of alternative secretion in yeasts.     

Benchmarking Human Protein Complexes to Investigate Drug-Related Systems and Evaluate Predicted Protein Complexes

Protein complexes are key entities to perform cellular functions. Human diseases are also revealed to associate with some specific human protein complexes. In fact, human protein complexes are widely used for protein function annotation, inference of human protein interactome, disease gene prediction, and so on. Therefore, it is highly desired to build an up-to-date catalogue of human complexes to support the research in these applications. Protein complexes from different databases are as expected to be highly redundant. In this paper, we designed a set of concise operations to compile these redundant human complexes and built a comprehensive catalogue called CHPC2012 (Catalogue of Human Protein Complexes). CHPC2012 achieves a higher coverage for proteins and protein complexes than those individual databases. It is also verified to be a set of complexes with high quality as its co-complex protein associations have a high overlap with protein-protein interactions (PPI) in various existing PPI databases. We demonstrated two distinct applications of CHPC2012, that is, investigating the relationship between protein complexes and drug-related systems and evaluating the quality of predicted protein complexes. In particular, CHPC2012 provides more insights into drug development. For instance, proteins involved in multiple complexes (the overlapping proteins) are potential drug targets; the drug-complex network is utilized to investigate multi-target drugs and drug-drug interactions; and the disease-specific complex-drug networks will provide new clues for drug repositioning. With this up-to-date reference set of human protein complexes, we believe that the CHPC2012 catalogue is able to enhance the studies for protein interactions, protein functions, human diseases, drugs, and related fields of research. CHPC2012 complexes can be downloaded from http://www1.i2r.a-star.edu.sg/xlli/CHPC2012/CHPC2012.htm.     

(Intrinsically disordered) splice variants in the proteome: implications for novel drug discovery

In higher eukaryotes, proteomes are typically larger than corresponding genomes mostly due to alternative spicing (AS) which is affecting more than 86 % of human genes. Tissue specificity of many proteins is often determined by the AS of pre-mRNAs that generates multiple proteins from a single gene. Aberrations in AS are found in numerous human diseases. This article shows that AS expanded the classic “one-gene–one-protein” paradigm to the “one-gene–many-proteins” concept. AS is intimately associated with protein intrinsic disorder. There is a tight connection between the altered AS of some key intrinsically disordered proteins and pathogenesis of neurodegenerative diseases, cardiovascular disease, cancer, and diabetes. The development of drugs for splice variants is a challenging endeavor. AS has tremendous influence on the applicability of several drugs developed to affect functions typically attributed to the ordered parts of disease-related proteins. Although there are several means for the differential targeting of splice variants, new approaches are obviously needed. It is expected that better understanding of the molecular mechanisms underlying modulation of biological activities of numerous spliced variants will be achieved and new drug design approaches for proteins with multiple splice variants will be developed.     

Transdifferentiation of human somatic cells by ribosome

Ribosomes are intracellular organelles ubiquitous in all organisms, which translate information from mRNAs to synthesize proteins. They are complex macromolecules composed of dozens of proteins and ribosomal RNAs. Other than translation, some ribosomal proteins also have side‐jobs called “Moonlighting” function. The majority of these moonlighting functions influence cancer progression, early development and differentiation. Recently, we discovered that ribosome is involved in the regulation of cellular transdifferentiation of human dermal fibroblasts (HDFs). In vitro incorporation of ribosomes into HDFs arrests cell proliferation and induces the formation of cell clusters, that differentiate into three germ layer derived cells upon induction by differentiation mediums. The discovery of ribosome induced transdifferentiation, that is not based on genetic modification, find new possibilities for the treatment of cancer and congenital diseases, as well as to understand early development and cellular lineage differentiation.     

Role of the microtubule-associated TPPP/p25 in Parkinson’s and related diseases and its therapeutic potential

Introduction: The discovery and development of therapeutic strategies for the treatments of Parkinson’s disease (PD) and other synucleinopathies are limited by a lack of understanding of the pathomechanisms and their connection with different diseases such as cancers.

Areas covered: The hallmarks of these diseases are frequently multifunctional disordered proteins displaying moonlighting and/or chameleon features, which are challenging drug targets. A representative of these proteins is the disordered Tubulin Polymerization Promoting Protein (TPPP/p25) expressed specifically in oligodendrocytes (OLGs) in normal brain. Its non-physiological level is tightly related to the etiology of PD and Multiple System Atrophy (TPPP/p25 enrichment in inclusions of neurons and OLGs, respectively), multiple sclerosis (TPPP/p25-positive OLG destruction), as well as glioma (loss of TPPP/p25 expression). The established anti-proliferative potency of TPPP/p25 may raise its influence in cancer development. The recognition that whereas too much TPPP/p25 could kill neurons in PD, but its loss keeps cells alive in cancer could contribute to our understanding of the interrelationship of ‘TPPP/p25 diseases’.

Expert commentary: The knowledge accumulated so far underlines the key roles of the multifunctional TPPP/p25 in both physiological and diverse pathological processes, consequently its validation as drug target sorely needs a new innovative strategy that is briefly reviewed here.     

Molecular evolution of the moonlighting protein SMN in metazoans

The spinal muscular atrophy (SMA) associated protein survival of motor neuron (SMN) is known to be a moonlighting protein: having one primary, ancestral function (presumed to be involvement in U snRNP assembly) along with one or more secondary functions. One hypothesis for the evolution of moonlighting proteins is that regions of a structure under relatively weak negative selection could gain new functions without interfering with the primary function. To test this hypothesis, we investigated sequence conservation and dN/dS, which reflects the selection acting on a coding sequence, in SMN and a related protein, splicing factor 30 (SPF30), which is not currently known to be multifunctional. We found very different patterns of evolution in the two genes, with SPF30 characterized by strong sequence conservation and negative selection in most animal taxa investigated, and SMN with much lower sequence conservation, and much weaker negative selection at many sites. Evidence was found of positive selection acting on some sites in primate genes for SMN. SMN was also found to have been duplicated in a number of species, and with patterns that indicate reduced negative selection following some of these duplications. There were also several animal species lacking an SMN gene.     

Target discovery and validation in the post-genomic era

The recent publication of the human genome sequence provides an opportunity both to combat diseases that are presently considered as pharmaceutically intractable and also to improve current therapies for many common human diseases. The identification of every human gene by ongoing bioinformatic efforts has the potential, when combined with functional genomic approaches, to pinpoint the molecular basis of every human disease, and to discover appropriate intervention points. This exciting prospect is directly relevant to the successful development of effective therapeutics because the past record of drug discovery suggests that 30%–40% of experimental drugs fail because an inappropriate biological target was pursued. The major impact of genomic information may therefore be to reduce this biological failure rate by earlier definition of drug targets related to disease susceptibility or progression. This paper briefly reviews some of the approaches that can be used to identify biologically relevant drug targets.     

Quantitative analysis on the characteristics of targets with FDA approved drugs

Accumulated knowledge of genomic information, systems biology, and disease mechanisms provide an unprecedented opportunity to elucidate the genetic basis of diseases, and to discover new and novel therapeutic targets from the wealth of genomic data. With hundreds to a few thousand potential targets available in the human genome alone, target selection and validation has become a critical component of drug discovery process. The explorations on quantitative characteristics of the currently explored targets (those without any marketed drug) and successful targets (targeted by at least one marketed drug) could help discern simple rules for selecting a putative successful target. Here we use integrative in silico (computational) approaches to quantitatively analyze the characteristics of 133 targets with FDA approved drugs and 3120 human disease genes (therapeutic targets) not targeted by FDA approved drugs. This is the first attempt to comparatively analyze targets with FDA approved drugs and targets with no FDA approved drug or no drugs available for them. Our results show that proteins with 5 or fewer number of homologs outside their own family, proteins with single-exon gene architecture and proteins interacting with more than 3 partners are more likely to be targetable. These quantitative characteristics could serve as criteria to search for promising targetable disease genes.     

Proteomic tools for the analysis of transient interactions between metalloproteins

Metalloproteins play major roles in cell metabolism and signalling pathways. In many cases, they show moonlighting behaviour, acting in different processes, depending on the physiological state of the cell. To understand these multitasking proteins, we need to discover the partners with which they carry out such novel functions. Although many technological and methodological tools have recently been reported for the detection of protein interactions, specific approaches to studying the interactions involving metalloproteins are not yet well developed. The task is even more challenging for metalloproteins, because they often form short-lived complexes that are difficult to detect. In this review, we gather the different proteomic techniques and biointeractomic tools reported in the literature. All of them have shown their applicability to the study of transient and weak protein-protein interactions, and are therefore suitable for metalloprotein interactions.