Exploring the structural landscape of DNA maintenance proteins.

Evolutionary annotation of genome maintenance (GM) proteins has conventionally been established by remote relationships within protein sequence databases. However, often no significant relationship can be established. Highly sensitive approaches to attain remote homologies based on iterative profile-to-profile methods have been developed. Still, these methods have not been systematically applied in the evolutionary annotation of GM proteins. Here, by applying profile-to-profile models, we systematically survey the repertoire of GM proteins from bacteria to man. We identify multiple GM protein candidates and annotate domains in numerous established GM proteins, among other PARP, OB-fold, Macro, TUDOR, SAP, BRCT, KU, MYB (SANT), and nuclease domains. We experimentally validate OB-fold and MIS18 (Yippee) domains in SPIDR and FAM72 protein families, respectively. Our results indicate that, surprisingly, despite the immense interest and long-term research efforts, the repertoire of genome stability caretakers is still not fully appreciated.

Localisation and function of key axonemal microtubule inner proteins and dynein docking complex members reveal extensive diversity among vertebrate motile cilia.

Vertebrate motile cilia are classified as (9+2) or (9+0), based on the presence or absence of the central pair apparatus, respectively. Cryogenic electron microscopy analyses of (9+2) cilia have uncovered an elaborate axonemal protein composition. The extent to which these features are conserved in (9+0) cilia remains unclear. CFAP53, a key axonemal filamentous microtubule inner protein (fMIP) and a centriolar satellites component, is essential for motility of (9+0), but not (9+2) cilia. Here, we show that in (9+2) cilia, CFAP53 functions redundantly with a paralogous fMIP, MNS1. MNS1 localises to ciliary axonemes, and combined loss of both proteins in zebrafish and mice caused severe outer dynein arm loss from (9+2) cilia, significantly affecting their motility. Using immunoprecipitation, we demonstrate that, whereas MNS1 can associate with itself and CFAP53, CFAP53 is unable to self-associate. We also show that additional axonemal dynein-interacting proteins, two outer dynein arm docking (ODAD) complex members, show differential localisation between types of motile cilia. Together, our findings clarify how paralogous fMIPs, CFAP53 and MNS1, function in regulating (9+2) versus (9+0) cilia motility, and further emphasise extensive structural diversity among these organelles.

Uncovering structural themes across cilia microtubule inner proteins with implications for human cilia function.

Centrosomes and cilia are microtubule-based superstructures vital for cell division, signaling, and motility. The once thought hollow lumen of their microtubule core structures was recently found to hold a rich meshwork of microtubule inner proteins (MIPs). To address the outstanding question of how distinct MIPs evolved to recognize microtubule inner surfaces, we applied computational sequence analyses, structure predictions, and experimental validation to uncover evolutionarily conserved microtubule- and MIP-binding modules named NWE, SNYG, and ELLEn, and PYG and GFG-repeat by their signature motifs. These modules intermix with MT-binding DM10-modules and Mn-repeats in 24 Chlamydomonas and 33 human proteins. The modules molecular characteristics provided keys to identify elusive cross-species homologs, hitherto unknown human MIP candidates, and functional properties for seven protein subfamilies, including the microtubule seam-binding NWE and ELLEn families. Our work defines structural innovations that underpin centriole and axoneme assembly and demonstrates that MIPs co-evolved with centrosomes and cilia.

Human RTEL1 associates with Poldip3 to facilitate responses to replication stress and R-loop resolution.

RTEL1 helicase is a component of DNA repair and telomere maintenance machineries. While RTEL1's role in DNA replication is emerging, how RTEL1 preserves genomic stability during replication remains elusive. Here we used a range of proteomic, biochemical, cell, and molecular biology and gene editing approaches to provide further insights into potential role(s) of RTEL1 in DNA replication and genome integrity maintenance. Our results from complementary human cell culture models established that RTEL1 and the Polδ subunit Poldip3 form a complex and are/function mutually dependent in chromatin binding after replication stress. Loss of RTEL1 and Poldip3 leads to marked R-loop accumulation that is confined to sites of active replication, enhances endogenous replication stress, and fuels ensuing genomic instability. The impact of depleting RTEL1 and Poldip3 is epistatic, consistent with our proposed concept of these two proteins operating in a shared pathway involved in DNA replication control under stress conditions. Overall, our data highlight a previously unsuspected role of RTEL1 and Poldip3 in R-loop suppression at genomic regions where transcription and replication intersect, with implications for human diseases including cancer.

IFT20 modulates ciliary PDGFRα signaling by regulating the stability of Cbl E3 ubiquitin ligases.

Primary cilia have pivotal roles as organizers of many different signaling pathways, including platelet-derived growth factor receptor α (PDGFRα) signaling, which, when aberrantly regulated, is associated with developmental disorders, tumorigenesis, and cancer. PDGFRα is up-regulated during ciliogenesis, and ciliary localization of the receptor is required for its appropriate ligand-mediated activation by PDGF-AA. However, the mechanisms regulating sorting of PDGFRα and feedback inhibition of PDGFRα signaling at the cilium are unknown. Here, we provide evidence that intraflagellar transport protein 20 (IFT20) interacts with E3 ubiquitin ligases c-Cbl and Cbl-b and is required for Cbl-mediated ubiquitination and internalization of PDGFRα for feedback inhibition of receptor signaling. In wild-type cells treated with PDGF-AA, c-Cbl becomes enriched in the cilium, and the receptor is subsequently ubiquitinated and internalized. In contrast, in IFT20-depleted cells, PDGFRα localizes aberrantly to the plasma membrane and is overactivated after ligand stimulation because of destabilization and degradation of c-Cbl and Cbl-b.

KIF13B establishes a CAV1-enriched microdomain at the ciliary transition zone to promote Sonic hedgehog signalling.

Ciliary membrane composition is controlled by transition zone (TZ) proteins such as RPGRIP1, RPGRIPL and NPHP4, which are vital for balanced coordination of diverse signalling systems like the Sonic hedgehog (Shh) pathway. Activation of this pathway involves Shh-induced ciliary accumulation of Smoothened (SMO), which is disrupted by disease-causing mutations in TZ components. Here we identify kinesin-3 motor protein KIF13B as a novel member of the RPGRIP1N-C2 domain-containing protein family and show that KIF13B regulates TZ membrane composition and ciliary SMO accumulation. KIF13B is upregulated during ciliogenesis and is recruited to the ciliary base by NPHP4, which binds to two distinct sites in the KIF13B tail region, including an RPGRIP1N-C2 domain. KIF13B and NPHP4 are both essential for establishment of a CAV1 membrane microdomain at the TZ, which in turn is required for Shh-induced ciliary SMO accumulation. Thus KIF13B is a novel regulator of ciliary TZ configuration, membrane composition and Shh signalling.

Ins and outs of GPCR signaling in primary cilia.

Primary cilia are specialized microtubule-based signaling organelles that convey extracellular signals into a cellular response in most vertebrate cell types. The physiological significance of primary cilia is underscored by the fact that defects in assembly or function of these organelles lead to a range of severe diseases and developmental disorders. In most cell types of the human body, signaling by primary cilia involves different G protein-coupled receptors (GPCRs), which transmit specific signals to the cell through G proteins to regulate diverse cellular and physiological events. Here, we provide an overview of GPCR signaling in primary cilia, with main focus on the rhodopsin-like (class A) and the smoothened/frizzled (class F) GPCRs. We describe how such receptors dynamically traffic into and out of the ciliary compartment and how they interact with other classes of ciliary GPCRs, such as class B receptors, to control ciliary function and various physiological and behavioral processes. Finally, we discuss future avenues for developing GPCR-targeted drug strategies for the treatment of ciliopathies.

Identification of conserved, centrosome-targeting ASH domains in TRAPPII complex subunits and TRAPPC8.

BACKGROUND: Assembly of primary cilia relies on vesicular trafficking towards the cilium base and intraflagellar transport (IFT) between the base and distal tip of the cilium. Recent studies have identified several key regulators of these processes, including Rab GTPases such as Rab8 and Rab11, the Rab8 guanine nucleotide exchange factor Rabin8, and the transport protein particle (TRAPP) components TRAPPC3, -C9, and -C10, which physically interact with each other and function together with Bardet Biedl syndrome (BBS) proteins in ciliary membrane biogenesis. However, despite recent advances, the exact molecular mechanisms by which these proteins interact and target to the basal body to promote ciliogenesis are not fully understood. RESULTS: We surveyed the human proteome for novel ASPM, SPD-2, Hydin (ASH) domain-containing proteins. We identified the TRAPP complex subunits TRAPPC8, -9, -10, -11, and -13 as novel ASH domain-containing proteins. In addition to a C-terminal ASH domain region, we predict that the N-terminus of TRAPPC8, -9, -10, and -11, as well as their yeast counterparts, consists of an α-solenoid bearing stretches of multiple tetratricopeptide (TPR) repeats. Immunofluorescence microscopy analysis of cultured mammalian cells revealed that exogenously expressed ASH domains, as well as endogenous TRAPPC8, localize to the centrosome/basal body. Further, depletion of TRAPPC8 impaired ciliogenesis and GFP-Rabin8 centrosome targeting. CONCLUSIONS: Our results suggest that ASH domains confer targeting to the centrosome and cilia, and that TRAPPC8 has cilia-related functions. Further, we propose that the yeast TRAPPII complex and its mammalian counterpart are evolutionarily related to the bacterial periplasmic trafficking chaperone PapD of the usher pili assembly machinery.

A divergent calponin homology (NN-CH) domain defines a novel family: implications for evolution of ciliary IFT complex B proteins.

MOTIVATION: Microtubules are dynamic polymers of tubulin dimers that undergo continuous assembly and disassembly. A mounting number of microtubule-associated proteins (MAPs) regulate the dynamic behavior of microtubules and hence the assembly and disassembly of disparate microtubule structures within the cell. Despite recent advances in identification and functional characterization of MAPs, a substantial number of microtubule accessory factors have not been functionally annotated. Here, using profile-to-profile comparisons and structure modeling, we show that the yeast outer kinetochore components NDC80 and NUF2 share evolutionary ancestry with a novel protein family in mammals comprising, besides NDC80/HEC1 and NUF2, three Intraflagellar Transport (IFT) complex B subunits (IFT81, IFT57, CLUAP1) as well as six proteins with poorly defined function (FAM98A-C, CCDC22, CCDC93 and C14orf166). We show that these proteins consist of a divergent N-terminal calponin homology (CH)-like domain adjoined to an array of C-terminal heptad repeats predicted to form a coiled-coil arrangement. We have named the divergent CH-like domain NN-CH after the founding members NDC80 and NUF2.

HCLK2 is required for activity of the DNA damage response kinase ATR.

ATR is a protein kinase that orchestrates the cellular response to replication problems and DNA damage. HCLK2 has previously been reported to stabilize ATR and Chk1. Here we provide evidence that human HCLK2 acts at an early step in the ATR signaling pathway and contributes to full-scale activation of ATR kinase activity. We show that HCLK2 forms a complex with ATR-ATRIP and the ATR activator TopBP1. We demonstrate that HCLK2-induced ATR kinase activity toward substrates requires TopBP1 and vice versa and provides evidence that HCLK2 facilitates efficient ATR-TopBP1 association. Consistent with its role in ATR activation, HCLK2 depletion severely impaired phosphorylation of multiple ATR targets including Chk1, Nbs1, and Smc1 after DNA damage. We show that HCLK2 is required for and stimulates ATR autophosphorylation and activity toward different substrates in vitro. Furthermore, HCLK2 depletion abrogated the G(2) checkpoint and decreased survival of cells after exposure to DNA damaging agents and replicative stress. Overall, our data suggest that HCLK2 facilitates ATR activation and, therefore, contributes to ATR-mediated checkpoint signaling. Importantly, our results suggest that HCLK2 functions in the same pathway as TopBP1 but that the two proteins regulate different steps in ATR activation.

Does the intracellular ionic concentration or the cell water content (cell volume) determine the activity of TonEBP in NIH3T3 cells?

The transcription factor, tonicity-responsive enhancer binding protein (TonEBP), is involved in the adaptive response against hypertonicity. TonEBP regulates the expression of genes that catalyze the accumulation of osmolytes, and its transcriptional activity is increased by hypertonicity. The goal of the present investigation was to investigate whether cell shrinkage or high intracellular ionic concentration induced the activation of TonEBP. We designed a model system for isotonically shrinking cells over a prolonged period of time. Cells swelled in hypotonic medium and performed a regulatory volume decrease. Upon return to the original isotonic medium, cells shrank initially, followed by a regulatory volume increase. To maintain cell shrinkage, the RVI process was inhibited as follows: ethyl-isopropyl-amiloride inhibited the Na(+)/H(+) antiport, bumetanide inhibited the Na(+)-K(+)-2Cl(-) cotransporter, and gadolinium inhibited shrinkage-activated Na(+) channels. Cells remained shrunken for at least 4 h (isotonically shrunken cells). The activity of TonEBP was investigated with a Luciferase assay after isotonic shrinkage and after shrinkage in a high-NaCl hypertonic medium. We found that TonEBP was strongly activated after 4 and 16 h in cells in high-NaCl hypertonic medium, but not after 4 or 16 h in isotonically shrunken cells. Cells treated with high-NaCl hypertonic medium for 4 h had significantly higher intracellular concentrations of both K(+) and Na(+) than isotonically shrunken cells. This strongly suggested that an increase in intracellular ionic concentration and not cell shrinkage is involved in TonEBP activation.

Early-stage apoptosis is associated with DNA-damage-independent ATM phosphorylation and chromatin decondensation in NIH3T3 fibroblasts.

Chromatin condensation and degradation of DNA into internucleosomal DNA fragments are key hallmarks of apoptosis. The phosphorylation of protein kinase ataxia telangiectasia mutated (ATM) and histone H2A.X was recently shown to occur concurrently with apoptotic DNA fragmentation. We have used immunofluorescence microscopy, Western blot analysis and alkali comet assays to show that phosphorylation of ATM in NIH3T3 fibroblasts occurs prior to apoptotic DNA fragmentation, nuclease degradation and phosphorylation of histone H2A.X in cells treated with low levels of either staurosporine (STS) or tumor necrosis factor-alpha mixed with cycloheximide (TNF-alpha/CHX). In extension to previous findings, ATM phosphorylation was associated with chromatin decondensation, i.e., by loss of dense foci of constitutive heterochromatin. These results suggest that chromatin is decondensed and that ATM is activated independently of DNA damage signaling pathways during the very early stages of apoptosis.