Moonlighting Proteins: Diverse Functions Found in Fungi.

Moonlighting proteins combine multiple functions in one polypeptide chain. An increasing number of moonlighting proteins are being found in diverse fungal taxa that vary in morphology, life cycle, and ecological niche. In this mini-review we discuss examples of moonlighting proteins in fungi that illustrate their roles in transcription and DNA metabolism, translation and RNA metabolism, protein folding, and regulation of protein function, and their interaction with other cell types and host proteins.

Current successes and remaining challenges in protein function prediction.

In recent years, improvements in protein function prediction methods have led to increased success in annotating protein sequences. However, the functions of over 30% of protein-coding genes remain unknown for many sequenced genomes. Protein functions vary widely, from catalyzing chemical reactions to binding DNA or RNA or forming structures in the cell, and some types of functions are challenging to predict due to the physical features associated with those functions. Other complications in understanding protein functions arise due to the fact that many proteins have more than one function or very small differences in sequence or structure that correspond to different functions. We will discuss some of the recent developments in predicting protein functions and some of the remaining challenges.

MSFP: undergraduate 'collaborate-from-home' research in macromolecular structure and function.

Summary: When the COVID-19 crisis shut down most undergraduate research opportunities, the Macromolecular Structure and Function Research Experiences for Undergraduates Program provided a mentored research experience on the topic of Macromolecular Structure and Function and training in professional skills to assist the participants in pursuing a degree and a future career in STEM. The fully online, remote, computer-based program was funded by the USA National Science Foundation. It involved faculty at four geographically distributed institutions specializing in diverse but complementary approaches to study macromolecular structure and function. Importantly, its online 'collaborate-from-home' format made it accessible to students during the pandemic to participate fully in the research, professional development and other activities of the program. This project can also serve as an example for future remote, online projects that would especially be helpful for students who do not have access to similar programs at their universities, cannot travel to attend a summer program, have physical challenges that make it difficult for them to work in a lab or students whose research opportunities are limited due to the war in Ukraine. The lessons learned with the Macromolecular Structure and Function REU program can provide helpful information for ISCB members to set up similar programs to serve additional students. Availability and implementation: More information and resources are available on the project web site http://jefferylab.moonlightingproteins.org. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Promoting a More Integrated Approach to Structure and Function.

The connection between structure and function is one of the fundamental tenets of biology: a biological unit's structure determines its function, and, conversely, its function depends upon its structure. Historically, important advances have been made either when understanding of structure leads to questions about function or when understanding of function raises questions about the structures involved. Consequently, considering the connections between structure and function from a broader perspective might lead to the development of novel hypotheses that move our understanding of the fundamental connections between structure and function forward. Better integration of structure and function is a key component in the broader goal of reintegrating biology within and across scales. Here, we provide examples of how integrating studies of structure and function as well as comparing structure-function relationships across biological scales can lead to scientific advances. We also emphasize the potential of integrating studies of structure and function across scales for bio-inspired design and for improving biology education.

The expanding world of metabolic enzymes moonlighting as RNA binding proteins.

RNA binding proteins play key roles in many aspects of RNA metabolism and function, including splicing, transport, translation, localization, stability and degradation. Within the past few years, proteomics studies have identified dozens of enzymes in intermediary metabolism that bind to RNA. The wide occurrence and conservation of RNA binding ability across distant branches of the evolutionary tree suggest that these moonlighting enzymes are involved in connections between intermediary metabolism and gene expression that comprise far more extensive regulatory networks than previously thought. There are many outstanding questions about the molecular structures and mechanisms involved, the effects of these interactions on enzyme and RNA functions, and the factors that regulate the interactions. The effects on RNA function are likely to be wider than regulation of translation, and some enzyme-RNA interactions have been found to regulate the enzyme's catalytic activity. Several enzyme-RNA interactions have been shown to be affected by cellular factors that change under different intracellular and environmental conditions, including concentrations of substrates and cofactors. Understanding the molecular mechanisms involved in the interactions between the enzymes and RNA, the factors involved in regulation, and the effects of the enzyme-RNA interactions on both the enzyme and RNA functions will lead to a better understanding of the role of the many newly identified enzyme-RNA interactions in connecting intermediary metabolism and gene expression.

Moonlighting Proteins in the Fuzzy Logic of Cellular Metabolism.

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.

Enzymes, pseudoenzymes, and moonlighting proteins: diversity of function in protein superfamilies.

As more genome sequences are elucidated, there is an increasing need for information about the functions of the millions of proteins they encode. The function of a newly sequenced protein is often estimated by sequence alignment with the sequences of proteins with known functions. However, protein superfamilies can contain members that share significant amino acid sequence and structural homology yet catalyze different reactions or act on different substrates. Some homologous proteins differ by having a second or even third function, called moonlighting proteins. More recently, it was found that most protein superfamilies also include pseudoenzymes, a protein, or a domain within a protein, that has a three-dimensional fold that resembles a conventional catalytically active enzyme, but has no catalytic activity. In this review, we discuss several examples of protein families that contain enzymes, pseudoenzymes, and moonlighting proteins. It is becoming clear that pseudoenzymes and moonlighting proteins are widespread in the evolutionary tree, and in many protein families, and they are often very similar in sequence and structure to their monofunctional and catalytically active counterparts. A greater understanding is needed to clarify when similarities and differences in amino acid sequences and structures correspond to similarities and differences in biochemical functions and cellular roles. This information can help improve programs that identify protein functions from sequence or structure and assist in more accurate annotation of sequence and structural databases, as well as in our understanding of the broad diversity of protein functions.

Multitalented actors inside and outside the cell: recent discoveries add to the number of moonlighting proteins.

During the past few decades, it's become clear that many enzymes evolved not only to act as specific, finely tuned and carefully regulated catalysts, but also to perform a second, completely different function in the cell. In general, these moonlighting proteins have a single polypeptide chain that performs two or more distinct and physiologically relevant biochemical or biophysical functions. This mini-review describes examples of moonlighting proteins that have been found within the past few years, including some that play key roles in human and animal diseases and in the regulation of biochemical pathways in food crops. Several belong to two of the most common subclasses of moonlighting proteins: trigger enzymes and intracellular/surface moonlighting proteins, but a few represent less often observed combinations of functions. These examples also help illustrate some of the current methods used for identifying proteins with multiple functions. In general, a greater understanding about the functions and molecular mechanisms of moonlighting proteins, their roles in the regulation of cellular processes, and their involvement in health and disease could aid in many areas including developing new antibiotics, predicting the functions of the millions of proteins being identified through genome sequencing projects, designing novel proteins, using biological circuitry analysis to construct bacterial strains that are better producers of materials for industrial use, and developing methods to tweak biochemical pathways for increasing yields of food crops.

Intracellular/surface moonlighting proteins that aid in the attachment of gut microbiota to the host.

The gut microbiota use proteins on their surface to form and maintain interactions with host cells and tissues. In recent years, many of these cell surface proteins have been found to be identical to intracellular enzymes and chaperones. When displayed on the cell surface these moonlighting proteins help the microbe attach to the host by interacting with receptors on the surface of host cells, components of the extracellular matrix, and mucin in the mucosal lining of the digestive tract. Binding of these proteins to the soluble host protein plasminogen promotes the conversion of plasminogen to an active protease, plasmin, which activates other host proteins that aid in infection and virulence. In this mini-review, we discuss intracellular/surface moonlighting proteins of pathogenic and probiotic bacteria and eukaryotic gut microbiota.

An enzyme in the test tube, and a transcription factor in the cell: Moonlighting proteins and cellular factors that affect their behavior.

In the cell, expression levels, allosteric modulators, post-translational modifications, sequestration, and other factors can affect the level of protein function. For moonlighting proteins, cellular factors like these can also affect the kind of protein function. This minireview discusses examples of moonlighting proteins that illustrate how a single protein can have different functions in different cell types, in different intracellular locations, or under varying cellular conditions. This variability in the kind of protein activity, added to the variability in the amount of protein activity, contributes to the difficulty in predicting the behavior of proteins in the cell.

The demise of catalysis, but new functions arise: pseudoenzymes as the phoenixes of the protein world.

Pseudoenzymes are noncatalytic homologues of enzymes and are found in most enzyme families. Although lacking catalytic activity and sometimes referred to as 'dead' enzymes, they instead resemble phoenixes because the loss of a catalytic function during evolution was associated with the development of vital new functions. They are important in regulating the activity and location of catalytically active homologues, scaffolding the assembly of signaling complexes, and regulating transcription or translation. They are key actors in cell proliferation and differentiation, proteostasis, and many other biochemical pathways and processes. They perform their functions in diverse ways, but many retain some aspects of the function of their catalytically active homologues. In some pseudoenzymes, their functions are very different from other members of their protein families, suggesting some arose from ancient moonlighting proteins during evolution. Much less is known about pseudoenzymes than their catalytically active counterparts, but a growing appreciation of their key roles in many important biochemical processes and signaling pathways has led to increased investigation in recent years. It is clear that there is still much more to learn about the structures, functions, and cellular roles of these phoenix-like proteins.

Open Conformation of the Escherichia coli Periplasmic Murein Tripeptide Binding Protein, MppA, at High Resolution.

Periplasmic ligand-binding proteins (PBPs) bind ligands with a high affinity and specificity. They undergo a large conformational change upon ligand binding, and they have a robust protein fold. These physical features have made them ideal candidates for use in protein engineering projects to develop novel biosensors and signaling molecules. The Escherichia coli MppA (murein peptide permease A) PBP binds the murein tripeptide, l-alanyl-γ-d-glutamyl-meso-diaminopimelate, (l-Ala-γ-d-Glu-meso-Dap), which contains both a D-amino acid and a gamma linkage between two of the amino acids. We have solved a high-resolution X-ray crystal structure of E. coli MppA at 1.5 Šresolution in the unliganded, open conformation. Now, structures are available for this member of the PBP protein family in both the liganded/closed form and the unliganded/open form.

Protein moonlighting: what is it, and why is it important?

Members of the GroEL/HSP60 protein family have been studied for many years because of their critical roles as ATP-dependent molecular chaperones, so it might come as a surprise that some have important functions in ATP-poor conditions, for example, when secreted outside the cell. At least some members of each of the HSP10, HSP70, HSP90, HSP100 and HSP110 heat shock protein families are also 'moonlighting proteins'. Moonlighting proteins exhibit more than one physiologically relevant biochemical or biophysical function within one polypeptide chain. In this class of multifunctional proteins, the multiple functions are not due to gene fusions or multiple proteolytic fragments. Several hundred moonlighting proteins have been identified, and they include a diverse set of proteins with a large variety of functions. Some participate in multiple biochemical processes by using an active site pocket for catalysis and a different part of the protein's surface to interact with other proteins. Moonlighting proteins play a central role in many diseases, and the development of novel treatments would be aided by more information addressing current questions, for example, how some are targeted to multiple cellular locations and how a single function can be targeted by therapeutics without targeting a function not involved in disease.This article is part of the theme issue 'Heat shock proteins as modulators and therapeutic targets of chronic disease: an integrated perspective'.

Moonlighting proteins - nature's Swiss army knives.

The human body is a complex biological machine with billions of cells and vast numbers of biochemical processes - but our genome only contains 22,000 protein-encoding genes. Moonlighting proteins provide one way to increase the number of cellular activities. Moonlighting proteins exhibit more than one physiologically relevant biochemical or biophysical function within one polypeptide chain. Already more than 300 moonlighting proteins have been identified, and they include a diverse set of proteins with a large variety of functions. This article discusses examples of moonlighting proteins, how one protein structure can perform two different functions, and how the multiple functions can be regulated. In addition to learning more about what our proteins do and how they work together in complex multilayered interaction networks and processes in our bodies, the study of moonlighting proteins can inform future synthetic biology projects in making proteins that perform new functions and new combinations of functions, for example, for synthesising new materials, delivering drugs into cells, and in bioremediation.

Disruption of the monocarboxylate transporter-4-basigin interaction inhibits the hypoxic response, proliferation, and tumor progression.

We have previously shown that glioblastoma stem cells (GSCs) are enriched in the hypoxic tumor microenvironment, and that monocarboxylate transporter-4 (MCT4) is critical for mediating GSC signaling in hypoxia. Basigin is involved in many physiological functions during early stages of development and in cancer and is required for functional plasma membrane expression of MCT4. We sought to determine if disruption of the MCT-Basigin interaction may be achieved with a small molecule. Using a cell-based drug-screening assay, we identified Acriflavine (ACF), a small molecule that inhibits the binding between Basigin and MCT4. Surface plasmon resonance and cellular thermal-shift-assays confirmed ACF binding to basigin in vitro and in live glioblastoma cells, respectively. ACF significantly inhibited growth and self-renewal potential of several glioblastoma neurosphere lines in vitro, and this activity was further augmented by hypoxia. Finally, treatment of mice bearing GSC-derived xenografts resulted in significant inhibition of tumor progression in early and late-stage disease. ACF treatment inhibited intratumoral expression of VEGF and tumor vascularization. Our work serves as a proof-of-concept as it shows, for the first time, that disruption of MCT binding to their chaperon, Basigin, may be an effective approach to target GSC and to inhibit angiogenesis and tumor progression.

Keeping good friends close - The surface and secreted proteomes of a probiotic bacterium provide candidate proteins for intestinal attachment and communication with the host.

Bacteria use cell surface proteins and secreted proteins to interact with host tissues. Several dozen previously published proteomics studies have identified cell surface proteins for pathogens. In this issue, Celebioglu and Svensson (Proteomics 2017, 17, 1700019) use 2D gel electrophoresis and mass spectrometry to identify secreted and cell surface proteins of a commensual gut bacterium, Lactobacillus acidophilus NCFM. Some of the proteins are known to have functions in the cytoplasm, and their presence on the cell surface suggests they might be moonlighting proteins. In addition, comparisons of proteins used by pathogenic and probiotic species to interact with their hosts could lead to improved treatments of infections and chronic diseases that are associated with an imbalance of pathogenic and probiotic gut bacteria.

An analysis of surface proteomics results reveals novel candidates for intracellular/surface moonlighting proteins in bacteria.

Proteins expressed on the bacterial cell surface play important roles in infection and virulence and can be targets for vaccine development or used as biomarkers. Surprisingly, an increasing number of surface proteins are being found to be identical to intracellular enzymes and chaperones, and a few dozen intracellular/surface moonlighting proteins have been found that have different functions inside the cell and on the cell surface. The results of twenty-two published bacterial surface proteomics studies were analyzed using bioinformatics tools to consider how many additional intracellular proteins are also found on the cell surface. More than 1000 out of the 3619 proteins observed on the cell surface lack the transmembrane alpha-helices or transmembrane beta-barrels found in integral membrane proteins and also lack the signal peptides found in proteins secreted through the Sec pathway. Many of the proteins found on the cell surface are intracellular chaperones or enzymes involved in central metabolic pathways, including some that have previously been shown to have a moonlighting function on the cell surface in at least one species, such as Hsp60/GroEL, DnaK, glyceraldehyde 3-phosphate dehydrogenase, enolase, and fructose 1,6-bisphosphate aldolase. The results of the proteomics studies suggest they could also be moonlighting on the surface of many other species. Hundreds of other intracellular proteins are also found on the cell surface, although a second function on the surface has not yet been demonstrated, for example, glutamine synthetase, gamma-glutamyl phosphate reductase, and cysteine desulfurase. The presence of intracellular proteins on the cell surface is more common than previously expected and suggests that many additional proteins might be candidates for being intracellular/surface moonlighting proteins.

Expression, Solubilization, and Purification of Bacterial Membrane Proteins.

Bacterial integral membrane proteins play many important roles, including sensing changes in the environment, transporting molecules into and out of the cell, and in the case of commensal or pathogenic bacteria, interacting with the host organism. Working with membrane proteins in the lab can be more challenging than working with soluble proteins because of difficulties in their recombinant expression and purification. This protocol describes a standard method to express, solubilize, and purify bacterial integral membrane proteins. The recombinant protein of interest with a 6His affinity tag is expressed in E. coli. After harvesting the cultures and isolating cellular membranes, mild detergents are used to solubilize the membrane proteins. Protein-detergent complexes are then purified using IMAC column chromatography. Support protocols are included to help select a detergent for protein solubilization and for use of gel filtration chromatography for further purification.

Protein species and moonlighting proteins: Very small changes in a protein's covalent structure can change its biochemical function.

In the past few decades, hundreds of moonlighting proteins have been identified that perform two or more distinct and physiologically relevant biochemical or biophysical functions that are not due to gene fusions, multiple RNA splice variants, or pleiotropic effects. For this special issue on protein species, this article discusses three topics related to moonlighting proteins that illustrate how small changes or differences in protein covalent structures can result in different functions. Examples are given of moonlighting proteins that switch between functions after undergoing post-translational modifications (PTMs), proteins that share high levels of amino acid sequence identity to a moonlighting protein but share only one of its functions, and several "neomorphic moonlighting proteins" in which a single amino acid mutation results in the addition of a new function. BIOLOGICAL SIGNIFICANCE: For this special issue on protein species, this article discusses three topics related to moonlighting proteins : Post-translational modifications (PTMs) that can cause a switch between functions, homologs that share only one of multiple functions, and proteins in which a single amino acid mutation results in the creation of a new function. The examples included illustrate that even in an average protein of hundreds of amino acids, a relatively small difference in sequence or PTMs can result in a large difference in function, which can be important in predicting protein functions, regulation of protein functions, and in the evolution of new functions.