C-terminal Coiled-coil Domain Research Articles

Abstract 4373: Molecular interactions of human receptor tyrosine kinase oncogenic fusions

Abstract Oncogenic fusions (OFs) play a critical role in cancer development. Despite the exponential increase in identifications, only a small number have been extensively characterized functionally. In this study, we are focused on receptor tyrosine kinase OFs (RTK-OFs) prevalent in aggressive cancers. Our primary objective is to elucidate the underlying molecular mechanisms of 182 in-frame RTK-OFs identified in The Cancer Genome Atlas. An automated high-throughput PCR technique will be used to clone individual gene fusions using the sequences obtained from RNA-Seq data sourced from the TCGA dataset. These clones will be used to establish an RTK-OF-expressing isogenic cell line library. Multiple Approaches Combined (MAC)-tag workflow combining Affinity Purification (AP) and proximity biotin-dependent (BioID) purification within a single construct will be used. Subsequent high-throughput EvoSep One Liquid Chromatography coupled to ultra-fast TimsTOF Pro 2 Mass Spectrometry sample processing enables sensitive and robust analysis. These methods enable the capture of stable protein complexes and transient interactors associated with RTK-OFs. In our proof of concept study, we have identified four in-frame variants of FGFR3::TACC3 oncogenic fusions.FGFR3, part of the fibroblast growth factor (FGF) RTK family, activates cellular proliferation and growth through FGF binding and receptor dimerization. In FGFR3-TACC3 OFs, diverse isoforms are observed, with prevalent rearrangements linking FGFR3 exon 17 to various TACC3 exons. TACC3 is vital for mitotic microtubule organization and stabilizes kinetochore fibers and the mitotic spindle through its conserved C-terminal coiled-coil domain (TACC domain). TACC3 contributes to the fusion with varying sizes of the C-terminal coiled-coil domain which all constitutively activate the FGFR3 tyrosine kinase. In our studies, we show that FGFR3::TACC3 variants have a different localization to their wild-type counterparts. While FGFR3 and TACC3 are found in the cytoplasm all the OF variants strongly localize around the nucleus. Our identified high-confidence interactors represent a comprehensive FGFR3::TACC3 interaction network and reveal central pathways and key interactors related to microtubule assembly and possible deregulation of the cell cycle. The resultant data from these advanced technologies enable the construction of comprehensive interaction networks, shedding light on critical signaling pathways and subnetworks relevant to RTK-OFs. Further in-depth analysis of 182 RTK-OFs may uncover actionable pathways and druggable interactors within these networks. Our multi-faceted research strategy holds significant promise for advancing targeted drug discovery, ultimately contributing to improved cancer treatment strategies. Citation Format: Giovanna Dashi, Markku Varjosalo. Molecular interactions of human receptor tyrosine kinase oncogenic fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4373.

Coiled-coil domain containing 159 is required for spermatid head and tail assembly in mice†.

The centrosome is critical for maintaining the sperm head-tail connection and the formation of flagellar microtubules. In this study, we found that in mouse testes, CCDC159 (coiled-coil domain-containing protein 159) is specifically localized to the head-tail coupling apparatus (HTCA) of spermatids, a structure that ensures sperm head-tail tight conjunction. CCDC159 contains a C-terminal coiled-coil domain that functions as the centrosomal localization signal. Gene knockout (KO) of Ccdc159 in mice resulted in acephalic spermatozoa, abnormal flagella, and male infertility. To explore the mechanism behind CCDC159 regulating spermatogenesis, we identified CCDC159-binding proteins using a yeast two-hybrid screen and speculated that CCDC159 participates in HTCA assembly by regulating protein phosphatase PP1 activity. Further RNA-sequencing analyses of Ccdc159 KO testes revealed numerous genes involved in male gamete generation that were downregulated. Together, our results show that CCDC159 in spermatids is a novel centrosomal protein anchoring the sperm head to the tail. Considering the limitation of KO mouse model in clarifying the biological function of CCDC159 in spermatogenesis, a gene-rescue experiment will be performed in the future.

Molecular and functional characterization of golden pompano (Trachinotus blochii) TBK1 on IFN regulation

The golden pompano (Trachinotus blochii), a pivotal commercial marine species in China, has gained significant popularity worldwide. However, accompanied with rapid growth and high density aquaculture, golden pompano has been seriously threatened by Nervous necrosis virus (NNV), while its molecular biology research regarding the innate immune system remains unexplored, which is crucial for understanding the activation of interferon (IFN) production and antiviral responses. In this study, we aimed to identify the characterization and function of golden pompano TANK-binding kinase 1 (gpTBK1), thereby providing evidence of the conservation of this classical factor in the RLR pathway among marine fish. Initially, we found the expression of gpTBK1 upregulation in diseased golden pompano with NNV infection and we successfully cloned the full-length open reading frame (ORF) of gpTBK1, consisting of 2172 nucleotides encoding 723 amino acids, from the head kidney. Subsequent analysis of the amino acid sequence revealed homology between gpTBK1 and other fish TBK1 proteins, with conserved N-terminal Serine/Threonine protein kinases catalytic domain (S_TKc) and C-terminal coiled coil domain (CCD). Moreover, the expression pattern showed that gpTBK1 exhibited ubiquitous expression across all evaluated tissues. Furthermore, functional identification experiments indicated that gpTBK1 activated interferon promoters’ activity in golden pompano and induced the expression of downstream IFN-stimulated genes (ISGs). Notably, gpTBK1 was found to co-localize and interact with gpIRF3 in the cytoplasm. Collectively, these data provide a comprehensive analysis of the characterization and functional role of gpTBK1 in promoting interferon production. This research may facilitate the further study of the innate antiviral response, particularly the anti-NNV mechanisms, in golden pompano.

Salmonella YqiC exerts its function through an oligomeric state.

Protein oligomerization occurs frequently both in vitro and in vivo, with specific functionalities associated with different oligomeric states. The YqiC protein from Salmonella Typhimurium forms a homotrimer through its C-terminal coiled-coil domain, and the protein is closely linked to the colonization and invasion of the bacteria to the host cells. To elucidate the importance of the oligomeric state of YqiC in vivo and its relation with bacterial infection, we mutated crucial residues in YqiC's coiled-coil region and confirmed the loss of trimer formation using chemical crosslinking and Size Exclusion Chromatography coupled with Multiple Angle Light Scattering (SEC-MALS) techniques. The yqiC-knockout strain complemented with mutant YqiC showed significantly reduced colonization and invasion of Salmonella to host cells, demonstrating the critical role of YqiC oligomerization in bacterial pathogenesis. Furthermore, we conducted a protein-protein interaction study of YqiC using a pulled-down assay coupled with mass spectrometry analysis to investigate the protein's role in bacterial virulence. The results reveal that YqiC interacts with subunits of Complex II of the electron transport chain (SdhA and SdhB) and the β-subunit of F0 F1 -ATP synthase. These interactions suggest that YqiC may modulate the energy production of Salmonella and subsequently affect the assembly of crucial virulence factors, such as flagella. Overall, our findings provide new insights into the molecular mechanisms of YqiC's role in S. Typhimurium pathogenesis and suggest potential therapeutic targets for bacterial infections. This article is protected by copyright. All rights reserved.

Cryo-EM structures of African swine fever virus topoisomerase.

African swine fever virus (ASFV) is a highly contagious virus that causes lethal hemorrhagic diseases known as African swine fever (ASF) with a case fatality rate of 100%. There is an urgent need to develop anti-ASFV drugs. We determine the first high-resolution structures of viral topoisomerase ASFV P1192R in both the closed and open C-gate forms. P1192R shows a similar overall architecture with eukaryotic and prokaryotic type II topoisomerases, which have been successful targets of many antimicrobials and anticancer drugs, with the most similarity to yeast topo II. P1192R also exhibits differences in the details of active site configuration, which are important to enzyme activity. These two structures offer useful structural information for antiviral drug design and provide structural evidence to support that eukaryotic type IIA topoisomerase likely originated from horizontal gene transfer from the virus.

#4317 CLINICAL DIVERSITY OF STEROID-RESISTANT NEPHROTIC SYNDROME CAUSED BY TRPC6 MUTATIONS

Abstract Background and Aims Steroid-resistant nephrotic syndrome (SRNS) is a clinically and genetically heterogeneous disorder caused by either genetic or immunological factors or their combination. Approximately 30–40% of patients with SRNS make a fast progression to ESRD. SRNS represents the second leading cause of end-stage renal disease in individuals under the age of 25 years worldwide. More than 60 podocyte-related gene mutations have thus far been reported in monogenic SRNS. Recent studies indicate that almost 30% of patients with childhood-onset SRNS have monogenic causative variants in one of the 27 SRNS genes. Among these, a mutation in Transient Receptor Potential Canonical 6 (TRPC6) gene, encoding non-selective cation channel, accounts for 6% of familial SRNS and approximately 2% of sporadic cases. We recently detected eight patients with TRPC6 mutations in our SRNS cohort (n = 39, 21%) and found inter- and intra-familial heterogeneity of clinical phenotypes in terms of disease onset and progression. Method A total of 39 Japanese SRNS patients were studied according to the protocol approved by the institutional review board of each affiliation. Subjects who manifested SRNS at age one to 14-year-old with biopsy-proven FSGS were enrolled. A total of 39 SRNS patients, whose average onset of proteinuria at median age 4 (range 0.9 to 17 yr) and ESRD at median age 7 (49% of total n = 19, range 3.0 to 16.0 yr) were studied. Family history was found in 44% (n = 17), including 13 autosomal recessive, 3 dominant, and one X-linked transmission. Sixteen percent (n = 10) of the patients who underwent renal transplantation win no recurrence. Sequence analysis revealed the pathogenic variants in 62% (n = 24) of all cases. Library for whole-exome sequencing or targeted panel sequencing were prepared from genomic DNA by use of SureSelect Human All Exon V5 (Agilent) or a HaloPlex target enrichment system kit (Agilent Technologies), or Ampli-Seq (Thermo), Prepared samples were run on a HiSeq 2000, or MiSeq (Illumina), or Ion PGM (Thermo). The sequence reads with 101-bp paired-end reads and 7-bp index reads were mapped to the human reference sequence (hg19, GRCh38.p7). After filtering the superfluous begin variants, only variants that fulfill the “pathogenic” criteria of ACMG and/or ClinVar, Varsome were selected. Results Sequence analysis revealed the pathogenic variants in 62% (n = 24) of all cases. TRPC6 mutations were the most frequent cause of SRNS (n = 8), which was followed by NUP107 (n = 5), PLCE1 (n = 3), COL4A3 (n = 2), COL4A5 (n = 1), and others were identified in only single case. The eight TRPC6 mutations clustered into the two distinctive cytoplasmic domains; N-terminal ANK (Y173D, R175W, R175G) and C-terminal coiled-coil domain (p.E875V, p.867_868Del, p.S893N, p.R895C). The p.R895C is deposited as the pathogenic in ClinVar and may be a gain-of-function. The remaining 7 variants are of unknown functions: six have not yet been deposited in ClinVar, whereas one is VUS. Four patients were de novo occurrence, three followed the dominant transmission, and one arose from gonadal mosaicism. Two index patients exhibited SRNS in early childhood (< age 3), four manifested it in childhood (age 3–12), while the remaining two developed it in adolescence (age >12). In familial cases, for example, affected ones harboring R175W or p.E875V showed a remarkable discordant age onset and disease severity. Conclusion TRPC6 channelopathy was found to be the most frequent cause in our SRNS cohort, of which age onset ranges from age 0.9 to 17 year. Heterogeneity of disease severity may reflect that channel activity is regulated by the mode of action (gain- or loss-of-function), the combination of channel subunit assembly, and interaction with other modifying factors. Mutations in these TRPCs are associated with relatively common kidney diseases as well as other pathologies. Further expression studies will improve our mechanistic understanding of TRP channel pathology and may help the development of novel therapeutic targets.

The Effect of Prohibitins on Mitochondrial Function during Octopus tankahkeei Spermiogenesis.

Mitochondria are essential for spermiogenesis. Prohibitins (PHBs; prohibitin 1, PHB1 or PHB, and prohibitin 2, PHB2) are evolutionarily conserved and ubiquitously expressed mitochondrial proteins that act as scaffolds in the inner mitochondrial membrane. In this study, we analyzed the molecular structure and dynamic expression characteristics of Ot-PHBs, observed the colocalization of Ot-PHB1 with mitochondria and polyubiquitin, and studied the effect of phb1 knockdown on mitochondrial DNA (mtDNA) content, reactive oxygen species (ROS) levels, and apoptosis-related gene expression in spermatids. Our aim was to explore the effect of Ot-PHBs on mitochondrial function during the spermiogenesis of Octopus tankahkeei (O. tankahkeei), an economically important species in China. The predicted Ot-PHB1/PHB2 proteins contained an N-terminal transmembrane, a stomatin/prohibitin/flotillin/HflK/C (SPFH) domain (also known as the prohibitin domain), and a C-terminal coiled-coil domain. Ot-phb1/phb2 mRNA were widely expressed in the different tissues, with elevated expression in the testis. Further, Ot-PHB1 and Ot-PHB2 were highly colocalized, suggesting that they may function primarily as an Ot-PHB compiex in O. tankahkeei. Ot-PHB1 proteins were mainly expressed and localized in mitochondria during spermiogenesis, implying that their function may be localized to the mitochondria. In addition, Ot-PHB1 was colocalized with polyubiquitin during spermiogenesis, suggesting that it may be a polyubiquitin substrate that regulates mitochondrial ubiquitination during spermiogenesis to ensure mitochondrial quality. To further investigate the effect of Ot-PHBs on mitochondrial function, we knocked down Ot-phb1 and observed a decrease in mtDNA content, along with increases in ROS levels and the expressions of mitochondria-induced apoptosis-related genes bax, bcl2, and caspase-3 mRNA. These findings indicate that PHBs might influence mitochondrial function by maintaining mtDNA content and stabilizing ROS levels; in addition, PHBs might affect spermatocyte survival by regulating mitochondria-induced apoptosis during spermiogenesis in O. tankahkeei.

WNK1, a molecular crowding sensor, links phase separation to cellular physiological stress.

A recently published study in Cell by Boyd-Shiwarski et al.1 revealed that WNK kinases sense intracellular molecular crowding and restore cell volume via phase separation (PS). Cells are suffer from hyperosmotic stress, which leads to volume shrinkage and a decrease in intracellular space, resulting in molecular crowding (Figure 1A). WNK1 rapidly senses molecular crowding, triggers PS via the C-terminal domain (CTD), and recruits downstream effectors to form condensates. The SPAK and OSR1 effector kinases are activated in condensates and transported to the cytosol to regulate the NKCC1 and KCC ion channels. Eventually, resulting in the obligate reclamation of water and cell volume is recovered, this rapid response termed regulatory volume increase1 (RVI) (Figure 1B). PS can be triggered by various cellular stresses, including temperature, pH, and hyperosmolarity.2 Meanwhile, PS can produce membrane-free biomolecular condensates, in which specific biomolecules are concentrated and serve biological functions.3 For instance, the phosphorylation of the Alzheimer's disease-related protein tau by cyclin-dependent kinase 2 is accelerated in condensates,4 and biomolecular condensates are implicated in the regulation of postsynaptic density at neuronal synapses in synaptic signaling and plasticity.3 Numerous studies have demonstrated the significance of biomolecular condensates in physiology and pathophysiology.2, 3 In addition to triggering PS, hyperosmotic stress can also activate WNK1.5 WNK1 belongs to the with-no-lysine (K) protein kinase family and is associated with Cl−, K+, and Na+ ion transport. When cells are subjected to hyperosmotic stress, WNK1 is activated, mediates ion flow, and triggers RVI to prevent cell death and triggers RVI to prevent cell death. When WNK1 is activated by hyperosmotic stress, it colocalizes with downstream effector kinases in cytoplasmic puncta, when these puncta were first described in 2007, it was assumed that these puncta were membrane-bound intracellular vesicles; however, recent work by Boyd-Shiwarski has characterized these WNK1 puncta as membrane-less. This is consistent with other proteins that undergo PS and form membrane-less compartments.5 However, PS triggered by molecular crowding caused by hyperosmotic stress appears to be a common mechanism by which multimeric proteomes exert biological functions.3 Boyd-Shiwarski et al.1 are the first to report how molecular crowding causes WNK1 to phase separate into cytoplasmic puncta, and these findings have major implications for the physiologic regulation of RVI. Surprisingly, the formation of WNK1 cytoplasmic puncta was found to be dependent on the degree of hyperosmotic stress, and electron hyperdensities observed under correlative light and electron microscopy experiments were typical of condensates formed through PS. These findings suggest that WNK1 cytoplasmic puncta were not vesicular membranes but rather membraneless condensates formed by PS triggered by hyperosmotic stress. Meanwhile, the authors demonstrated that the WNK1 CTD is critical in mediating WNK1 PS and condensate formation. Interestingly, the authors found that, while C-terminal coiled-coil domains (CT CCDs) are not required for PS, the presence of CT CCDs enable WNK1 condensates to form at the appropriate time. Second, the authors conducted a bioinformatics analysis to determine the WNK CTD amino acid composition during the evolution from protists to humans. Although the primary amino acid composition of the WNK CTD differs between vertebrates and invertebrates, both mediate condensate formation in a similar manner. Then, studies on the WNK1 homolog Drosophila melanogaster WNK (Dm WNK) revealed that, similar to mammalian WNK1, Dm WNK CTD also generates condensates in response to hyperosmotic stress. These findings suggest that, while the amino acid compositions of vertebrate and invertebrate WNK CTDs are not identical, the ability of the WNK CTD to mediate PS to generate condensates is evolutionarily conserved. Subsequently, Boyd-Shiwarski et al.1 demonstrated that WNK1 CTD is required for RVI. In addition, the influx of the K+ congener rubidium (Rb+) was studied under isotonic and hypertonic conditions in WNK1/WNK3 double knockout cells expressing full-length or the 1–494 WNK1 fragment. The authors demonstrated that WNK1 CTD plays an important role in NKCC1 transport. Another interesting finding is that after introducing a sequence capable of causing PS to replace the original CTD sequence of WNK1, the WNK1 kinase domain can still phosphorylate downstream effector kinases, thereby mediating RVI. Finally, Boyd-Shiwarski et al.1 utilized the molecular crowding agent Ficoll, which can trigger the protein PS in vitro, to investigate the effect of molecular crowding on WNK1 condensate formation. Following intracytoplasmic microinjection of Ficoll, the degree of molecular crowding in the cytoplasm increased, and WNK1 condensate formed. In addition, when WT HEK293T cells transiently expressing either full-length WNK1 or an N-terminal fragment of WNK1 (1-494), only the full-length WNK1 expressing cells exhibited PS. This suggests that WNK1 is an intracellular molecular crowding sensor that triggers PS to regulate cell volume. Boyd-Shiwarski et al.1 first demonstrated that cytoplasmic puncta are membraneless condensates formed by hyperosmotic stress-induced PS via WNK1. Meanwhile, the authors found that WNK1 responds to hyperosmotic stress by sensing molecular crowding and that its CTD is indispensable in the process of condensate formation. Furthermore, the WNK1 CTD is evolutionarily conserved, suggesting that PS may be fundamental cell behavior. The authors demonstrated the significance of protein phase behavior in cellular physiological processes, which was previously believed to correlate little with physiological relevance. In addition, the authors refuted the notion that WNK1 cytoplasmic puncta are membrane vesicles, which has significant implications for further studies of the dynamics of WNK1 condensates and whether additional regulatory mechanisms exist in the condensates. Several issues remain to be resolved in future research. For instance, previous studies have shown that phosphorylation of different WNK1 amino acid residues can activate or inhibit its activity during hyperosmotic stress.5 However, there is no direct evidence for the involvement of other proteins in this process. It will be critical to elucidate the underlying mechanism of WNK1 phosphorylation in condensates. This will not only be beneficial for exploring relevant physiological regulatory mechanisms but also be important for the development of WNK1-SPAK/OSR1 pathway targeted-drugs. In addition, the transport mechanism of SPAK/OSR1 following phosphorylation in the cytosol is also worth investigating, which will facilitate the discovery of relevant transporters as well as regulatory targets. In summary, the authors established for the first time a direct link between WNK1-mediated PS and physiological stress responses, which is critical for future research into the relationship between PS and cell physiology. Tinghui Lan performed the drawing of the images and the writing of the article. Fangfang Zhou and Long Zhang provided modifications. All authors have read and approved the final manuscript. The current work was supported by the the Chinese National Natural Science Funds (grant number: U20A20393) and the National Science Fund for Distinguished Young Scholars (grant numbers: 32125016 and 31925013). The authors declare no competing interests. No ethical approval was necessary for this work. No.

Structural insights into substrate recognition and translocation of human peroxisomal ABC transporter ALDP

Dysfunctions of ATP-binding cassette, subfamily D, member 1 (ABCD1) cause X-linked adrenoleukodystrophy, a rare neurodegenerative disease that affects all human tissues. Residing in the peroxisome membrane, ABCD1 plays a role in the translocation of very long-chain fatty acids for their β-oxidation. Here, the six cryo-electron microscopy structures of ABCD1 in four distinct conformational states were presented. In the transporter dimer, two transmembrane domains form the substrate translocation pathway, and two nucleotide-binding domains form the ATP-binding site that binds and hydrolyzes ATP. The ABCD1 structures provide a starting point for elucidating the substrate recognition and translocation mechanism of ABCD1. Each of the four inward-facing structures of ABCD1 has a vestibule that opens to the cytosol with variable sizes. Hexacosanoic acid (C26:0)-CoA substrate binds to the transmembrane domains (TMDs) and stimulates the ATPase activity of the nucleotide-binding domains (NBDs). W339 from the transmembrane helix 5 (TM5) is essential for binding substrate and stimulating ATP hydrolysis by substrate. ABCD1 has a unique C-terminal coiled-coil domain that negatively modulates the ATPase activity of the NBDs. Furthermore, the structure of ABCD1 in the outward-facing state indicates that ATP molecules pull the two NBDs together and open the TMDs to the peroxisomal lumen for substrate release. The five structures provide a view of the substrate transport cycle and mechanistic implication for disease-causing mutations.

The heme-regulated inhibitor kinase requires dimerization for heme-sensing activity

The heme-regulated inhibitor (HRI) is a heme-sensing kinase that regulates mRNA translation in erythroid cells. In heme deficiency, HRI is activated to phosphorylate eukaryotic initiation factor 2α and halt production of globins, thus avoiding accumulation of heme-free globin chains. HRI is inhibited by heme via binding to one or two heme-binding domains within the HRI N-terminal and kinase domains. HRI has recently been found to inhibit fetal hemoglobin (HbF) production in adult erythroid cells. Depletion of HRI increases HbF production, presenting a therapeutically exploitable target for the treatment of patients with sickle cell disease or thalassemia, which benefit from elevated HbF levels. HRI is known to be an oligomeric enzyme that is activated through autophosphorylation, although the exact nature of the HRI oligomer, its relation to autophosphorylation, and its mode of heme regulation remain unclear. Here, we employ biochemical and biophysical studies to demonstrate that HRI forms a dimeric species that is not dependent on autophosphorylation, the C-terminal coiled-coil domain in HRI is essential for dimer formation, and dimer formation facilitates efficient autophosphorylation and activation of HRI. We also employ kinetic studies to demonstrate that the primary avenue by which heme inhibits HRI is through the heme-binding site within the kinase domain, and that this inhibition is relatively independent of binding of ATP and eukaryotic initiation factor 2α substrates. Together, these studies highlight the mode of heme inhibition and the importance of dimerization in human HRI heme-sensing activity.

Structural basis of human PDZD8\u2013Rab7 interaction for the ER-late endosome tethering

The membrane contact sites (MCSs) between the ER and late endosomes (LEs) are essential for the regulation of endosomal protein sorting, dynamics, and motility. PDZD8 is an ER transmembrane protein containing a Synaptotagmin-like Mitochondrial lipid-binding Proteins (SMP) domain. PDZD8 tethers the ER to late endosomes and lysosomes by associating its C-terminal coiled-coil (CC) with the LE Rab7. To identify the structural determinants for the PDZD8–Rab7 interaction, we determined the crystal structure of the human PDZD8 CC domain in complex with the GTP-bound form of Rab7. The PDZD8 CC contains one short helix and the two helices forming an antiparallel coiled-coil. Two Rab7 molecules bind to the opposite sides of the PDZD8 CC in a 2:1 ratio. The switch I/II and interswitch regions of the GTP-loaded Rab7 form the binding interfaces, which correlates with the GTP-dependent interaction of PDZD8 and Rab7. Analysis of the protein interaction by isothermal titration calorimetry confirms that two Rab7 molecules bind the PDZD8 CC in a GTP-dependent manner. The structural model of the PDZD8 CC–Rab7 complex correlates with the recruitment of PDZD8 at the LE–ER interface and its role in lipid transport and regulation.

Jujube witches' broom phytoplasma effectors SJP1 and SJP2 induce lateral bud outgrowth by repressing the ZjBRC1-controlled auxin efflux channel.

Comprehensively controlling phytoplasma-associated jujube witches' broom (JWB) disease is extremely challenging for the jujube industry. Although the pathogenesis of phytoplasma disease has been highlighted in many plant species, the release of lateral buds from dormancy under JWB phytoplasma infection has not been characterized in woody perennial jujube. Here, two 16SrV-B group phytoplasma effectors, SJP1 and SJP2, were experimentally determined to induce witches' broom with increased lateral branches. In vivo interaction and subcellular localization analyses showed that both SJP1 and SJP2 were translocated from the cytoplasm to the nucleus to target the CYC/TB1-TCP transcription factor ZjBRC1. The N- and C-terminal coiled-coil domains of SJP1 and SJP2 were required for the TCP-binding ability. ZjBRC1 bound directly to the auxin efflux carrier ZjPIN1c/3 promoters and down-regulated their expression to promote the accumulation of endogenous auxin indole-3-acetic acid in jujube calli. Furthermore, JWB phytoplasma infection suppressed ZjBRC1 accumulation and induced ZjPIN1c/3 expression to stimulate lateral bud outgrowth. Therefore, SJP1 and SJP2 stimulate lateral bud outgrowth, at least partly, by repressing the ZjBRC1-controlled auxin efflux channel in jujube, representing a potential strategy for comprehensive phytoplasma-associated disease control and a resource for gene editing breeding to create new cultivars with varying degrees of shoot branching.

MO049FUNCTIONAL IMPORTANCE OF MAPKBP1 PROTEIN DOMAINS IN NEPHRONOPHTHISIS

Abstract Background and Aims Nephronophthisis is an autosomal-recessive kidney disease that accounts for a significant proportion of end-stage renal disease (ESRD) in childhood, adolescence and early adulthood. Biallelic pathogenic variants in MAPKBP1, encoding the c-Jun N-terminale kinase (JNK)-binding protein 1, are associated with development of Nephronophthisis and subsequent chronic kidney disease (CKD) (Macia et al, AJHG, 2017). We recently characterized MAPKBP1 as microtubule-associated protein that is able to localize to centrioles and the base of primary cilia depending on dimerization via its C-terminal coiled-coil domain (Schönauer et al, Kidney Int, 2020). However, the physiological function of its N-terminal WD40 and intermediate JNK-binding domain is still poorly understood. By in vitro comparison of artificial domain deletions with known and novel patient variants, we aim at pinpointing functional consequences of pathogenic MAPKBP1 in cilia and cell cycle control. Method N-terminally GFP-tagged MAPKBP1 constructs with either full-domain deletions or patient-derived variants were expressed in non-ciliated HeLa and ciliated H69 cells for fluorescence microscopy studies. Furthermore, RNA-seq analysis using primary patient cells was conducted to investigate differentially regulated molecular pathways compared to healthy control individuals. Results Immunofluorescence microscopy revealed inappropriate intracellular localization upon single or combined deletion of any MAPKBP1 protein domain. Compared to wild type, all deletion variants showed reduced intensity at the centrosome and ciliary base. Despite preserved dimerization ability, loss of the intermediate JNK-binding domain (JBD) most effectively abolished centrosomal or ciliary targeting, whereas loss of the N-terminal WD40-domain induced strongest mitotic aberrations. Unlike wild type, both, artificial and patient-derived truncating variants were able to enter the nucleus. RNA-seq analysis using primary patient fibroblasts with varying C-terminal truncations will allow important insights into common gene expression profiles unveiling consequences of aberrant intracellular trafficking. Conclusion In the present work, we demonstrate that all protein domains are indispensable for appropriate MAPKBP1 intracellular localization and function. Most of clinically reported patient variants exhibiting C-terminal truncation of varying lengths resulted in comparable intracellular behavior in presence of an intact N-terminal WD40 domain. Surprisingly, deletion of the JNK-binding domain alone aggravated functional disturbances hinting at a prominent regulatory role of this protein part interdepending with dimerization. Further insights into domain-specific functions will explain molecular disease mechanisms of MAPKBP1.

The dimeric Golgi protein Gorab binds to Sas6 as a monomer to mediate centriole duplication.

The duplication and ninefold symmetry of the Drosophila centriole requires that the cartwheel molecule, Sas6, physically associates with Gorab, a trans-Golgi component. How Gorab achieves these disparate associations is unclear. Here, we use hydrogen-deuterium exchange mass spectrometry to define Gorab's interacting surfaces that mediate its subcellular localization. We identify a core stabilization sequence within Gorab's C-terminal coiled-coil domain that enables homodimerization, binding to Rab6, and thereby trans-Golgi localization. By contrast, part of the Gorab monomer's coiled-coil domain undergoes an antiparallel interaction with a segment of the parallel coiled-coil dimer of Sas6. This stable heterotrimeric complex can be visualized by electron microscopy. Mutation of a single leucine residue in Sas6's Gorab-binding domain generates a Sas6 variant with a sixteenfold reduced binding affinity for Gorab that cannot support centriole duplication. Thus, Gorab dimers at the Golgi exist in equilibrium with Sas6-associated monomers at the centriole to balance Gorab's dual role.

Targeting novel human transient receptor potential ankyrin 1 splice variation with splice-switching antisense oligonucleotides.

Activation of transient receptor potential ankyrin 1 (TRPA1) channels by both environmental irritants and endogenous inflammatory mediators leads to excitation of the nerve endings, resulting in acute sensation of pain, itch, or chronic neurogenic inflammation. As such, TRPA1 channels are actively pursued as therapeutic targets for various pathological nociception and pain disorders. We uncovered that exon 27 of human TRPA1 (hTRPA1) could be alternatively spliced into hTRPA1_27A and hTRPA1_27B splice variants. The resulting channel variants displayed reduced expression, weakened affinity to interact with WT, and suffered from complete loss of function because of disruption of the C-terminal coiled-coil domain. Using a human minigene construct, we revealed that binding of splicing factor serine/arginine-rich splicing factor 1 (SRSF1) to the exonic splicing enhancer was critical for the inclusion of intact exon 27. Knockdown of SRSF1, mutation within exonic splicing enhancer, or masking SRSF1 binding with antisense oligonucleotides promoted alternative splicing within exon 27. Finally, antisense oligonucleotides-induced alternative splicing produced transcript and protein variants that could be functionally determined as diminished endogenous TRPA1 activity in human Schwann cell-line SNF96.2 and hiPSCs-derived sensory neurons. The outcome of the work could potentially offer a novel therapeutic strategy for treating pain by targeting alternative splicing of hTRPA1.

Structural insights into kinetoplastid coronin oligomerization domain and F-actin interaction.

The two-domain actin associated protein coronin interacts with filamentous (F-) actin, facilitating diverse biological processes including cell proliferation, motility, phagocytosis, host-parasite interaction and cargo binding. The conserved N-terminal β-propeller domain is involved in protein: protein interactions, while the C-terminal coiled-coil domain mediates oligomerization, transducing conformational changes. The L. donovani coronin coiled-coil (LdCoroCC) domain exhibited a novel topology and oligomer association with an inherent asymmetry, caused primarily by three a residues of successive heptads. In the T.brucei homolog (TbrCoro), two of these ‘a’ residues are different (Val 493 & 507 replacing LdCoroCC Ile 486 and Met 500 respectively). The elucidated structure possesses a similar topology and assembly while comparative structural analysis shows that the T.brucei coronin coiled-coil domain (TbrCoroCC) too possesses the asymmetry though its magnitude is smaller. Analysis identifies that the asymmetric state is stabilized via cyclic salt bridges formed by Arg 497 and Glu 504. Co-localization studies (LdCoro, TbrCoro and corresponding mutant coiled coil constructs) with actin show that there are subtle differences in their binding patterns, with the double mutant V493I–V507M showing maximal effect. None of the constructs have an effect on F-actin length. Taken together with LdCoroCC, we therefore conclude that the inherent asymmetric structures are essential for kinetoplastids, and are of interest in understanding and exploiting actin dynamics.

Abstract A27: Increasing proximity triggers Mst2 autophosphorylation

Abstract The Hippo pathway limits cell number and differentiation by blocking the transcriptional activity of YAP. The fidelity of signal transduction relies on the coordinated activity of a core kinase cassette that includes the kinases Mst1/2 and Lats1/2 and the accessory proteins Mob1 and hSalvador. Mst1/2 contains a kinase domain, linker region, and a C-terminal coiled-coil domain termed SARAH and is responsible for the phosphorylation of downstream components. Activation of Mst1/2 requires phosphorylation of the activation loop, a process believed to be a consequence of trans-autophosphorylation that is promoted by SARAH-domain mediated homodimerization. We aimed to understand what mechanistically triggers Mst1/2 autophosphorylation. Using purified proteins, we show that the kinase domain of Mst2 is sufficient for autophosphorylation but full-length Mst2 undergoes autophosphorylation faster than variants lacking the SARAH domain. This increased autophosphorylation could arise from either a specific contribution of a SARAH-domain mediated homodimer (allostery) or the increased proximity of kinase domains following homodimerization (effective local concentration). We investigated both possibilities using a series of biochemical assays and revealed that autophosphorylation of Mst2 variants lacking a SARAH domain could be stimulated by either chemically induced dimerization or increased localization following recruitment to the surface of a lipid-vesicle. Our results suggest that SARAH-domain mediated homodimerization is not the only event that promotes autophosphorylation of Mst1/2 and, instead, supports a more inclusive model that relies on colocalization. Any cellular event that increases the proximity of Mst1/2 could activate the kinase such as SARAH-domain mediated homodimerization, higher-order complex formation with hSalvador, or membrane recruitment. Citation Format: Thao Tran, Jennifer M. Kavran. Increasing proximity triggers Mst2 autophosphorylation [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A27.

ROP INTERACTIVE PARTNER b Interacts with RACB and Supports Fungal Penetration into Barley Epidermal Cells.

Rho of Plants (ROP) G-proteins are key components of cell polarization processes in plant development. The barley (Hordeum vulgare) ROP protein RACB is a susceptibility factor in the interaction of barley with the barley powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh). RACB also drives polar cell development, and this function might be coopted during the formation of fungal haustoria in barley epidermal cells. To understand RACB signaling during the interaction of barley with Bgh, we searched for potential downstream interactors of RACB. Here, we show that ROP INTERACTIVE PARTNER b (RIPb; synonym: INTERACTOR OF CONSTITUTIVE ACTIVE ROP b) directly interacts with RACB in yeast and in planta. Overexpression of RIPb supports the susceptibility of barley to Bgh RIPb further interacts with itself at microtubules. However, the interaction with activated RACB largely takes place at the plasma membrane. Both RIPb and RACB are recruited to the site of fungal attack around the neck of developing haustoria, suggesting locally enhanced ROP activity. We further assigned different functions to different domains of the RIPb protein. The N-terminal coiled-coil CC1 domain is required for microtubule localization, while the C-terminal coiled-coil CC2 domain is sufficient to interact with RACB and to fulfill a function in susceptibility at the plasma membrane. Hence, RIPb appears to be localized at microtubules and is then recruited by activated RACB for a function at the plasma membrane during formation of the haustorial complex.

Novel nephronophthisis-associated variants reveal functional importance of MAPKBP1 dimerization for centriolar recruitment

Biallelic mutations in MAPKBP1 were recently associated with late-onset cilia-independent nephronophthisis. MAPKBP1 was found at mitotic spindle poles but could not be detected at primary cilia or centrosomes. Here, by identification and characterization of novel MAPKBP1 variants, we aimed at further investigating its role in health and disease. Genetic analysis was done by exome sequencing, homozygosity mapping, and a targeted kidney gene panel while coimmunoprecipitation was used to explore wild-type and mutant protein-protein interactions. Expression of MAPKBP1 in non-ciliated HeLa and ciliated inner medullary collecting duct cells enabled co-localization studies by fluorescence microscopy. By next generation sequencing, we identified two novel homozygous MAPKBP1 splice-site variants in patients with nephronophthisis-related chronic kidney disease. Splice-site analyses revealed truncation of C-terminal coiled-coil domains and patient-derived deletion constructs lost their ability to homodimerize and heterodimerize with paralogous WDR62. While wild-type MAPKBP1 exhibited centrosomal, basal body, and microtubule association, mutant proteins lost the latter and showed reduced recruitment to cell cycle dependent centriolar structures. Wild-type and mutant proteins had no reciprocal influence upon co-expression excluding dominant negative effects. Thus, MAPKBP1 appears to be a novel microtubule-binding protein with cell cycle dependent centriolar localization. Truncation of its coiled-coil domain is enough to abrogate its dimerization and results in severely disturbed intracellular localizations. Delineating the impact of impaired dimerization on cell cycle regulation and intracellular kidney signaling may provide new insights into common mechanisms of kidney degeneration. Thus, due to milder clinical presentation, MAPKBP1-associated nephronophthisis should be considered in adult patients with otherwise unexplained chronic kidney disease.

Ectopic Overexpression of Coiled-Coil Domain Containing 110 Delays G2/M Entry in U2-OS Cells.

Coiled-coil domain containing 110 (CCDC110, KM-HN-1) is a protein containing C-terminal coiled-coil domain (CCD) which was previously discovered as a member of the human cancer/testis antigen (CTA). In addition, CCDC110 has both nuclear localization signal sequence and the leucine zipper motif. Although the functional role of CCDC110 has yet to be fully identified, the mRNA expression levels of CCDC110 are known to be highly elevated in various cancer types including testis, implying its relevance to cancer pathogenesis. In this study, we first developed several monoclonal antibody (mAb) hybridoma clones targeting CCDC110 and further isolated clone by characterizing for its specificity using immunoblotting and immunoprecipitation approaches with basal parenchymal sperm cells in testis tissue. Next, using these mAbs, we showed that the Tet-inducible overexpression of CCDC110 protein delayed the entry of G2/M phase in U2-OS osteosarcoma cells. Based on these results, we propose that CCDC110 plays a crucial role in cell cycle progression.