Systematic Discovery of Short Linear Motifs Decodes Calcineurin Phosphatase Signaling.

Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.

Substrate and phosphorylation site selection by phosphoprotein phosphatases.

Dynamic protein phosphorylation and dephosphorylation are essential regulatory mechanisms that ensure proper cellular signaling and biological functions. Deregulation of either reaction has been implicated in several human diseases. Here, we focus on the mechanisms that govern the specificity of the dephosphorylation reaction. Most cellular serine/threonine dephosphorylation is catalyzed by 13 highly conserved phosphoprotein phosphatase (PPP) catalytic subunits, which form hundreds of holoenzymes by binding to regulatory and scaffolding subunits. PPP holoenzymes recognize phosphorylation site consensus motifs and interact with short linear motifs (SLiMs) or structural elements distal to the phosphorylation site. We review recent advances in understanding the mechanisms of PPP site-specific dephosphorylation preference and substrate recruitment and highlight examples of their interplay in the regulation of cell division.

A Consensus Binding Motif for the PP4 Protein Phosphatase.

Dynamic protein phosphorylation constitutes a fundamental regulatory mechanism in all organisms. Phosphoprotein phosphatase 4 (PP4) is a conserved and essential nuclear serine and threonine phosphatase. Despite the importance of PP4, general principles of substrate selection are unknown, hampering the study of signal regulation by this phosphatase. Here, we identify and thoroughly characterize a general PP4 consensus-binding motif, the FxxP motif. X-ray crystallography studies reveal that FxxP motifs bind to a conserved pocket in the PP4 regulatory subunit PPP4R3. Systems-wide in silico searches integrated with proteomic analysis of PP4 interacting proteins allow us to identify numerous FxxP motifs in proteins controlling a range of fundamental cellular processes. We identify an FxxP motif in the cohesin release factor WAPL and show that this regulates WAPL phosphorylation status and is required for efficient cohesin release. Collectively our work uncovers basic principles of PP4 specificity with broad implications for understanding phosphorylation-mediated signaling in cells.

Intrinsic and extrinsic regulators of Aux/IAA protein degradation dynamics.

The plant hormone auxin acts through regulated degradation of Auxin/INDOLE-3-ACETIC ACID (Aux/IAA) proteins to regulate transcriptional events. In this review, we examine the composition and function of each Aux/IAA structural motif. We then focus on recent characterization of Aux/IAA N-terminal disordered regions, formation of secondary structure within these disordered regions, and post-translational modifications (PTMs) that affect Aux/IAA function and stability. We propose how structural variations between Aux/IAA family members may be tuned for differential transcriptional repression and degradation dynamics.

Conditionally Deleterious Mutation Load Accumulates in Genomic Islands of Local Adaptation but Can Be Purged with Sufficient Genotypic Redundancy.

AbstractLocal adaptation frequently evolves in patches or environments that are connected via migration. In these cases, genomic regions that are linked to a locally adapted locus experience reduced effective migration rates. Via individual-based simulations of a two-patch system, we show that this reduced effective migration results in the accumulation of conditionally deleterious mutations, but not universally deleterious mutations, adjacent to adaptive loci. When there is redundancy in the genetic basis of local adaptation (i.e., genotypic redundancy), turnover of locally adapted polymorphisms allows conditionally deleterious mutation load to be purged. The amount of mutational load that accumulates adjacent to locally adapted loci is dependent on redundancy, recombination rate, migration rate, population size, strength of selection, and the phenotypic effect size of adaptive alleles. Our results highlight the need to be cautious when interpreting patterns of local adaptation at the level of phenotype or fitness, as the genetic basis of local adaptation can be transient, and evolution may confer a degree of maladaptation to nonlocal environments.

A long-term growth stable Halomonas sp. deleted with multiple transposases guided by its metabolic network model Halo-ecGEM.

Microbial instability is a common problem during bio-production based on microbial hosts. Halomonas bluephagenesis has been developed as a chassis for next generation industrial biotechnology (NGIB) under open and unsterile conditions. However, the hidden genomic information and peculiar metabolism have significantly hampered its deep exploitation for cell-factory engineering. Based on the freshly completed genome sequence of H. bluephagenesis TD01, which reveals 1889 biological process-associated genes grouped into 84 GO-slim terms. An enzyme constrained genome-scale metabolic model Halo-ecGEM was constructed, which showed strong ability to simulate fed-batch fermentations. A visible salt-stress responsive landscape was achieved by combining GO-slim term enrichment and CVT-based omics profiling, demonstrating that cells deploy most of the protein resources by force to support the essential activity of translation and protein metabolism when exposed to salt stress. Under the guidance of Halo-ecGEM, eight transposases were deleted, leading to a significantly enhanced stability for its growth and bioproduction of various polyhydroxyalkanoates (PHA) including 3-hydroxybutyrate (3HB) homopolymer PHB, 3HB and 3-hydroxyvalerate (3HV) copolymer PHBV, as well as 3HB and 4-hydroxyvalerate (4HB) copolymer P34HB. This study sheds new light on the metabolic characteristics and stress-response landscape of H. bluephagenesis, achieving for the first time to construct a long-term growth stable chassis for industrial applications. For the first time, it was demonstrated that genome encoded transposons are the reason for microbial instability during growth in flasks and fermentors.

Gene regulatory networks underlying sulfate deficiency responses in plants.

Sulfur (S) is an essential macronutrient for plants and its availability in soils is an important determinant for growth and development. Current regulatory policies aimed at reducing industrial S emissions together with changes in agronomical practices have led to a decline in S contents in soils worldwide. Deficiency of sulfate-the primary form of S accessible to plants in soil-has adverse effects on both crop yield and nutritional quality. Hence, recent research has increasingly focused on unraveling the molecular mechanisms through which plants detect and adapt to a limiting supply of sulfate. A significant part of these studies involves the use of omics technologies and has generated comprehensive catalogs of sulfate deficiency-responsive genes and processes, principally in Arabidopsis together with a few studies centering on crop species such as wheat, rice, or members of the Brassica genus. Although we know that sulfate deficiency elicits an important reprogramming of the transcriptome, the transcriptional regulators orchestrating this response are not yet well understood. In this review, we summarize our current knowledge of gene expression responses to sulfate deficiency and recent efforts towards the identification of the transcription factors that are involved in controlling these responses. We further compare the transcriptional response and putative regulators between Arabidopsis and two important crop species, rice and tomato, to gain insights into common mechanisms of the response to sulfate deficiency.

Retrospective comparative study of new slim-delivery and conventional large-cell stents for stent-in-stent methods for hilar malignant biliary obstruction.

OBJECTIVES: Endoscopic management of unresectable hilar malignant biliary obstruction (HMBO) is technically challenging, and effectiveness of stent-in-stent using large-cell, metal stents was reported. A new, large-cell stent with a 6F tapered delivery system was recently developed. The aim of this study was to compare clinical outcomes of slim-delivery and conventional large-cell stents. METHODS: This was a multicenter retrospective comparative study of stent-in-stent methods using slim-delivery stents (Niti-S Large Cell SR Slim Delivery [LC slim-delivery]) and conventional stents (Niti-S large-cell D-type; LCD) for unresectable HMBO. RESULTS: Eighty-three patients with HMBO were included; 31 LC slim-delivery and 52 LCD. Overall technical and clinical success rates were 100% and 90% in LC slim-delivery group and 98% and 88% in LCD group. Use of the LC slim-delivery was associated with shorter stent placement time in the multiple regression analysis, with a stent placement time of 18 and 23 min in LC slim-delivery and LCD groups, respectively. The early adverse event (AE) rate of LC slim-delivery was 10%, with no cholangitis or cholecystitis as compared to 23% in the LCD group. Recurrent biliary obstruction (RBO) rates and time to RBO were comparable between the two groups: 35% and 44%, and 8.5 and 8.0 months in LC slim-delivery and LCD groups, respectively. The major cause of RBO was tumor ingrowth (82%) in the LC slim-delivery group and sludge (43%) and ingrowth (48%) in LCD group. CONCLUSION: Stent-in-stent methods using LC slim-delivery shortened stent placement time with low early AE rates and comparable time to RBO in patients with HMBO.

Exploring Adolescent girls' Food-Talk in Kathmandu Valley, Nepal: A Qualitative Study.

This study aimed to analyze how adolescent girls residing in Kathmandu valley, Nepal, talk about food within the context of their everyday experiences. We conducted 10 in-depth and four focus group interviews. Qualitative thematic analysis based on the constructivist paradigm was used to organize the interviews. The Utilitarian domain contained health statements using biomedical language and lay theories on health. Hedonic talk emphasized the taste of food, but notions about enjoyment were limited. Collective talk constructed an ideal family. In agency talk, the interviewees described their active role in achieving a slim body. Participants were not concerned about food insecurity but about eating too much.

In silico analysis of cis-elements and identification of transcription factors putatively involved in the regulation of the OAS cluster genes SDI1 and SDI2.

Arabidopsis thaliana sulfur deficiency-induced 1 and sulfur deficiency-induced 2 (SDI1 and SDI2) are involved in partitioning sulfur among metabolite pools during sulfur deficiency, and their transcript levels strongly increase in this condition. However, little is currently known about the cis- and trans-factors that regulate SDI expression. We aimed at identifying DNA sequence elements (cis-elements) and transcription factors (TFs) involved in regulating expression of the SDI genes. We performed in silico analysis of their promoter sequences cataloging known cis-elements and identifying conserved sequence motifs. We screened by yeast-one-hybrid an arrayed library of Arabidopsis TFs for binding to the SDI1 and SDI2 promoters. In total, 14 candidate TFs were identified. Direct association between particular cis-elements in the proximal SDI promoter regions and specific TFs was established via electrophoretic mobility shift assays: sulfur limitation 1 (SLIM1) was shown to bind SURE cis-element(s), the basic domain/leucine zipper (bZIP) core cis-element was shown to be important for HY5-homolog (HYH) binding, and G-box binding factor 1 (GBF1) was shown to bind the E box. Functional analysis of GBF1 and HYH using mutant and over-expressing lines indicated that these TFs promote a higher transcript level of SDI1 in vivo. Additionally, we performed a meta-analysis of expression changes of the 14 TF candidates in a variety of conditions that alter SDI expression. The presented results expand our understanding of sulfur pool regulation by SDI genes.

A SLiM-dependent conformational change in baculovirus IE1 controls its focus formation ability.

The baculovirus IE1 gene encodes a multifunctional protein that is essential for both DNA replication and RNA transcription of the virus. Prior to viral DNA replication, IE1 promotes early gene transcription when localized in hr-dependent foci. During viral DNA replication, the IE1 foci expand and fuse to generate the virogenic stroma (VS) with IE1 found in the VS reticulum. To explore the IE1 structural features essential for this coordinated localization, we constructed various IE1 mutants based on three putative domains (N, I, and C). We determined that a BDI motif located in the intrinsic disorder region (IDR) between the N and I domains acts as a nuclear localization signal, whereas BDII and HLH in the C domain are required for VS localization in infected cells or for chromosomal association in uninfected mitotic cells. Deletion of the SLiM (short linear motif) located in the I domain restrains both nuclear- and VS localization. Intra-molecular fluorescence resonance energy transfer (FRET) probes of IE1 mutants revealed a conformational change of the I-C two-domain fragment during infection, which was inhibited by aphidicolin, suggesting that IE1 undergoes a stage-dependent conformational change. Further, homo-dimerization of the I domain and stage-dependent conformational changes require an intact SLiM. Mutational analysis of SLiM revealed that VS localization and chromosomal association were retained following S291A and S291E substitutions, but hr-dependent focus formation differed between the two mutations. These results suggest that coordinated IE1 localization is controlled by SLiM-dependent conformational changes that are potentially switched by the phosphorylation state of the SLiM.

Compartment-based reconstruction of 3D acquisition-weighted 31 P cardiac magnetic resonance spectroscopic imaging at 7 T: A reproducibility study.

Even at 7 T, cardiac 31 P magnetic resonance spectroscopic imaging (MRSI) is fundamentally limited by low signal-to-noise ratio (SNR), leading to long scan times and poor temporal and spatial resolutions. Compartment-based reconstruction algorithms such as magnetic resonance spectroscopy with linear algebraic modeling (SLAM) and spectral localization by imaging (SLIM) may improve SNR or reduce scan time without changes to acquisition. Here, we compare the repeatability and SNR performance of these compartment-based methods, applied to three different acquisition schemes at 7 T. Twelve healthy volunteers were scanned twice. Each scan session consisted of a 6.5-min 3D acquisition-weighted (AW) cardiac 31 P phase encode-based MRSI acquisition and two 6.5-min truncated k-space acquisitions with increased averaging (4 × 4 × 4 central k-space phase encodes and fractional SLAM [fSLAM] optimized k-space phase encodes). Spectra were reconstructed using (i) AW Fourier reconstruction; (ii) AW SLAM; (iii) AW SLIM; (iv) 4 × 4 × 4 SLAM; (v) 4 × 4 × 4 SLIM; and (vi) fSLAM acquisition-reconstruction combinations. The phosphocreatine-to-adenosine triphosphate (PCr/ATP) ratio, the PCr SNR, and spatial response functions were computed, in addition to coefficients of reproducibility and variability. Using the compartment-based reconstruction algorithms with the AW 31 P acquisition resulted in a significant increase in SNR compared with previously published Fourier-based MRSI reconstruction methods while maintaining the measured PCr/ATP ratio and improving interscan reproducibility. The alternative acquisition strategies with truncated k-space performed no better than the common AW approach. Compartment-based spectroscopy approaches provide an attractive reconstruction method for cardiac 31 P spectroscopy at 7 T, improving reproducibility and SNR without the need for a dedicated k-space sampling strategy.

New insights into the regulation of plant metabolism by O-acetylserine: sulfate and beyond.

Under conditions of sulfur deprivation, O-acetylserine (OAS) accumulates, which leads to the induction of a common set of six genes, called OAS cluster genes. These genes are induced not only under sulfur deprivation, but also under other conditions where OAS accumulates, such as shift to darkness and stress conditions leading to reactive oxygen species (ROS) or methyl-jasmonate accumulation. Using the OAS cluster genes as a query in ATTED-II, a co-expression network is derived stably spanning several hundred conditions. This allowed us not only to describe the downstream function of the OAS cluster genes but also to score for functions of the members of the co-regulated co-expression network and hence the effects of the OAS signal on the sulfate assimilation pathway and co-regulated pathways. Further, we summarized existing knowledge on the regulation of the OAS cluster and the co-expressed genes. We revealed that the known sulfate deprivation-related transcription factor EIL3/SLIM1 exhibits a prominent role, as most genes are subject to regulation by this transcription factor. The role of other transcription factors in response to OAS awaits further investigation.

Evaluation of affinity-purification coupled to mass spectrometry approaches for capture of short linear motif-based interactions.

Low affinity and transient protein-protein interactions, such as short linear motif (SLiM)-based interactions, require dedicated experimental tools for discovery and validation. Here, we evaluated and compared biotinylated peptide pulldown and protein interaction screen on peptide matrix (PRISMA) coupled to mass-spectrometry (MS) using a set of peptides containing interaction motifs. Eight different peptide sequences that engage in interactions with three distinct protein domains (KEAP1 Kelch, MDM2 SWIB, and TSG101 UEV) with a wide range of affinities were tested. We found that peptide pulldown can be an effective approach for SLiM validation, however, parameters such as protein abundance and competitive interactions can prevent the capture of known interactors. The use of tandem peptide repeats improved the capture and preservation of some interactions. When testing PRISMA, it failed to provide comparable results for model peptides that successfully pulled down known interactors using biotinylated peptide pulldown. Overall, in our hands, we find that albeit more laborious, biotin-peptide pulldown was more successful in terms of validation of known interactions. Our results highlight that the tested affinity-capture MS-based methods for validation of SLiM-based interactions from cell lysates are suboptimal, and we identified parameters for consideration for method development.

A context-dependent and disordered ubiquitin-binding motif.

Ubiquitin is a small, globular protein that is conjugated to other proteins as a posttranslational event. A palette of small, folded domains recognizes and binds ubiquitin to translate and effectuate this posttranslational signal. Recent computational studies have suggested that protein regions can recognize ubiquitin via a process of folding upon binding. Using peptide binding arrays, bioinformatics, and NMR spectroscopy, we have uncovered a disordered ubiquitin-binding motif that likely remains disordered when bound and thus expands the palette of ubiquitin-binding proteins. We term this motif Disordered Ubiquitin-Binding Motif (DisUBM) and find it to be present in many proteins with known or predicted functions in degradation and transcription. We decompose the determinants of the motif showing it to rely on features of aromatic and negatively charged residues, and less so on distinct sequence positions in line with its disordered nature. We show that the affinity of the motif is low and moldable by the surrounding disordered chain, allowing for an enhanced interaction surface with ubiquitin, whereby the affinity increases ~ tenfold. Further affinity optimization using peptide arrays pushed the affinity into the low micromolar range, but compromised context dependence. Finally, we find that DisUBMs can emerge from unbiased screening of randomized peptide libraries, featuring in de novo cyclic peptides selected to bind ubiquitin chains. We suggest that naturally occurring DisUBMs can recognize ubiquitin as a posttranslational signal to act as affinity enhancers in IDPs that bind to folded and ubiquitylated binding partners.

Orchestrating serine/threonine phosphorylation and elucidating downstream effects by short linear motifs.

Cellular function is based on protein-protein interactions. A large proportion of these interactions involves the binding of short linear motifs (SLiMs) by folded globular domains. These interactions are regulated by post-translational modifications, such as phosphorylation, that create and break motif binding sites or tune the affinity of the interactions. In addition, motif-based interactions are involved in targeting serine/threonine kinases and phosphatases to their substrate and contribute to the specificity of the enzymatic actions regulating which sites are phosphorylated. Here, we review how SLiM-based interactions assist in determining the specificity of serine/threonine kinases and phosphatases, and how phosphorylation, in turn, affects motif-based interactions. We provide examples of SLiM-based interactions that are turned on/off, or are tuned by serine/threonine phosphorylation and exemplify how this affects SLiM-based protein complex formation.

Load Estimation of Moving Passenger Cars Using Inductive-Loop Technology.

Due to their lack of driving controllability, overweight vehicles are a big threat to road safety. The proposed method for a moving passenger car load estimation is capable of detecting an overweight vehicle, and thus it finds its application in road safety improvement. The weight of a car's load entering or leaving a considered zone, e.g., industrial facility, a state, etc., is also of concern in many applications, e.g., surveillance. Dedicated vehicle weight-in-motion measurement systems generally use expensive load sensors that also require deep intervention in the road while being installed and also are calibrated only for heavy trucks. In this paper, a vehicle magnetic profile (VMP) is used for defining a load parameter proportional to the passenger vehicle load. The usefulness of the proposed load parameter is experimentally demonstrated in field tests. The sensitivity of the VMP to the load change results from the fact that the higher load decreases the vehicle clearance value which in turn increases the VMP. It is also shown that a slim inductive-loop sensors allows the building of a load estimation system, with a maximum error around 30 kg, which allows approximate determination of the number of passengers in the car. The presented proof of concept extends the functionality of inductive loops, already installed in the road, for acquiring other traffic parameters, e.g., moving vehicle axle-to-axle distance measurement, to road safety and surveillance related applications.

[Assembly and Disassembly of the Nuclear Pore Complex: A View from the Structural Side].

Nucleocytoplasmic exchange in the cell occurs through the nuclear pore complexes (NPCs). NPCs are large multiprotein complexes with octagonal symmetry about their axis and imperfect mirror symmetry about a plane parallel with the nuclear envelop (NE). NPC fuses the inner and outer nuclear membranes and opens up a channel between nucleus and cytoplasm. NPC is built of nucleoporins. Each nucleoporin occurs in at least eight copies per NPC. Inside the NPC a permeability barrier forms by which NPCs can provide fast and selectable transport of molecules from one side of the nuclear membrane to the other. NPC architecture is based on hierarchical principle of organization. Nucleoporins are integrated into complexes that oligomerizes into bigger octomeric high-order structures. These structures are the main components of NPCs. In the first part of this work, the main attention is paid to NPC structure and nucleoporin properties. The second part is dedicated to mechanisms of NPC assembly and disassembly at different stages of the cell cycle.

αα-Hub domains and intrinsically disordered proteins: A decisive combo.

Hub proteins are central nodes in protein-protein interaction networks with critical importance to all living organisms. Recently, a new group of folded hub domains, the αα-hubs, was defined based on a shared αα-hairpin supersecondary structural foundation. The members PAH, RST, TAFH, NCBD, and HHD are found in large proteins such as Sin3, RCD1, TAF4, CBP, and harmonin, which organize disordered transcriptional regulators and membrane scaffolds in interactomes of importance to human diseases and plant quality. In this review, studies of structures, functions, and complexes across the αα-hubs are described and compared to provide a unified description of the group. This analysis expands the associated molecular concepts of "one domain-one binding site", motif-based ligand binding, and coupled folding and binding of intrinsically disordered ligands to additional concepts of importance to signal fidelity. These include context, motif reversibility, multivalency, complex heterogeneity, synergistic αα-hub:ligand folding, accessory binding sites, and supramodules. We propose that these multifaceted protein-protein interaction properties are made possible by the characteristics of the αα-hub fold, including supersite properties, dynamics, variable topologies, accessory helices, and malleability and abetted by adaptability of the disordered ligands. Critically, these features provide additional filters for specificity. With the presentations of new concepts, this review opens for new research questions addressing properties across the group, which are driven from concepts discovered in studies of the individual members. Combined, the members of the αα-hubs are ideal models for deconvoluting signal fidelity maintained by folded hubs and their interactions with intrinsically disordered ligands.

Roles of constitutive and signal-dependent protein phosphatase 2A docking motifs in burst attenuation of the cyclic AMP response element-binding protein.

The cAMP response element-binding protein (CREB) is an important regulator of cell growth, metabolism, and synaptic plasticity. CREB is activated through phosphorylation of an evolutionarily conserved Ser residue (S133) within its intrinsically disordered kinase-inducible domain (KID). Phosphorylation of S133 in response to cAMP, Ca2+, and other stimuli triggers an association of the KID with the KID-interacting (KIX) domain of the CREB-binding protein (CBP), a histone acetyl transferase (HAT) that promotes transcriptional activation. Here we addressed the mechanisms of CREB attenuation following bursts in CREB phosphorylation. We show that phosphorylation of S133 is reversed by protein phosphatase 2A (PP2A), which is recruited to CREB through its B56 regulatory subunits. We found that a B56-binding site located at the carboxyl-terminal boundary of the KID (BS2) mediates high-affinity B56 binding, while a second binding site (BS1) located near the amino terminus of the KID mediates low affinity binding enhanced by phosphorylation of adjacent casein kinase (CK) phosphosites. Mutations that diminished B56 binding to BS2 elevated both basal and stimulus-induced phosphorylation of S133, increased CBP interaction with CREB, and potentiated the expression of CREB-dependent reporter genes. Cells from mice harboring a homozygous CrebE153D mutation that disrupts BS2 exhibited increased S133 phosphorylation stoichiometry and elevated transcriptional bursts to cAMP. These findings provide insights into substrate targeting by PP2A holoenzymes and establish a new mechanism of CREB attenuation that has implications for understanding CREB signaling in cell growth, metabolism, synaptic plasticity, and other physiologic contexts.