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Short sequence motifs are ubiquitous across the three major types of biomolecules: hundreds of classes and thousands of instances of DNA regulatory elements, RNA motifs and protein short linear motifs (SLiMs) have been characterised. The increase in complexity of transcriptional, post-transcriptional and post-translational regulation in higher Eukaryotes has coincided with a significant expansion of motif use. But how did the eukaryotic cell acquire such a vast repertoire of motifs? In this review, we curate the available literature on protein motif evolution and discuss the evidence that suggests SLiMs can be acquired by mutations, insertions and deletions in disordered regions. We propose a mechanism of ex nihilo SLiM evolution – the evolution of a novel SLiM from “nothing” – adding a functional module to a previously non-functional region of protein sequence. In our model, hundreds of motif-binding domains in higher eukaryotic proteins connect simple motif specificities with useful functions to create a large functional motif space. Accessible peptides that match the specificity of these motif-binding domains are continuously created and destroyed by mutations in rapidly evolving disordered regions, creating a dynamic supply of new interactions that may have advantageous phenotypic novelty. This provides a reservoir of diversity to modify existing interaction networks. Evolutionary pressures will act on these motifs to retain beneficial instances. However, most will be lost on an evolutionary timescale as negative selection and genetic drift act on deleterious and neutral motifs respectively. In light of the parallels between the presented model and the evolution of motifs in the regulatory segments of genes and (pre-)mRNAs, we suggest our understanding of regulatory networks would benefit from the creation of a shared model describing the evolution of transcriptional, post-transcriptional and post-translational regulation.

Ser/thr phosphatases dephosphorylate their targets with high specificity, yet the structural and sequence determinants of phosphosite recognition are poorly understood. Calcineurin (CN) is a conserved Ca(2+)/calmodulin-dependent ser/thr phosphatase and the target of immunosuppressants, FK506 and cyclosporin A (CSA). To investigate CN substrate recognition we used X-ray crystallography, biochemistry, modeling, and in vivo experiments to study A238L, a viral protein inhibitor of CN. We show that A238L competitively inhibits CN by occupying a critical substrate recognition site, while leaving the catalytic center fully accessible. Critically, the 1.7 Å structure of the A238L-CN complex reveals how CN recognizes residues in A238L that are analogous to a substrate motif, \"LxVP.\" The structure enabled modeling of a peptide substrate bound to CN, which predicts substrate interactions beyond the catalytic center. Finally, this study establishes that \"LxVP\" sequences and immunosuppressants bind to the identical site on CN. Thus, FK506, CSA, and A238L all prevent \"LxVP\"-mediated substrate recognition by CN, highlighting the importance of this interaction for substrate dephosphorylation. Collectively, this work presents the first integrated structural model for substrate selection and dephosphorylation by CN and lays the groundwork for structure-based development of new CN inhibitors.

Calcineurin, the Ca 2+ /calmodulin-activated protein phosphatase, recognizes substrates and regulators via short linear motifs, PxIxIT and LxVP, which dock to distinct sites on calcineurin to determine enzyme distribution and catalysis, respectively. Calcimembrin/C16orf74 (CLMB), an intrinsically disordered microprotein whose expression correlates with poor cancer outcomes, targets calcineurin to membranes where it may promote oncogenesis by shaping calcineurin signaling. We show that CLMB associates with membranes via lipidation, i.e., N-myristoylation and reversible S-acylation. Furthermore, CLMB contains an unusual composite ‘LxVPxIxIT’ motif, that binds the PxIxIT-docking site on calcineurin with extraordinarily high affinity when phosphorylated, 33 LDVPDIIITPP(p)T 44 . Calcineurin dephosphorylates CLMB to decrease this affinity, but Thr44 is protected from dephosphorylation when PxIxIT-bound. We propose that CLMB is dephosphorylated in multimeric complexes, where one PxIxIT-bound CLMB recruits calcineurin to membranes, allowing a second CLMB to engage via its LxVP motif to be dephosphorylated. In vivo and in vitro data, including nuclear magnetic resonance (NMR) analyses of CLMB-calcineurin complexes, support this model. Thus, CLMB with its composite motif imposes distinct properties to calcineurin signaling at membranes including sensitivity to CLMB:calcineurin ratios, CLMB phosphorylation and dynamic S-acylation. C16orf74/ calcimembrin (CLMB), a lipidated microprotein with roles in cancer, targets the calcineurin phosphatase to membranes. Here, authors show how CLMB uses a composite motif to form multimeric complexes with calcineurin that are required for dephosphorylation.

Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca 2+ signaling. Here, to discover calcineurin-regulated processes we examined an understudied isoform, CNAβ1. We show that unlike canonical cytosolic calcineurin, CNAβ1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase. Palmitoylation targets CNAβ1 to a distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange reveals multiple calcineurin-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which we establish as a calcineurin substrate. Calcineurin inhibitors decrease PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. In sum, this work discovers a calcineurin-regulated signaling pathway which highlights the PI4KA complex as a regulatory target and reveals that dynamic palmitoylation confers unique localization, substrate specificity and regulation to CNAβ1. Calcineurin — the Ca2+ regulated phosphatase and target of immunosuppressants — regulates GPCR-mediated phospholipid signaling at the plasma membrane. Here the authors show that CNAβ1 (a poorly studied isoform of the calcineurin catalytic subunit) is targeted to the plasma membrane through palmitoylation to dephosphorylate and promote PI4KA complex activity.

Transient, regulated binding of globular protein domains to Short Linear Motifs (SLiMs) in disordered regions of other proteins drives cellular signaling. Mapping the energy landscapes of these interactions is essential for deciphering and perturbing signaling networks but is challenging due to their weak affinities. We present a powerful technology (MRBLE-pep) that simultaneously quantifies protein binding to a library of peptides directly synthesized on beads containing unique spectral codes. Using MRBLE-pep, we systematically probe binding of calcineurin (CN), a conserved protein phosphatase essential for the immune response and target of immunosuppressants, to the PxIxIT SLiM. We discover that flanking residues and post-translational modifications critically contribute to PxIxIT-CN affinity and identify CN-binding peptides based on multiple scaffolds with a wide range of affinities. The quantitative biophysical data provided by this approach will improve computational modeling efforts, elucidate a broad range of weak protein-SLiM interactions, and revolutionize our understanding of signaling networks.

All living organisms require nutrient minerals for growth and have developed mechanisms to acquire, utilize, and store nutrient minerals effectively. In the aqueous cellular environment, these elements exist as charged ions that, together with protons and hydroxide ions, facilitate biochemical reactions and establish the electrochemical gradients across membranes that drive cellular processes such as transport and ATP synthesis. Metal ions serve as essential enzyme cofactors and perform both structural and signaling roles within cells. However, because these ions can also be toxic, cells have developed sophisticated homeostatic mechanisms to regulate their levels and avoid toxicity. Studies in Saccharomyces cerevisiae have characterized many of the gene products and processes responsible for acquiring, utilizing, storing, and regulating levels of these ions. Findings in this model organism have often allowed the corresponding machinery in humans to be identified and have provided insights into diseases that result from defects in ion homeostasis. This review summarizes our current understanding of how cation balance is achieved and modulated in baker’s yeast. Control of intracellular pH is discussed, as well as uptake, storage, and efflux mechanisms for the alkali metal cations, Na+ and K+, the divalent cations, Ca2+ and Mg2+, and the trace metal ions, Fe2+, Zn2+, Cu2+, and Mn2+. Signal transduction pathways that are regulated by pH and Ca2+ are reviewed, as well as the mechanisms that allow cells to maintain appropriate intracellular cation concentrations when challenged by extreme conditions, i.e., either limited availability or toxic levels in the environment.

Calmodulin, a small, ubiquitous Ca 2+ -binding protein, regulates a wide variety of proteins and processes in all eukaryotes. CMD1 , the single gene encoding calmodulin in S. cerevisiae , is essential, and this review discusses studies that identified many of calmodulin's physiological targets and their functions in yeast cells. Calmodulin performs essential roles in mitosis, through its regulation of Nuf1p/Spc110p, a component of the spindle pole body, and in bud growth, by binding Myo2p, an unconventional class V myosin required for polarized secretion. Surprisingly, mutant calmodulins that fail to bind Ca 2+ can perform these essential functions. Calmodulin is also required for endocytosis in yeast and participates in Ca 2+ -dependent, stress-activated signaling pathways through its regulation of a protein phosphatase, calcineurin, and the protein kinases, Cmk1p and Cmk2p. Thus, calmodulin performs important physiological functions in yeast cells in both its Ca 2+ -bound and Ca 2+ -free form.

RNA binding proteins (RBPs) are vital to the regulation of mRNA transcripts, and can alter mRNA localization, degradation, translation, and storage. Whi3 was originally identified in a screen for small cell size mutants, and has since been characterized as an RBP. The identification of Whi3-interacting mRNAs involved in mediating cellular responses to stress suggested that Whi3 might be involved in stress-responsive RNA processing. We show that Whi3 localizes to stress granules in response to glucose deprivation or heat shock. The kinetics and pattern of Whi3 localization in response to a range of temperatures were subtly but distinctly different from those of known components of RNA processing granules. Deletion of Whi3 resulted in an increase in the relative abundance of Whi3 target RNAs, either in the presence or absence of heat shock. Increased levels of the CLN3 mRNA in whi3Δ cells may explain their decreased cell size. Another mRNA target of Whi3 encodes the zinc-responsive transcription factor Zap1, suggesting a role for Whi3 in response to zinc stress. Indeed, we found that whi3Δ cells have enhanced sensitivity to zinc toxicity. Together our results suggest an expanded model for Whi3 function: in addition to its role as a regulator of the cell cycle, Whi3 may have a role in stress-dependent RNA processing and responses to a variety of stress conditions.

A major factor in the progression to heart failure in humans is the inability of the adult heart to repair itself after injury. We recently demonstrated that the early postnatal mammalian heart is capable of regeneration following injury through proliferation of preexisting cardiomyocytes 1 , 2 and that Meis1, a three amino acid loop extension (TALE) family homeodomain transcription factor, translocates to cardiomyocyte nuclei shortly after birth and mediates postnatal cell cycle arrest 3 . Here we report that Hoxb13 acts as a cofactor of Meis1 in postnatal cardiomyocytes. Cardiomyocyte-specific deletion of Hoxb13 can extend the postnatal window of cardiomyocyte proliferation and reactivate the cardiomyocyte cell cycle in the adult heart. Moreover, adult Meis1–Hoxb13 double-knockout hearts display widespread cardiomyocyte mitosis, sarcomere disassembly and improved left ventricular systolic function following myocardial infarction, as demonstrated by echocardiography and magnetic resonance imaging. Chromatin immunoprecipitation with sequencing demonstrates that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and cell cycle. Finally, we show that the calcium-activated protein phosphatase calcineurin dephosphorylates Hoxb13 at serine-204, resulting in its nuclear localization and cell cycle arrest. These results demonstrate that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and proliferation and provide mechanistic insights into the link between hyperplastic and hypertrophic growth of cardiomyocytes. Hoxb13 acts as a cofactor of Meis1 in regulating cardiomyocyte maturation and cell cycle, and knockout of both proteins enables regeneration of postnatal cardiac tissue in a mouse model of heart injury.

Calcineurin, the Ca /calmodulin-activated protein phosphatase, recognizes substrates and regulators via short linear motifs, PxIxIT and LxVP, which dock to distinct sites on calcineurin to determine enzyme distribution and catalysis, respectively. Calcimembrin/C16orf74 (CLMB), an intrinsically disordered microprotein whose expression correlates with poor cancer outcomes, targets calcineurin to membranes where it may promote oncogenesis by shaping calcineurin signaling. We show that CLMB associates with membranes via lipidation, i.e. N-myristoylation and reversible S-acylation. Furthermore, CLMB contains an unusual composite 'LxVPxIxIT' motif, that binds the PxIxIT-docking site on calcineurin with extraordinarily high affinity when phosphorylated, LDVPDIIITPP(p)T . Calcineurin dephosphorylates CLMB to decrease this affinity, but Thr44 is protected from dephosphorylation when PxIxIT-bound. We propose that CLMB is dephosphorylated in multimeric complexes, where one PxIxIT-bound CLMB recruits calcineurin to membranes, allowing a second CLMB to engage via its LxVP motif to be dephosphorylated. and data, including nuclear magnetic resonance (NMR) analyses of CLMB-calcineurin complexes, support this model. Thus, CLMB with its composite motif imposes unique properties to calcineurin signaling at membranes including sensitivity to CLMB:calcineurin ratios, CLMB phosphorylation and dynamic S-acylation.