The Roles of Pseudophosphatases in Disease.

The pseudophosphatases, atypical members of the protein tyrosine phosphatase family, have emerged as bona fide signaling regulators within the past two decades. Their roles as regulators have led to a renaissance of the pseudophosphatase and pseudoenyme fields, catapulting interest from a mere curiosity to intriguing and relevant proteins to investigate. Pseudophosphatases make up approximately fourteen percent of the phosphatase family, and are conserved throughout evolution. Pseudophosphatases, along with pseudokinases, are important players in physiology and pathophysiology. These atypical members of the protein tyrosine phosphatase and protein tyrosine kinase superfamily, respectively, are rendered catalytically inactive through mutations within their catalytic active signature motif and/or other important domains required for catalysis. This new interest in the pursuit of the relevant functions of these proteins has resulted in an elucidation of their roles in signaling cascades and diseases. There is a rapid accumulation of knowledge of diseases linked to their dysregulation, such as neuropathies and various cancers. This review analyzes the involvement of pseudophosphatases in diseases, highlighting the function of various role(s) of pseudophosphatases involvement in pathologies, and thus providing a platform to strongly consider them as key therapeutic drug targets.

Genome-wide and structural analyses of pseudokinases encoded in the genome of Arabidopsis thaliana provide functional insights.

Protein Kinase-Like Non-Kinases (PKLNKs), commonly known as "pseudokinases", are homologous to eukaryotic Ser/Thr/Tyr protein kinases (PKs) but lack the crucial aspartate residue in the catalytic loop, indispensable for phosphotransferase activity. Therefore, they are predicted to be "catalytically inactive" enzyme homologs. Analysis of protein-kinase like sequences from Arabidopsis thaliana led to the identification of more than 120 pseudokinases lacking catalytic aspartate, majority of which are closely related to the plant-specific receptor-like kinase family. These pseudokinases engage in different biological processes, enabled by their diverse domain architectures and specific subcellular localizations. Structural comparison of pseudokinases with active and inactive conformations of canonical PKs, belonging to both plant and animal origin, revealed unique structural differences. The currently available crystal structures of pseudokinases show that the loop topologically equivalent to activation segment of PKs adopts a distinct-folded conformation, packing against the pseudoenzyme core, in contrast to the extended and inhibitory geometries observed for active and inactive states, respectively, of catalytic PKs. Salt-bridge between ATP-binding Lys and DFG-Asp as well as hydrophobic interactions between the conserved nonpolar residue C-terminal to the equivalent DFG motif and nonpolar residues in C-helix mediate such a conformation in pseudokinases. This results in enhanced solvent accessibility of the pseudocatalytic loop in pseudokinases that can possibly serve as an interacting surface while associating with other proteins. Specifically, our analysis identified several residues that may be involved in pseudokinase regulation and hints at the repurposing of pseudocatalytic residues to achieve mechanistic control over noncatalytic functions of pseudoenzymes.

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.

Pseudo-DUBs as allosteric activators and molecular scaffolds of protein complexes.

The ubiquitin (Ub) proteasome system and Ub signalling networks are crucial to cell biology and disease development. Deubiquitylases (DUBs) control cell signalling by removing mono-Ub and polyubiquitin chains from substrates. DUBs take part in almost all processes that regulate cellular life and are frequently dysregulated in disease. We have catalogued 99 currently known DUBs in the human genome and sequence conservation analyses of catalytic residues suggest that 11 lack enzyme activity and are classed as pseudo-DUBs. These pseudoenzymes play important biological roles by allosterically activating catalytically competent DUBs as well as other active enzymes. Additionally, pseudoenzymes act as assembly scaffolds of macromolecular complexes. We discuss how pseudo-DUBs have lost their catalytic activity, their diverse mechanisms of action and their potential as therapeutic targets. Many known pseudo-DUBs play crucial roles in cell biology and it is likely that unstudied and overlooked pseudo-DUB genes will have equally important functions.

Computational tools and resources for pseudokinase research.

Pseudokinases regulate diverse cellular processes associated with normal cellular functions and disease. They are defined bioinformatically based on the absence of one or more catalytic residues that are required for canonical protein kinase functions. The ability to define pseudokinases based on primary sequence comparison has enabled the systematic mapping and cataloging of pseudokinase orthologs across the tree of life. While these sequences contain critical information regarding pseudokinase evolution and functional specialization, extracting this information and generating testable hypotheses based on integrative mining of sequence and structural data requires specialized computational tools and resources. In this chapter, we review recent advances in the development and application of open-source tools and resources for pseudokinase research. Specifically, we describe the application of an interactive data analytics framework, KinView, for visualizing the patterns of conservation and variation in the catalytic domain motifs of pseudokinases and evolutionarily related canonical kinases using a consistent set of curated alignments organized based on the widely used kinome evolutionary hierarchy. We also demonstrate the application of an integrated Protein Kinase Ontology (ProKinO) and an interactive viewer, ProtVista, for mapping and analyzing primary sequence motifs and annotations in the context of 3D structures and AlphaFold2 models. We provide examples and protocols for generating testable hypotheses on pseudokinase functions both for bench biologists and advanced users.

Current Challenges for IDO2 as Target in Cancer Immunotherapy.

Immune checkpoint inhibitors have revolutionized the clinical approach of untreatable tumors and brought a breath of fresh air in cancer immunotherapy. However, the therapeutic effects of these drugs only cover a minority of patients and alternative immunotherapeutic targets are required. Metabolism of l-tryptophan (Trp) via the kynurenine pathway represents an important immune checkpoint mechanism that controls adaptive immunity and dampens exaggerated inflammation. Indoleamine 2,3-dioxygenase 1 (IDO1), the enzyme catalyzing the first, rate-limiting step of the pathway, is expressed in several human tumors and IDO1 catalytic inhibitors have reached phase III clinical trials, unfortunately with disappointing results. Although much less studied, the IDO1 paralog IDO2 may represent a valid alternative as drug target in cancer immunotherapy. Accumulating evidence indicates that IDO2 is much less effective than IDO1 in metabolizing Trp and its functions are rather the consequence of interaction with other, still undefined proteins that may vary in distinct inflammatory and neoplastic contexts. As a matter of fact, the expression of IDO2 gene variants is protective in PDAC but increases the risk of developing tumor in NSCLC patients. Therefore, the definition of the IDO2 interactome and function in distinct neoplasia may open innovative avenues of therapeutic interventions.

A novel predicted ADP-ribosyltransferase-like family conserved in eukaryotic evolution.

The presence of many completely uncharacterized proteins, even in well-studied organisms such as humans, seriously hampers full understanding of the functioning of the living cells. ADP-ribosylation is a common post-translational modification of proteins; also nucleic acids and small molecules can be modified by the covalent attachment of ADP-ribose. This modification, important in cellular signalling and infection processes, is usually executed by enzymes from the large superfamily of ADP-ribosyltransferases (ARTs). Here, using bioinformatics approaches, we identify a novel putative ADP-ribosyltransferase family, conserved in eukaryotic evolution, with a divergent active site. The hallmark of these proteins is the ART domain nestled between flanking leucine-rich repeat (LRR) domains. LRRs are typically involved in innate immune surveillance. The novel family appears as putative novel ADP-ribosylation-related actors, most likely pseudoenzymes. Sequence divergence and lack of clearly detectable "classical" ART active site suggests the novel domains are pseudoARTs, yet atypical ART activity, or alternative enzymatic activity cannot be excluded. We propose that this family, including its human member LRRC9, may be involved in an ancient defense mechanism, with analogies to the innate immune system, and coupling pathogen detection to ADP-ribosyltransfer or other signalling mechanisms.

Dimeric Structure of the Pseudokinase IRAK3 Suggests an Allosteric Mechanism for Negative Regulation.

Interleukin-1 receptor associated kinases (IRAKs) are key players in innate immune signaling that mediate the host response to pathogens. In contrast to the active kinases IRAK1 and IRAK4, IRAK2 and IRAK3 are pseudokinases lacking catalytic activity and their functions are poorly understood. IRAK3 is thought to be a negative regulator of innate immune signaling and mutations in IRAK3 are associated with asthma and cancer. Here, we report the crystal structure of the human IRAK3 pseudokinase domain in a closed, pseudoactive conformation. IRAK3 dimerizes in a unique way through a head-to-head arrangement not observed in any other kinases. Multiple conserved cysteine residues imply a potential redox control of IRAK3 conformation and dimerization. By analyzing asthma-associated mutations, we identify an evolutionarily conserved surface on IRAK3 that could form an interaction interface with IRAK4, suggesting a model for the negative regulation of IRAK4 by IRAK3.

The dead phosphatases society: a review of the emerging roles of pseudophosphatases.

Phosphatases are a diverse family of enzymes, comprising at least 10 distinct protein folds. Like most other enzyme families, many have sequence variations that predict an impairment or loss of catalytic activity classifying them as pseudophosphatases. Research on pseudoenzymes is an emerging area of interest, with new biological functions repurposed from catalytically active relatives. Here, we provide an overview of the pseudophosphatases identified to date in all major phosphatase families. We will highlight the degeneration of the various catalytic sequence motifs and discuss the challenges associated with the experimental determination of catalytic inactivity. We will also summarize the role of pseudophosphatases in various diseases and discuss the major challenges and future directions in this field.

FLIP(L): the pseudo-caspase.

Possessing structural homology with their active enzyme counterparts but lacking catalytic activity, pseudoenzymes have been identified for all major enzyme groups. Caspases are a family of cysteine-dependent aspartate-directed proteases that play essential roles in regulating cell death and inflammation. Here, we discuss the only human pseudo-caspase, FLIP(L), a paralog of the apoptosis-initiating caspases, caspase-8 and caspase-10. FLIP(L) has been shown to play a key role in regulating the processing and activity of caspase-8, thereby modulating apoptotic signaling mediated by death receptors (such as TRAIL-R1/R2), TNF receptor-1 (TNFR1), and Toll-like receptors. In this review, these canonical roles of FLIP(L) are discussed. Additionally, a range of nonclassical pseudoenzyme roles are described, in which FLIP(L) functions independently of caspase-8. These nonclassical pseudoenzyme functions enable FLIP(L) to play key roles in the regulation of a wide range of biological processes beyond its canonical roles as a modulator of cell death.

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.

Using Pseudoenzymes to Probe Evolutionary Design Principles of Enzymes.

Enzymes are governed by unique evolutionary design principles as their catalytic sites were shown to induce long-range evolutionary conservation gradients. We have recently used a comparative bioinformatics approach to disentangle structural determinants from other possible determinants of the evolutionary conservation gradients. The approach is based on comparing the evolutionary patterns of enzymes to those of pseudoenzymes with the same tertiary structure where the catalytic functionality is turned off. This approach provides a way to evaluate several hypotheses regarding the origin of the observed evolutionary conservation gradient in enzymes. The conclusions from such comparative analyses are important for a better understanding of the unique evolutionary design principles of enzymes, which can in turn potentially guide the design of new and improved enzymes.

Crystal Structure of Aldehyde Dehydrogenase 16 Reveals Trans-Hierarchical Structural Similarity and a New Dimer.

The aldehyde dehydrogenase (ALDH) superfamily is a vast group of enzymes that catalyze the NAD+-dependent oxidation of aldehydes to carboxylic acids. ALDH16 is perhaps the most enigmatic member of the superfamily, owing to its extra C-terminal domain of unknown function and the absence of the essential catalytic cysteine residue in certain non-bacterial ALDH16 sequences. Herein we report the first production of recombinant ALDH16, the first biochemical characterization of ALDH16, and the first crystal structure of ALDH16. Recombinant expression systems were generated for the bacterial ALDH16 from Loktanella sp. and human ALDH16A1. Four high-resolution crystal structures of Loktanella ALDH16 were determined. Loktanella ALDH16 is found to be a bona fide enzyme, exhibiting NAD+-binding, ALDH activity, and esterase activity. In contrast, human ALDH16A1 apparently lacks measurable aldehyde oxidation activity, suggesting that it is a pseudoenzyme, consistent with the absence of the catalytic Cys in its sequence. The fold of ALDH16 comprises three domains: NAD+-binding, catalytic, and C-terminal. The latter is unique to ALDH16 and features a Rossmann fold connected to a protruding ß-flap. The tertiary structural interactions of the C-terminal domain mimic the quaternary structural interactions of the classic ALDH superfamily dimer, a phenomenon we call "trans-hierarchical structural similarity." ALDH16 forms a unique dimer in solution, which mimics the classic ALDH superfamily dimer-of-dimer tetramer. Small-angle X-ray scattering shows that human ALDH16A1 has the same dimeric structure and fold as Loktanella ALDH16. We suggest that the Loktanella ALDH16 structure may be considered to be the archetype of the ALDH16 family.

The role of pseudophosphatases as signaling regulators.

Pseudophosphatases are atypical members of the protein tyrosine phosphatase superfamily. Mutations within their catalytic signature motif render them catalytically inactive. Despite this lack of catalytic function, pseudophosphatases have been implicated in various diseases such as Charcot Marie-Tooth disorder, cancer, metabolic disorder, and obesity. Moreover, they have roles in various signaling networks such as spermatogenesis, apoptosis, stress response, tumorigenesis, and neurite differentiation. This review highlights the roles of pseudophosphatases as essential regulators in signaling cascades, providing insight into the function of these catalytically inactive enzymes.

Natural Infection of C. elegans by an Oomycete Reveals a New Pathogen-Specific Immune Response.

In its natural habitat, the nematode Caenorhabditis elegans encounters a plethora of other organisms, including many that are pathogenic [1, 2]. The study of interactions between C. elegans and various pathogens has contributed to characterizing key mechanisms of innate immunity [2-4]. However, how C. elegans recognizes different pathogens to mount pathogen-specific immune responses remains still largely unknown [3, 5-8]. Expanding the range of known C. elegans-infecting pathogens and characterizing novel pathogen-specific immune responses are key steps toward answering this question. We report here that the oomycete Myzocytiopsis humicola is a natural pathogen of C. elegans, and we describe its infection strategy. We identify a new host immune response to pathogen exposure that involves induction of members of a previously uncharacterized gene family encoding chitinase-like (CHIL) proteins. We demonstrate that this response is highly specific against M. humicola and antagonizes the infection. We propose that CHIL proteins may diminish the ability of the oomycete to infect by hindering pathogen attachment to the host cuticle. This work expands our knowledge of natural eukaryotic pathogens of C. elegans and introduces a new pathosystem to address how animal hosts recognize and respond to oomycete infections.

Review: The Arabidopsis ß-amylase (BAM) gene family: Diversity of form and function.

Multi-gene families present a rich research area to study how related proteins evolve to acquire new structures and functions. The ß-amylase (BAM) gene family is named for catalytic members' ability to hydrolyze starch into maltose units. However, the family also contains proteins that are catalytically inactive, have additional domains, or are not localized with a starch substrate. Here we review the current knowledge of each of the nine Arabidopsis BAMs, including information on their localization, structural features, expression patterns, regulation and potential functions. We also discuss unique characteristics of studying multi-gene families, such as the consideration of different kinetic parameters when performing assays on leaf extracts, and suggest approaches that may be fruitful in learning more about their unique functions.

Structure of Fam20A reveals a pseudokinase featuring a unique disulfide pattern and inverted ATP-binding.

Mutations in FAM20A cause tooth enamel defects known as Amelogenesis Imperfecta (AI) and renal calcification. We previously showed that Fam20A is a secretory pathway pseudokinase and allosterically activates the physiological casein kinase Fam20C to phosphorylate secreted proteins important for biomineralization (Cui et al., 2015). Here we report the nucleotide-free and ATP-bound structures of Fam20A. Fam20A exhibits a distinct disulfide bond pattern mediated by a unique insertion region. Loss of this insertion due to abnormal mRNA splicing interferes with the structure and function of Fam20A, resulting in AI. Fam20A binds ATP in the absence of divalent cations, and strikingly, ATP is bound in an inverted orientation compared to other kinases. Fam20A forms a dimer in the crystal, and residues in the dimer interface are critical for Fam20C activation. Together, these results provide structural insights into the function of Fam20A and shed light on the mechanism by which Fam20A mutations cause disease.