Biology subjects

Smolka, M. B.

Publications and source records attributed to Smolka, M. B..

31 records · Page 2Linked to original sources

bioRxiv2021

Fork Slowing and Reversal as an Adaptive Response to Chronic ATR Inhibition

Inhibitors of the replication stress response kinase ATR are currently being explored in anti-cancer therapy. Acute ATR inhibition is known to impair the proper control of origin firing, DNA repair, and cell cycle, resulting in DNA breaks and mitotic catastrophe. Less is understood about the effects of clinically relevant regimes of ATR inhibition, which involve chronic and low doses of ATR inhibitors (cATRi) to cells. Here we report unexpected molecular effects of cATRi on replication dynamics. cATRi strongly reduces fork speed but has minimal effects on the accumulation of DNA breaks or cell survival. cATRi promotes extensive fork reversal and RAD51- and PARP-mediated fork slowing that correlate with the accumulation of DNA-RNA hybrids. Our work shows that fork reversal is a critical adaptive response ensuring cell survival during cATRi and that the manipulation of fork reversal causes hypersensitivity to cATRi, increasing the effectiveness of ATR inhibitors in anti-cancer therapies.

cancer biology↗

bioRxiv2021

Phosphoproteomics of ATR Signaling in Prophase I of Mouse Meiosis

During mammalian meiosis, the ATR kinase plays crucial roles in the coordination of DNA repair, meiotic sex chromosome inactivation and checkpoint signaling. Despite the importance of ATR in meiosis, the meiotic ATR signaling network remains largely unknown. Here we defined ATR signaling during prophase I in mice. Quantitative analysis of phosphoproteomes obtained after genetic ablation of the ATR-activating 9-1-1 complex or chemical inhibition of ATR revealed over 12,000 phosphorylation sites, of which 863 phosphorylation sites were dependent on both 9-1-1 and ATR. ATR and 9-1-1-dependent signaling was enriched for S/T-Q and S/T-X-X-K motifs and included proteins involved in DNA damage signaling, DNA repair, and piRNA and mRNA metabolism. We find that ATR targets the RNA processing factors SETX and RANBP3 and regulate their localization to the sex body. Overall, our analysis establishes a comprehensive map of ATR signaling in spermatocytes and highlights potential meiotic-specific actions of ATR during prophase I.

developmental biology↗

bioRxiv2021

Multiple 9-1-1 complexes promote homolog synapsis, DSB repair, and ATR signaling during mammalian meiosis

DNA damage response mechanisms have meiotic roles that ensure successful gamete formation. While completion of meiotic double-strand break (DSB) repair requires the canonical RAD9A-RAD1-HUS1 (9A-1-1) complex, mammalian meiocytes also express RAD9A and HUS1 paralogs, RAD9B and HUS1B, predicted to form alternative 9-1-1 complexes. The RAD1 subunit is shared by all predicted 9-1-1 complexes and localizes to meiotic chromosomes even in the absence of HUS1 and RAD9A. Here we report that testis-specific RAD1 disruption resulted in impaired DSB repair, germ cell depletion and infertility. Unlike Hus1 or Rad9a disruption, Rad1 loss also caused defects in homolog synapsis, ATR signaling and meiotic sex chromosome inactivation. Comprehensive testis phosphoproteomics revealed that RAD1 and ATR coordinately regulate numerous proteins involved in DSB repair, meiotic silencing, synaptonemal complex formation, and cohesion. Together, these results establish critical roles for both canonical and alternative 9-1-1 complexes in meiotic ATR activation and successful prophase I completion.

genetics↗

bioRxiv2020

Structure and mechanism of TRAPPIII-mediated Rab1 activation

The GTPase Rab1 is a master regulator of both the early secretory pathway and autophagy. Rab1 activation is controlled by its GEF (guanine nucleotide exchange factor), the multi-subunit TRAPPIII complex. The Trs85 regulatory subunit is critical for robust activation of Rab1 but its mechanistic role within the complex has remained unclear. Here we report the cryo-EM structure of the intact yeast TRAPPIII complex bound to its substrate Rab1/Ypt1. The orientation of the Rab1/Ypt1 hypervariable domain when bound to the complex leads to a model for how TRAPPIII associates with and activates Rab1/Ypt1 at the membrane surface. We identify a conserved amphipathic -helix motif within Trs85 and demonstrate that this helix is required for stable membrane binding and Rab1/Ypt1 activation by TRAPPIII. Taken together, our results provide a comprehensive analysis of the structure and function of the yeast TRAPPIII complex and reveal that the key function of Trs85 is to serve as a membrane anchor, via its amphipathic helix, for the entire TRAPPIII complex.

cell biology↗

bioRxiv2020

Phosphoproteomics Reveals a Distinct Mode of Mec1/ATR Signaling in Response to DNA End Hyper-Resection

The Mec1/ATR kinase is crucial for genome maintenance in response to a range of genotoxic insults, although how it promotes context-dependent signaling and DNA repair remains elusive. Here we uncovered a specialized mode of Mec1/ATR signaling triggered by the extensive nucleolytic processing (resection) of DNA ends. Cells lacking RAD9, a checkpoint activator and an inhibitor of resection, exhibit a selective increase in Mec1-dependent phosphorylation of proteins associated with single strand DNA transactions, including the ssDNA binding protein Rfa2, the translocase/ubiquitin ligase Uls1 and the HR-regulatory Sgs1-Top3-Rmi1 (STR) complex. Extensive Mec1-dependent phosphorylation of the STR complex, mostly on the Sgs1 helicase subunit, promotes an interaction between STR and the DNA repair scaffolding protein Dpb11. Fusion of Sgs1 to phosphopeptide-binding domains of Dpb11 strongly impairs HR-mediated repair, supporting a model whereby Mec1 signaling regulates STR upon hyper-resection to influence recombination outcomes. Overall, the identification of a distinct mode of Mec1 signaling triggered by hyper-resection highlights the multi-faceted action of this kinase in the coordination of checkpoint signaling and HR-mediated DNA repair.

molecular biology↗

bioRxiv2020

Maximizing Quantitative Phosphoproteomics of Kinase Signaling Expands the Mec1 and Tel1 Networks

Global phosphoproteome analysis is crucial for comprehensive and unbiased investigation of kinase-mediated signaling. However, since each phosphopeptide represents a unique entity for defining identity, site-localization, and quantitative changes, phosphoproteomics often suffers from lack of redundancy and statistical power for generating high confidence datasets. Here we developed a phosphoproteomic approach in which data consistency among experiments using reciprocal stable isotope labeling defines a central filtering rule for achieving reliability in phosphopeptide identification and quantitation. We find that most experimental error or biological variation in phosphopeptide quantitation does not revert in quantitation once light and heavy media are swapped between two experimental conditions. Exclusion of non-reverting data-points from the dataset not only reduces quantitation error and variation, but also drastically reduces false positive identifications. Application of our approach in combination with extensive fractionation of phosphopeptides by HILIC identifies new substrates of the Mec1 and Tel1 kinases, expanding our understanding of the DNA damage signaling network regulated by these kinases. Overall, the proposed quantitative phosphoproteomic approach should be generally applicable for investigating kinase signaling networks with high confidence and depth.

molecular biology↗

bioRxiv2020

ATR Inhibitors as Potent Modulators of DNA End Resection Capacity

DNA end resection is a key step in homologous recombination-mediated DNA repair. The ability to manipulate resection capacity is expected to be a powerful strategy to rationally modulate DNA repair outcomes in cancer cells and induce selective cell lethality. However, clinically compatible strategies to manipulate resection are not yet well established. Here we find that long-term inhibition of the ATR kinase has a drastic effect on DNA end resection. Inhibition of ATR over multiple cell division cycles depletes the pool of pro-resection factors and prevents RAD51 as well as RAD52-mediated DNA repair, leading to toxic end-joining and hypersensitivity to PARP inhibitors. The effect is markedly distinct from acute ATR inhibition, which blocks RAD51-mediated repair but not resection and RAD52-mediated repair. Our findings reveal a key pro-resection function for ATR and define how ATR inhibitors can be used for effective manipulation of DNA end resection capacity and DNA repair outcomes in cancer cells.

cancer biology↗

bioRxiv2019

Deubiquitination of phosphoribosyl-ubiquitin conjugates by PDE domain-containing Legionella effectors

Posttranslational protein modification by ubiquitin (Ub) is a central eukaryotic mechanism that regulates a plethora of physiological processes. Recent studies unveiled an unconventional type of ubiquitination mediated by the SidE family of Legionella pneumophila effectors, such as SdeA, that catalyzes the conjugation of Ub to a serine residue of target proteins via a phosphoribosyl linker (hence named PR-ubiquitination). Comparable to the deubiquitinases (DUBs) in the canonical ubiquitination pathway, here we show that two Legionella effectors, named DupA (deubiquitinase for PR-ubiquitination) and DupB, reverse PR-ubiquitination by specific removal of phosphoribosyl-Ub (PR-Ub) from substrates. Both DupA and DupB are fully capable of rescuing the Golgi fragmentation phenotype caused by exogenous expression of SdeA in mammalian cells. We further show that deletion of these two genes results in significant accumulation of PR-ubiquitinated species in host cells infected with Legionella. In addition, we have identified a list of specific PR-ubiquitinated host targets and show that DupA and DupB play a role in modulating the association of PR-ubiquitinated host targets with Legionella containing vacuoles (LCV). Together, our data establish a complete PR-ubiquitination and deubiquitination cycle and demonstrate the intricate control that Legionella has over this unusual Ub-dependent posttranslational modification.\n\nStatement of significanceUbiquitination is a vital posttranslational modification in eukaryotes. A variety of microbial pathogens exploit this pathway during their infection. Legionella pneumophila, the causative bacterial pathogen of Legionnaires disease, has been show to hijack host ubiquitination pathway via a large number of effectors. Recent studies revealed a family of effectors catalyzing a novel type of Ub-dependent posttranslational modification, namely PR-ubiquitination. Here we report two new players, DupA and DupB, involved in this unconventional pathway. We found that DupA and DupB function as PR-Ub specific DUBs and play a role in regulating the PR-ubiquitination levels of host targets. Our results not only provide an expanding view of the PR-ubiquitination pathway, but may also facilitate the future identification of PR-ubiquitination pathways in eukaryotes.

biochemistry↗

bioRxiv2019

Protein polyglutamylation catalyzed by the bacterial Calmodulin-dependent pseudokinase SidJ

Pseudokinases are considered to be the inactive counterparts of conventional protein kinases and comprise approximately 10% of the human and mouse kinomes. Here we report the crystal structure of the Legionella pneumophila effector protein, SidJ, in complex with the eukaryotic Ca2+-binding regulator, Calmodulin (CaM). The structure reveals that SidJ contains a protein kinase-like fold domain, which retains a majority of the characteristic kinase catalytic motifs. However, SidJ fails to demonstrate kinase activity. Instead, mass spectrometry and in vitro biochemical analysis demonstrate that SidJ modifies another Legionella effector SdeA, an unconventional phosphoribosyl ubiquitin ligase, by adding glutamate molecules to a specific residue of SdeA in a CaM-dependent manner. Furthermore, we show that SidJ-mediated polyglutamylation suppresses the ADP-ribosylation activity. Our work further implies that some pseudokinases may possess ATP-dependent activities other than conventional phosphorylation.

biochemistry↗

bioRxiv2019

In-depth and 3-Dimensional Exploration of the Budding Yeast Phosphoproteome

Phosphorylation is one of the most dynamic and widespread post-translational modifications regulating virtually every aspect of eukaryotic cell biology. Here we present a comprehensive phosphoproteomic dataset for budding yeast, comprised of over 30,000 high confidence phosphorylation sites identified by mass spectrometry. This single dataset nearly doubles the size of the known phosphoproteome in budding yeast and defines a set of cell cycle-regulated phosphorylation events. With the goal of enhancing the identification of functional phosphorylation events, we performed computational positioning of phosphorylation sites on available 3D protein structures and systematically identified events predicted to regulate protein complex architecture. Results reveal a large number of phosphorylation sites mapping to or near protein interaction interfaces, many of which result in steric or electrostatic \"clashes\" predicted to disrupt the interaction. Phosphorylation site mutants experimentally validate our predictions and support a role for phosphorylation in negatively regulating protein-protein interactions. With the advancement of Cryo-EM and the increasing number of available structures, our approach should help drive the functional and spatial exploration of the phosphoproteome.

biochemistry↗

bioRxiv2019

The yeast Art1 arrestin domain contains disordered insertions that regulate its localization and activity

The protein composition of the plasma membrane is rapidly remodeled in response to changes in nutrient availability or cellular stress. This occurs, in part, through the selective ubiquitylation and endocytosis of plasma membrane proteins which, in the yeast Saccharomyces cerevisiae, is mediated by the HECT E3 ubiquitin ligase Rsp5 and arrestin-related trafficking (ART) adaptors. Here, we provide evidence that an ART family member, Art1, consists of an arrestin fold with extended N- and C-terminal tails, and interspersed with loop insertions. These loop and tail regions, while not strictly required for Art1 function, regulate its activity through two separate mechanisms. One loop mediates Art1 cargo specificity. Other loops are subjected to phosphorylation in a manner dependent on the Pho85 cyclins Clg1 and Pho80. Phosphorylation of the loops controls Art1s localization to the plasma membrane, which promotes cargo ubiquitylation and endocytosis, demonstrating a mechanism through which Art1 activity is regulated.

cell biology↗

bioRxiv2019

Progranulin deficiency leads to reduced glucocerebrosidase activity

Mutation in the GRN gene, encoding the progranulin (PGRN) protein, shows a dose-dependent disease correlation, wherein haploinsufficiency results in frontotemporal lobar degeneration (FTLD) and complete loss results in neuronal ceroid lipofuscinosis (NCL). Although the exact function of PGRN is unknown, it has been increasingly implicated in lysosomal physiology. Here we report that PGRN interacts with the lysosomal enzyme, glucocerebrosidase (GBA), and is essential for proper GBA activity. GBA activity is significantly reduced in tissue lysates from PGRN-deficient mice. This is further evidence that reduced lysosomal hydrolase activity may be a pathological mechanism in cases of GRN-related FTLD and NCL.

neuroscience↗

bioRxiv2019

MaXLinker: proteome-wide cross-link identifications with high specificity and sensitivity

Protein-protein interactions play a vital role in nearly all cellular functions. Hence, understanding their interaction patterns and three-dimensional structural conformations can provide crucial insights about various biological processes and underlying molecular mechanisms for many disease phenotypes. Cross-linking mass spectrometry has the unique capability to detect protein-protein interactions at a large scale along with spatial constraints between interaction partners. However, the current cross-link search algorithms follow an "MS2-centric" approach and, as a result, suffer from a high rate of mis-identified cross-links (~15%). We address this urgent problem, by designing a novel "MS3-centric" approach for cross-link identification and implemented it as a search engine called MaXLinker. MaXLinker significantly outperforms the current state of the art search engine with up to 18-fold lower false positive rate. Additionally, MaXLinker results in up to 31% more cross-links, demonstrating its superior sensitivity and specificity. Moreover, we performed proteome-wide cross-linking mass spectrometry using K562 cells. Employing MaXLinker, we unveiled the most comprehensive set of 9,319 unique cross-links at 1% false discovery rate, comprising 8,051 intraprotein and 1,268 interprotein cross-links. Finally, we experimentally validated the quality of a large number of novel interactions identified in our study, providing a conclusive evidence for MaXLinkers robust performance.

systems biology↗