Biology subjects

Kuper, J.

Publications and source records attributed to Kuper, J..

7 recordsLinked to original sources

bioRxiv2024

Trapped in translocation: Stalling of XPD on a crosslinked DNA substrate

The super family 2 (SF2) helicase XPD is a central component of the general transcription factor II H (TFIIH) which is essential for transcription and nucleotide excision DNA repair (NER)1. Within these two processes XPDs helicase function is vital for NER but not for transcription initiation, where XPD only acts as a scaffold for other factors 2. We deciphered one of the most enigmatic steps in XPD helicase action: the active separation of dsDNA and its stalling upon approaching an interstrand crosslink, one of the most severe DNA damages in the cell, using cryo EM. Furthermore, the structure clearly shows how dsDNA is separated and reveals a highly unusual involvement of the Arch domain in active dsDNA separation. Combined with mutagenesis and biochemical analyses, we identify distinct functional residues important for helicase activity. Surprisingly, those areas also affect core TFIIH translocase activity, revealing a yet unencountered function of XPD within the TFIIH scaffold. Importantly, our structure provides a basis for XPD damage recognition and further suggests how the NER bubble could be formed, leading to a model for the location of the XPG nuclease relative to the excised damage.

biochemistry↗

bioRxiv2020

Constitutively active TrkB kinase signalling reduces actin filopodia dynamics and cell migration

Trk receptors and gene fusions of NTRK are targets in precision oncology. Classical Trk signalling concepts fail to explain ligand-independent signalling of intracellular TrkB or NTRK fusion proteins. Here, we show that abundance of the intracellular domain of TrkB is sufficient for ligand-independent autophosphorylation. This constitutive TrkB signalling reduced actin filopodia dynamics, could phosphorylate FAK, and changed cell morphology. Mutating Y705 in the kinase domain of TrkB alone specifically blocked these pathways. Engineered intracellular kinase domain proteins and a cancer-related intracellular NTRK2-fusion protein (SQSTM1-NTRK2) also underwent constitutive activation. In migrating glioblastoma-like U87MG cells, self-active TrkB kinase reduced cell migration. Moreover, we found evidences for constitutively active, intracellular TrkB in tissue of human grade IV glioblastoma. Structural modelling of the kinase domain let us postulate that release from cis-autoinhibition by abundance is sufficient for TrkB/FAK/Actin signalling via Y705. These constitutive signalling pathways could be fully blocked within minutes by clinically approved, anti-tumorigenic Trk inhibitors. In conclusion, our data provide an explanation and biological function for TrkB kinase domain signalling in the absence of a ligand.

neuroscience↗

bioRxiv2020

How to limit the speed of a motor: The intricate regulation of the XPB ATPase and Translocase in TFIIH

The superfamily 2 helicase XPB is an integral part of the general transcription factor TFIIH and assumes essential catalytic functions in transcription initiation and nucleotide excision repair. In both processes the ATPase activity of XPB is essential. We investigated the interaction network that regulates XPB via the p52 and p8 subunits with functional mutagenesis based on a crystal structure of the full p52/p8 complex and current cryo-EM structures. Importantly, we show that XPBs ATPase can be activated either by DNA or by the interaction with the p52/p8 proteins. Intriguingly, we observe that the ATPase activation by p52/p8 is significantly weaker than the activation by DNA and when both p52/p8 and DNA are present, p52/p8 dominates the maximum activation. We therefore define p52/p8 as the master regulator of XPB that acts as an activator and speed limiter at the same time. A correlative analysis of the ATPase and translocase activities of XPB shows that XPB only acts as a translocase within the context of complete core TFIIH and that XPA increases the processivity of the translocase complex without altering XPBs ATPase activity. Our data unravel an intricate network that tightly controls the activity of XPB during transcription and nucleotide excision repair.

biochemistry↗

bioRxiv2020

Structural basis for the activation of CDK7 by MAT1 and Cyclin H

CDK7, Cyclin H, and MAT1 form the heterotrimeric CDK-activating kinase (CAK) complex. CAK is a vital factor for the two essential processes of transcription and cell cycle control. When associated with the general transcription factor II H (TFIIH) it activates RNA polymerase II by hyperphosphorylation of its C-terminal domain (CTD). In the absence of TFIIH it phosphorylates the T-loop of CDKs that control cell cycle progression. CAK holds a special position among the CDK branch due to this dual activity and the dependence on the MAT1 protein for activation in addition to Cyclin H. We solved the structure of the CAK complex from the model organism C. thermophilum at 2.6 [A] resolution. Our structure reveals an intricate network of interactions between MAT1 and its two binding partners CDK7 and Cyclin H providing a structural basis for the mechanism of CDK7 activation and CAK activity regulation. In vitro activity measurements combined with functional mutagenesis show that CDK7 activation can occur independently of T-loop phosphorylation and is thus exclusively MAT1 dependent by positioning the CDK7 T-loop in its active conformation. Finally, our structure of the active CAK with a peptide model provides a molecular rationale for heptad repeat phosphorylation. Significance StatementThe fundamental processes of cell cycle regulation and transcription are linked by the heterotrimeric CDK-activating kinase (CAK) complex. We have solved the crystal structure of the active CAK complex and provide a molecular rationale for CAK activation, regulation, and substrate recognition thereby highly advancing our understanding of this essential factor.

biochemistry↗

bioRxiv2020

Global discovery of bacterial RNA-binding proteins by RNase-sensitive gradient profiles reports a new FinO domain protein

RNA-binding proteins (RBPs) play important roles in bacterial gene expression and physiology but their true number and functional scope remain little understood even in model microbes. To advance global RBP discovery in bacteria, we here establish glycerol gradient sedimentation with RNase treatment and mass spectrometry (GradR). Applied to Salmonella enterica, GradR confirms many known RBPs such as CsrA, Hfq and ProQ by their RNase-sensitive sedimentation profiles, and discovers the FopA protein as a new member of the emerging family of FinO/ProQ-like RBPs. FopA, encoded on resistance plasmid pCol1B9, primarily targets a small RNA associated with plasmid replication. The target suite of FopA dramatically differs from the related global RBP ProQ, revealing context-dependent selective RNA recognition by FinO-domain RBPs. Numerous other unexpected RNase-induced changes in gradient profiles suggest that cellular RNA helps to organize macromolecular complexes in bacteria. By enabling poly(A)-independent generic RBP discovery, GradR provides an important element in the quest to build a comprehensive catalogue of microbial RBPs.

microbiology↗

bioRxiv2019

The TFIIH components p44/p62 act as a damage sensor during nucleotide excision repair

Nucleotide excision repair (NER) protects the genome following exposure to diverse types of DNA damage, including UV light and chemotherapeutics. Mutations in mammalian NER genes lead to diseases such as xeroderma pigmentosum, trichothiodystrophy, and Cockayne syndrome. In eukaryotes, the major transcription factor TFIIH is the central hub of NER. The core components of TFIIH include the helicases XPB, XPD, and five structural subunits. Two of these structural TFIIH proteins, p44 and p62 remain relatively unstudied; p44 is known to regulate the helicase activity of XPD during NER whereas p62s role is thought to be structural. However, a recent cryo-EM structure shows that p44, p62, and XPD make extensive contacts within TFIIH, with part of p62 occupying XPDs DNA binding site. This observation implies a more extensive role in DNA repair beyond the structural integrity of TFIIH. Here, we show that p44 stimulates XPDs ATPase but upon encountering DNA damage, further stimulation is only observed when p62 is part of the ternary complex; suggesting a role for the p44/p62 heterodimer in TFIIHs mechanism of damage detection. Using single molecule imaging, we demonstrate that p44/p62 independently interacts with DNA; it is seen to diffuse, however, in the presence of UV-induced DNA lesions the complex stalls. Combined with the analysis of a recent cryo-EM structure we suggest that p44/p62 acts as a novel DNA-binding entity within TFIIH that is capable of recognizing DNA damage. This revises our understanding of TFIIH and prompts more extensive investigation into the core subunits for an active role during both DNA repair and transcription.

biochemistry↗

bioRxiv2018

Fgf3 is crucial for the generation of monoaminergic cerebrospinal fluid contacting cells in zebrafish

In most vertebrates, including zebrafish, the hypothalamic serotonergic cerebrospinal fluid-contacting (CSF-c) cells constitute a prominent population. In contrast to the hindbrain serotonergic neurons, little is known about the development and function of these cells. Here, we identify Fibroblast growth factor (Fgf)3 as the main Fgf ligand controlling the ontogeny of serotonergic CSF-c cells. We show that fgf3 positively regulates the number of serotonergic CSF-c cells, as well as a subset of dopaminergic and neuroendocrine cells in the posterior hypothalamus. Further, expression of the ETS-domain transcription factor etv5b is downregulated after fgf3 impairment. Previous findings identified etv5b as critical for the proliferation of serotonergic progenitors in the hypothalamus, and therefore we now suggest that Fgf3 acts via etv5b during early development to ultimately control the number of mature serotonergic CSF-c cells. Moreover, our analysis of the developing hypothalamic transcriptome shows that the expression of fgf3 is upregulated upon fgf3 loss-of-function, suggesting activation of a self-compensatory mechanism. Together, these results highlight Fgf3 in a novel context as part of a signalling pathway of critical importance for hypothalamic development.\n\nSummary statementThis study highlights Fgf3 in a novel context where it is being part of a signalling pathway of critical importance for development of hypothalamic monoaminergic cells in zebrafish.

developmental biology↗