Journal of Biological Chemistry

Coenzyme A (CoA) is an essential molecule for the intraerythrocytic stage of Plasmodium falciparum. Pantothenate kinase (PanK) catalyses the first step of the CoA biosynthesis pathway and functions as a homodimer in most organisms investigated thus far. P. falciparum possesses a novel heteromeric PanK complex composed of PfPanK1, PfPanK2 and Pf14-3-3I. Using a mutagenesis approach, we generated 10 PfPanK mutants and demonstrate that the PfPanK complex has only one functional active site, with both PfPanK1 and PfPanK2 required for activity by the complex. We also show that PfPanK2 is essential for normal intraerythrocytic parasite proliferation using a conditional knockdown system. 14-3-3 binding motifs generally contain a phosphoserine/threonine residue. Mass spectrometry analyses of phospho-peptide enriched, immunoprecipitated PfPanK samples revealed phosphorylation sites in both PfPanK1 and PfPanK2 that were additional to the previously reported sites. To investigate the role of specific sites in PfPanK1 and PfPanK2 that may be involved in Pf14-3-3I binding, five additional mutants were generated. Mutagenesis of four predicted Pf14-3-3I binding sites in PfPanK1 resulted in a significant reduction in the amount of Pf14-3-3I bound to the PfPanK complex, with S334 being the most likely binding site. Heterologous expression of the PfPanK complex in an insect cell system yielded a small amount of soluble protein that assembled in situ into a functional complex. Combined results from heterologous expression and P. falciparum mutagenesis suggest that Pf14-3-3I may not be essential for PfPanK activity but may be important for stabilising the PfPanK complex.

The 100 kDa hexokinase (HK) enzyme family represents an attractive model to investigate the molecular origins of allosteric regulation in multidomain enzymes. Extant HK homologs are subject to various allosteric phenomena, including activation and inhibition by both homotropic and heterotropic ligands. Here, we report the results of a phylogenetic investigation of this enzyme family using the recently developed Topiary ancestral sequence reconstruction pipeline. The results agree with prior studies that used a smaller number of sequences from individual HK domains and suggest that modern HK3 isozymes diverged first from a 100 kDa ancestor, followed by gene duplication and divergence of the HK2 isozymes. A subsequent gene duplication event led to divergence of HK1 and the hexokinase domain containing protein 1 (HKDC1). To probe the ability of Topiary to yield functional, allosterically regulated ancestral enzymes, we resurrected and biochemically characterized two HKs from early vertebrate evolution, Anc1 and Anc2. Both enzymes were functionally similar to extant HK1, and possessed a low activity, regulatory N-terminal domain that governs allosteric regulation of the C-terminal active site by two heterotropic effectors, glucose 6-phosphate and inorganic phosphate. Neither ancestor was subject to homotropic regulation by substrate glucose, a characteristic observed in several extant HK3 family members. Our phylogenetic analysis provides a foundation for investigating the evolution of allostery in this enzyme family. It also demonstrates the need to sequence and biochemically characterize additional full-length HKs, especially those from jawless vertebrates, to enable more robust inferences of ancestral regulatory traits.

Leucine Rich Repeat Containing 8C (LRRC8C) anion channels modulate NADPH oxidase 1 activity and allow extracellular superoxide influx promoting inflammatory signaling. Here we studied chimeric 8C/8D channels and identified oxidant-dependent current modulation within the N-terminus (NT) and first transmembrane domain (TM1). Chloramine-T (ChlT) elicited inhibitory and activating current responses, whereas other redox agents had comparatively little impact. ChlT moderately inhibited wild-type (WT) 8C current and abrogated block by DCPIB. Substitution of the 8D NT (8D1-22) conferred ChlT-dependent current activation, as did 8D2-4, 8D5-11, I2F, and I2Y substitution. M48T (distal TM1) substitution enhanced WT 8C current inhibition and impaired activation in NT mutants. An M48D mutation diminished 8C current block by DCPIB by [~]50%. WT 8D currents were potently inhibited by ChlT. Substitution of the 8C first extracellular loop (EL1) weakened inhibition, while 8C EL1 + TM145-49 substitution produced ChlT-mediated current activation. 8C45-49 or T48M substitutions in 8D resulted in rapid disruption and loss of initial current inhibition, and a progressive increase of non-rectifying current. These results provide evidence that NT2-4, particularly I2/F2, in combination with M48 are primary determinants of activating vs. inhibitory current modulation by ChlT. M48 oxidation limits 8C inhibition and is required for activating responses, while T48 and 8D EL1 promote 8D signature current inhibition. ChlT exposure disrupts subsequent or preexisting channel block by DCPIB, consistent with a common site of interaction. Thus, factors that alter NT pore stability and mobility may regulate inhibition vs. activation of LRRC8C by redox stress.

Escherichia coli YicC enzyme is the founding member of a family of endoribonucleases that is encoded in virtually all bacterial species. Previous structural studies revealed that this ribonuclease binds RNA by a novel mechanism in which the hexameric apoprotein presents an open channel that undergoes a large rotation upon RNA binding and clamps down on the RNA. The current study follows up on these findings by examining the cleavage of various oligonucleotide substrates designed to probe recognition elements required for YicC binding and cleavage. A 26-nucleotide RNA oligomer (oligo), with a KD in the low micromolar range, was the standard to which numerous oligos with altered sequence were compared. In vitro RNase assays and fluorescence anisotropy binding measurements indicated that the preferred substrates for YicC were relatively small RNAs that contain some secondary structure. Larger RNAs or highly structured RNAs were less-than-optimal substrates. Similarly, RyhB RNA, a [~]90-nucleotide, iron-responsive RNA of E. coli, which has been described as a target of YicC binding and/or cleavage, was a poor YicC substrate in our assays. These results suggest that the native substrates for YicC-family members are very small RNAs or RNA fragments derived from larger RNAs.

The ubiquitin conjugating enzyme UBE2J1/Ubc6e localizes to the endoplasmic reticulum where it mediates the ubiquitination and proteasomal degradation of terminally misfolded proteins. Although the protein is known to undergo phosphorylation at serine S184, we have considered modification at an additional site and used a bespoke anti-phospho antibody to confirm phosphorylation also at serine residue S266. Despite the well-described role of UBE2J1 in ER associated degradation (ERAD), we found no evidence for regulation at S266 during Unfolded Protein Response (UPR) induction by thapsigargin. Instead, our studies suggest that phosphorylation occurs independently at the S184 and S266 sites, with mutation at one site failing to disrupt basal phosphorylation at the second. We identified several contexts in which these two phosphorylations were differentially regulated. For example, ER localization, which is important for phosphorylation at S184, was not required for modification at S266, and sensitivity to proteasome inhibitors, which is regarded as a distinguishing feature of the S184 phospho-variant, was unaltered by the S266A mutation. Regarding regulation at S266 on the other hand, we found that pharmacological activation of protein kinase A resulted in rapid phosphorylation, with differential use of phospho-specific antibodies confirming that phosphorylation at S184 was unchanged by this treatment. Hormonal stimulation by glucagon resulted in a similar pattern of UBE2J1 phosphorylation, which occurred exclusively at S266 and could be inhibited by H89. The differential regulation demonstrated in these studies extends our understanding of the UBE2J1 enzyme, and may indicate a role in the integration of energy metabolism with environmental stress conditions.

The Ube2J1 enzyme that mediates the ubiquitination and proteasomal degradation of misfolded proteins at the ER is phosphorylated at serine S184. Following anisomycin treatment of HEK293T cells, we observed an inverse relationship between phosphorylation and dephosphorylation at this site. This suggested a dynamic interchange between the two forms, and we show that S184 is a target for protein phosphatase 2A. The S184-phosphorylated protein is known to exhibit increased sensitivity to proteasomal degradation, and we found that mutation at K186R increased the ratio of S184-phosphorylated to S184-dephosphorylated protein. Although the K186R mutant retained some sensitivity to proteasomal inhibition, our results show that Ube2J1 steady state expression can be exercised at multiple levels, and can involve dynamic phosphorylation and dephosphorylation at S184.

Anchoring fibrils formed by collagen VII play a critical role in stabilizing the dermal-epidermal junction. The N-terminal non-collagenous (NC1) domain of collagen VII binds firmly to basement membrane components including collagen IV and has also been reported to interact with mesenchymal fibrillar collagens via its von Willebrand factor A-like domain 2 (A2 domain). To elucidate how collagen VII recognizes fibrillar collagen, we performed yeast two-hybrid screening using a triple-helical random peptide library, which resulted in the identification of a Met-Gly-{Phi} ({Phi}; aromatic amino acid residue) motif. Biochemical analysis with synthetic triple-helical peptides revealed a binding preference of Trp > Phe as the {Phi} residue by the A2 domain despite Trp being absent in native collagens. The crystal structure of the A2 domain in complex with the Nle (Met surrogate)-Gly-Trp-containing peptide revealed a unique mechanism by which two distinct hydrophobic pockets of the A2 domain accommodate the Nle and Trp residues corresponding to the Met-Gly-{Phi} motif, engaging all three chains of the triple helix. Subsequent molecular dynamics simulations demonstrated that the A2 domain recognizes the corresponding native Met-Gly-Phe motif in a similar manner, but with lower affinity, implying a transient interaction with mesenchymal collagens. The findings obtained in this work suggest models in which transient A2-triple helix interaction promotes the recruitment of collagen I and III fibrils into the arc-shaped structure of anchoring fibrils. This also provides a foundation for linking structural understanding to skin fragility diseases caused by collagen VII dysfunction.

Reversible inactivation of protein tyrosine phosphatases by reactive oxygen species (ROS) is essential to the phosphorylation of growth factor receptors. An important outcome of the inactivation of protein tyrosine phosphatase 1B (PTP1B) by ROS involves the conformational change of its phosphotyrosine binding loop which adopts a solvent exposed position in its oxidized form. We previously demonstrated that 14-3-3{zeta} binds to the phosphotyrosine binding loop of the oxidized form of PTP1B. Using a rational approach, we developed a unique protein-protein interaction (PPI) inhibitor peptide derived from the phosphotyrosine binding loop of PTP1B designed to disrupt the interaction between PTP1B and the 14-3-3{zeta}-complex. Exploiting this cell-permeable peptide, we showed decreased association between PTP1B and the 14-3-3{zeta}-complex in cells treated with epidermal growth factor (EGF). We also demonstrated that preventing the association of this 14-3-3{zeta}-complex to PTP1B deterred oxidation and inactivation of PTP1B following EGF receptor (EGFR) activation and generation of ROS. Treating cells with our PPI inhibitor decreased EGFR phosphorylation on PTP1B-specific sites. Furthermore, treating EGFR-driven epidermal cancer cells with our PPI inhibitor also significantly inhibited colony formation and cell viability, consitent with increased activation of PTP1B. These data highlight the ability of PTP1B to downregulate critical signaling pathways in cancer when activated using peptide drugs such as our protein-protein interaction inhibitor. We anticipate that preventing or destabilizing the reversible oxidation of other members of the protein tyrosine phosphatase superfamily using PPI inhibitors may offer a foundation for a broad therapeutic approach to rectify dysregulated signaling pathways in vivo. Significance StatementLimited understanding of redox mechanisms regulating PTP catalytic activity is a major knowledge gap that has hampered our efforts to develop activation strategies. In its reversibly oxidized and inactivated form, conformational changes of PTP1B influence its association with regulatory proteins. We demonstrate that designing a cell-permeable peptide based on a loop of PTP1B that becomes exposed during oxidation can block its interaction with the 14-3-3{zeta}-multiprotein complex and activate the phosphatase. Moreover, activating PTP1B using our protein-protein interaction inhibitor peptide decreases the phosphorylation of its substrate EGFR and decreases the effectiveness of cancer cells to form colonies. This study provides important insights into the therapeutic potential of protein-protein interaction inhibitors that regulate the redox cycle of PTPs to reestablish physiological signaling.

Excessive osteoclast formation is a key contributor to pathological bone loss in disorders such as osteoporosis and rheumatoid arthritis. Asarinin, a natural lignan, has not previously been examined in the context of osteoclast differentiation. Here, we investigated the anti-osteoclastogenic effects of asarinin using RANKL-stimulated RAW264.7 cells. Asarinin significantly suppressed TRAP-positive multinucleated osteoclast formation under the tested conditions. Mechanistically, asarinin selectively inhibited RANKL-induced phosphorylation of p38 and ERK MAPKs, leading to reduced c-Fos expression and inhibition of NFATc1 nuclear translocation. In addition, asarinin disrupted actin ring formation in mature osteoclasts. Collectively, these findings identify asarinin as a pathway-selective inhibitor of osteoclast differentiation, targeting the p38/ERK-c-Fos-NFATc1 axis while sparing parallel signaling pathways.

Glutamate racemase (MurI) catalyzes the stereochemical interconversion of L-glutamate to D-glutamate, a key element of bacterial peptidoglycan biosynthesis. In this study, we present the crystal structure of Helicobacter pylori glutamate racemase at 1.43 [A] and in monoclinic symmetry, as previously reported models, but different unit-cell parameters. The present model contains a single dimer and retains the previously described head-to-head dimer arrangement. The differences between the models arise from variations in unit-cell parameters, which lead to altered crystal packing interactions rather than changes in the quaternary assembly. The monomeric fold and active-site architecture remain conserved and are consistent with the catalytic features described for bacterial glutamate racemases. This structure provides an updated, high-resolution structural model for H. pylori glutamate racemase and highlights the variability in crystal packing within the same space group.

The surface of gram-positive bacteria is a highly regulated environment with specific attachment of proteins required for viability. Sortase enzymes are cysteine transpeptidases that recognize and ligate substrates to the peptidoglycan layer in these microorganisms, which can be highly pathogenic (e.g., Staphylococcus aureus, Streptococcus pyogenes, etc.). As such, sortases represent a potentially novel target for antibiotic development. In addition, the catalytic activity of sortase enzymes is utilized in sortase-mediated ligation (SML) engineering approaches for a variety of uses. In SML experiments, engineered variants of Staphylococcus aureus sortase A (saSrtA) are the most widely used enzymes. One of the mutated amino acids in the previously engineered pentamutant (or saSrtA5M) enzyme is P94. Structural analyses of experimental saSrtA structures revealed that P94 interacts directly with Y187 when saSrtA is in its inactive conformation. While saSrtA5M, developed via directed evolution, contains a P94R mutation, we wanted to interrogate this position further and ask if other single P94 mutations may reveal a greater effect on activity and/or substrate specificity. We created 18 P94X mutations (excluding P94C), and tested relative activity using a fluorescence resonance energy transfer (FRET) assay for 4 substrate sequences: LPATG, LPETG, LPKTG, and LPSTG. We identified several P94 variants that outperformed the single mutant P94R for all peptides tested, including P94A, P94D, P94E, P94G, P94H, P94N, P94Q, P94S, and P94T. We further observed that the reactivity of substrates with variations in the central position of the pentapeptide recognition motif (LPXTG) can be sensitive to the identity of the P94X residue. We tested P94A and P94D saSrtA5M variants and found that, depending on LPXTG sequence, these variants could outperform saSrtA5M in activity > 3-fold. Finally, we compared saSrtA5M and P94D saSrtA5M in a model sortase-mediated ligation reaction using a LPKTG substrate and saw [~]2-fold greater product formation. Taken together, we characterized an important position that modulates substrate access and activity in saSrtA. Furthermore, we argue that future studies which combine rational design and high throughput approaches, e.g., directed evolution, may result in sortase variants with increased SML potential.

BCL11A is a transcription factor crucial for neurodevelopment and hematopoiesis. It regulates the developmental switch from fetal hemoglobin (HbF) to adult hemoglobin and is a major therapeutic target for sickle cell disease and {beta}-thalassemia. BCL11A exists in multiple isoforms, including the L isoform (containing a single two-finger ZF2-3 DNA-binding domain) and the XL isoform (containing two arrays: the two-finger ZF2-3 and the three-finger ZF4-6). We used three approaches to investigate BCL11A functions. First, we examined DNA recognition by BCL11A, which preferentially binds the short 6-bp DNA motif TGNCCA. ZF4-6 recognizes all four variants of this motif with distinct strand-specific interactions: TGTCCA on the top strand, TG(A/C)CCA on the complementary strand, and the palindromic TGGCCA on either strand. ZF2-3 also binds TGTCCA from the top strand, featuring a unique thymine interaction by ZF2 Phe388. Motif multiplicity within BCL11A binding sites may promote BCL11A oligomerization by enabling multiple ZF arrays to engage DNA simultaneously. Second, we treated HUDEP-2 cells (which express adult hemoglobin) with inhibitors targeting three epigenetic silencing marks - DNA methylation, histone H3 lysine 9 methylation or H3 lysine 27 methylation. All treatments, individually or in combination, increased HbF expression to varying degrees. Notably, FTX6058 markedly reduced BCL11A transcription and translation (likely via effects on LIN28B), while EML741 caused a partial reduction. Third, we screened 213 pomalidomide- and lenalidomide-derived compounds and quantified proportions of HbF+ cells by flow cytometry. Effects of four compounds were analyzed by protein mass spectrometry. Although BCL11A levels themselves were unchanged, all four compounds selectively decreased levels of known pomalidomide targets, with consistently decreased levels of the zinc-finger proteins IKZF1 and ZFP91. Together, our studies clarify how BCL11A recognizes DNA, how its expression can be modulated epigenetically, and how small-molecule degraders influence its regulatory network, providing new avenues for HbF reactivation therapies.

We have determined the molecular structure and investigated the catalytic mechanism of two new ribozymes of the Hepatitis delta virus family, found in the nematode Caenorhabditis briggsae and virus Ackermannviridae. Crystal structures of both conform to the double-pseudoknot architecture adopted by the viral HDV ribozyme. The C. briggsae ribozyme has been determined both pre- and post-cleavage. In the former both nucleotides flanking the scissile phosphate are observed, along with a metal ion, and cytosine 75 N3 bound to the O5 leaving group. The pH dependence of cleavage rate reveals a pKa of 6.6 and together with the inactivity of a C75U mutant provides evidence for its role as general acid. In contrast to other nucleolytic ribozymes that use catalytic metal ions, reaction rate does not depend on the pKa of the divalent metal ion. Limited adjustment of structure of the active center is consistent with direct bonding of the metal ion to the O2 and non-bridging O, suggesting that the ion acts as a Lewis acid to activate nucleophilic attack. This mechanism appears to be general for the HDV ribozyme class, and distinguishes it from the majority of nucleolytic ribozymes that use general base catalysis.

Disulfide bond formation is crucial to the structure and function of many proteins. It is known that there is diversity in the pathways for disulfide bond formation in bacteria and that there are gaps in our knowledge of these pathways. Using a combination of experimental and bioinformatic approaches we show that some of these gaps can be filled by a newly discovered oxidative folding pathway centered on methylamine utilization protein E (MauE). MauE has previously been associated with the methylamine utilization (MAU) gene cluster, which is involved in methylamine metabolism, in particular it is associated with the maturation of the small subunit of methylamine dehydrogenase. Here we show MauE from Caldithrix abyssi and Desulfatibacillum alphaticivorans functionally replace disulfide bond formation protein B (DsbB) in E. coli using two independent disulfide bond dependent assays. Furthermore, MauE is found in 14 species from 2 bacterial phyla that lack known pathways for structural disulfide bond formation, but which have proteins with structural disulfide bonds in the protein data bank. The active site for MauE was determined to be a conserved CXC motif. Using molecular docking predictions, we demonstrate that MauE is likely to interact with ubiquinone, similarly to the well characterized bacterial DsbB. We also constructed a dataset across thirty-five different phyla to demonstrate that MauE is potentially the second most common disulfide bond formation protein in bacterial disulfide bond formation pathways after DsbB. In addition, the distribution of MauE largely differs from the distribution of other MAU gene cluster markers affirming its role as a newly discovered generalist disulfide bond formation protein rather than being a specialized maturation factor for methylamine dehydrogenase. We also reveal further gaps in disulfide bond pathways, as well as species which may contain redundancies in their disulfide bond pathways.

The NS3 helicases from the Flaviviridae family of viruses exhibit nucleotide-hydrolysis-dependent, nucleic-acid-unwinding activity. The RNA unwinding activity for NS3 helicases from the Orthoflavivirus genus has not been fully explored and contrasts with NS3 helicase from Hepatitis C virus (HCV) of the Hepacivirus genus, which has thus far served as the prototypical model enzyme from this family of viruses. To begin to understand the functional differences between flavivirus NS3 helicases, we first developed an expression and purification system for full-length untagged NS3 protein from West Nile virus (WNV) and Zika virus (ZIKV). Both enzymes exhibit RNA-stimulated ATPase activity and are dependent on the nucleoside triphosphatase active site of the enzyme. Unlike HCV NS3, orthoflavivirus NS3s do not efficiently pre-assemble on a 3-ssRNA-tailed dsRNA substrate in the absence of ATP-Mg which is a prerequisite for formation of a productive HCV NS3-RNA complex that can exhibit a rapid burst of RNA unwinding. Instead, to observe RNA unwinding by WNV and ZIKV NS3s, low Mg-ATP concentrations are required at a time coincident when NS3 encounters the RNA substrate. In addition, we find that orthoflavivirus NS3s require translocation beyond the displaced strand to completely unwind a dsRNA substrate. Last, we find that orthoflavivirus NS5 stimulates the ability of NS3 to unwind dsRNA. These results suggest that functional differences exist between the flavivirus NS3 helicases and illuminate that orthoflavivirus NS3s require a functional interaction with the NS5 protein for coordination of its activity, as it is believed these two proteins constitute the viral replicase.

G protein-coupled receptors rely on dynamic conformational changes to coordinate G protein activation and recruitment of regulatory transducers such as G protein-coupled receptor kinases and {beta}-arrestins. The chemotactic receptor GPR183 has been implicated in a context-dependent role in hematological malignancies. Here, we investigated the impact of A338V mutation located within the C-terminal tail of GPR183. This mutation is associated with acute myeloid leukaemia. Using bioluminescence resonance energy transfer-based assays in HEK293A cells, we assessed receptor-proximal signaling events. The A338V variant displayed preserved agonist potency and comparable agonist-induced Gi activation relative to wild type, although constitutive activity towards Gi was modestly reduced. In contrast, recruitment of GRK2 and {beta}-arrestin2 was consistently impaired across multiple assay configurations. These differences were not attributable to altered receptor abundance, as the C-tail untagged mutant exhibited increased plasma membrane expression despite reduced regulatory transducer engagement. While intramolecular conformational biosensor measurements revealed subtle differences in global receptor conformation between WT and A338V, extensive molecular dynamics simulations supported the altered conformational sampling of the C-terminal tail in the A338V variant. Together, these data support a model in which the A338V substitution selectively alters C-terminal structural dynamics, impairing GRK2 and {beta}-arrestin2 recruitment while preserving G protein activation.

TBCK-related encephalopathy (TBCKE) is a neurodevelopmental disorder associated with biallelic mutations in TBCK. Despite the increasing number of reported cases worldwide, the biochemical and biophysical properties of TBCK remain unclear, hindering molecular understanding of its role in disease. Here, we present the successful expression, purification, and biochemical characterization of full-length human TBCK produced in Spodoptera frugiperda cells. Biochemical and biophysical analyses reveal that the catalytically inactive pseudokinase domain of TBCK lacks nucleotide binding, consistent with the absence of the canonical VAIK, HRD, and DFG motifs required for catalysis. These findings support that TBCK is a class I pseudokinase and provide a foundation for future structural and functional studies to elucidate its biological role.

N-terminal methylation of proteins by the trimethylase NRMT1 plays important roles in oncogenesis, development, and aging. As N-terminal methylation has frequently been shown to regulate protein-DNA interactions, and many NRMT1 substrates are transcription factors or regulators of chromatin structure, previous research has focused on how transcriptional regulation by NRMT1 affects cell growth and differentiation. However, we have recently identified a new, cytoplasmic role for NRMT1, inhibiting the eukaryotic elongation factor 1 alpha (eEF1A) methyltransferase METTL13, which indicates NRMT1 could also be acting as a translational regulator. Here we further explore NRMT1 cytoplasmic functions and show that, unlike previously thought, NRMT1 can methylate substrates in the cytoplasm. We also show that while many of these substrates remain bound to NRMT1, it can also interact with a number of non-target ribosomal proteins and proteins associated with the endoplasmic reticulum (ER). To confirm NRMT1 interaction with the ribosome, we performed polysome profiling, which showed a portion of NRMT1 co-migrates with the 40S and 60S subunits but not with actively translating polysomes, indicating NRMT1 may play an early role in translation. To see if NRMT1 was affecting target mRNA selection of ribosomes, we also performed ribosome-sequencing experiments in proliferating and differentiating C2C12 mouse myoblasts. These results show a striking upregulation of translation of soluble proteins with NRMT1 loss and corresponding decrease in translation of transmembrane and signal sequence-containing proteins. We now propose a model where NRMT1 regulates the translation of transmembrane and secreted proteins by facilitating interactions between the ribosome and the ER.

Analysis of yeast and mammalian telomerase ribonucleoprotein (RNP) complexes reveals striking divergence in their biogenesis and protein complements. However, little is known about telomerase in plants. In addition to the catalytic subunit TERT and the templating RNA TR, we previously reported Arabidopsis thaliana contains two telomerase accessory factors, AtNAP57, a dyskerin homolog that in mammals is essential for telomerase activity, and the telomeric DNA binding protein AtPOT1a. Both proteins stimulate Arabidopsis telomerase repeat addition processivity. Here we employ quantitative mass spectrometry (MS) to further examine telomerase in Arabidopsis. Unexpectedly, dyskerin and AtPOT1a were not detected in our purified complexes, but AtLa1, an RNA-binding factor that recognizes the UUU-3'OH of RNA Pol III transcripts, was highly enriched. RNA-IP assays confirmed AtLa1 association with AtTR in vivo. RNAi-mediated knockdown of AtLa1 strongly diminished telomerase activity, indicating AtLa1 is required for its function in vivo. In vitro binding studies revealed that AtLa1 contacts AtTR via the UUU-3'OH and a plant-specific P1a-P1b-P4 three-way junction (TWJ). Since the TWJ is also required for AtNAP57 binding, the data suggesting that AtNAP57 and AtLa1 compete for AtTR binding or sequentially associate during RNP during biogenesis. In contrast to AtNAP57, AtLa1 did not stimulate telomerase activity when TERT and TR were assembled in vitro, consistent with function during a different step in telomerase assembly. We conclude Arabidopsis telomerase employs multiple accessory factors utilized by both mammalian and single-celled relatives. Further exploration of Arabidopsis telomerase may offer novel insight into telomerase evolution and mechanisms of biogenesis. Significance of ResearchQuantitative mass spectrometry of Arabidopsis telomerase uncovered AtLa1, a homolog of ciliate and yeast proteins that promotes telomerase maturation. AtLa1 is essential for telomerase function in vivo, and in vitro it engages the same region of AtTR bound by AtNAP57, homologous to a telomerase accessory from mammals.

Galectin-3 (Gal3) is one of the most pro-inflammatory proteins and a biomarker of inflammatory diseases and cancer. Previous studies showed that Gal3 binds to v and {beta}1 integrins but it is unclear how Gal3 binds to integrins. Here, we show that Gal3 bound to soluble v{beta}3 and IIb{beta}3 integrins in 1 mM Mn2+ in cell-free conditions in a glycan-independent manner. Docking simulation predicts that Gal3 binds to the classical RGD-binding site (site 1) of v{beta}3, but the predicted Gal3-binding site does not include galactose-binding site. RGDfV or eptifibatide inhibited Gal3 binding to v{beta}3 and IIb{beta}3, respectively, but lactose, pan-galectin inhibitor, did not inhibit Gal3 binding to integrins. Point mutations of the predicted site 1 binding interface of Gal3 effectively inhibited Gal3 binding to site 1. Site 2 is involved in pro-inflammatory signaling (e.g., TNF and IL-6 secretion) and we previously showed that pro-inflammatory cytokines (e.g., CCL5 and TNF) bind to site 2 and allosteric integrin activation. Docking simulation predicts that Gal3 binds to site 2 of v{beta}3 and 5{beta}1. We found that Gal3 induced allosteric activation of soluble integrins v{beta}3, IIb{beta}3, and 5{beta}1 in 1 mM Ca2+ in cell-free conditions. Point mutations in the predicted site 2-binding interface inhibited Gal3-induced integrin activation, suggesting that Gal3 binding to site 2 is required for Gal3-induced integrin activation. Known anti-inflammatory agents, Ivermectin, NRG1, and FGF1 inhibited integrin activation induced by Gal3 in v{beta}3 and IIb{beta}3. These findings suggest that Gal3 binding to site 2 may be a potential mechanism of pro-inflammatory and pro-thrombotic action of Gal3.