Biology subjects

Kreienkamp, H.-J.

Publications and source records attributed to Kreienkamp, H.-J..

7 recordsLinked to original sources

bioRxiv2026

DHX30 is a replication stress-responsive R-loop helicase that safeguards genome stability

R-loops are three-stranded nucleic acid structures formed primarily during transcription that can obstruct replication fork progression, leading to replication stress and genome instability. Although RNA helicases are increasingly implicated in R-loop metabolism, how individual helicases safeguard genome integrity remains incompletely understood. Here we identify DHX30, a neurodevelopmental disorder-associated DExH-box RNA helicase, as a replication stress-responsive regulator of R-loops. We show that DHX30 localizes to the nucleus in addition to its known cytoplasmic and mitochondrial pools, and that its nuclear abundance in-creases following aphidicolin or camptothecin treatment. DHX30 deficiency results in RNA:DNA hybrid accumulation in human cells and zebrafish, R-loop-dependent DNA double-strand breaks, micronucleus formation, reduced DNA synthesis, and altered expression of immediate-early stress genes under basal conditions and following replication perturbation. Biochemically, DHX30 binds branched nucleic acid structures and directly unwinds R-loop substrates containing a 3' single-stranded RNA overhang in an ATP- and polarity-dependent manner. Proteomic analyses place DHX30 within a nuclear network of RNA- and R-loop-associated factors, including the RNA helicase DHX9. These findings establish DHX30 as a conserved R-loop-resolving helicase that protects from R-loop-induced replication perturbations, contributes to transcriptional homeostasis, and maintains genome stability, thereby providing a mechanistic framework for understanding DHX30-associated neurodevelopmental disorders.

molecular biology↗

bioRxiv2026

An internal PDZ-binding motif in Densin-180 promotes activity-dependent SHANK scaffold remodelling

SHANK proteins form core postsynaptic density (PSD) scaffolds that organise synaptic signalling complexes through multiple protein-protein interaction domains, including PDZ domains that typically recognise C-terminal peptide motifs. Here, we identify an internal PDZ recognition mechanism that links the synaptic protein Densin-180 to SHANK and promotes SHANK scaffold assembly. We map SHANK binding to an internal PDZ-binding motif in Densin-180 (residues 843-863) and show by NMR spectroscopy and fluorescence polarisation that this motif binds SHANK1-3 PDZ domains with high affinity and specificity, competing with canonical C-terminal ligands. The crystal structure of the Densin-180-SHANK complex reveals that Phe858 inserts into the hydrophobic pocket of the PDZ domain despite the absence of a terminal carboxylate. In neurons, this interaction mediates Densin-180 recruitment to dendritic spines and drives activity-dependent reorganisation of postsynaptic SHANK3 assemblies, whereas mutation of Phe858 disrupts both processes. Disrupting the Densin-180-SHANK interaction using a Densin-180-derived peptide impairs activity-dependent structural plasticity of spines, accompanied by defects in PSD organisation and actin cytoskeleton remodelling. These findings define an internal mode of PDZ recognition and reveal how Densin-180 couples neuronal activity to the remodelling of postsynaptic SHANK scaffolds.

neuroscience↗

bioRxiv2023

The Shank/ProSAP N-terminal (SPN) domain of Shank3 regulates targeting to postsynaptic sites and postsynaptic signalling.

Members of the Shank family of postsynaptic scaffold proteins (Shank1-3) link neurotransmitter receptors to the actin cytoskeleton in dendritic spines through establishing numerous interactions within the postsynaptic density (PSD) of excitatory synapses. Large Shank isoforms carry at their N-termini a highly conserved domain termed the Shank/ProSAP N-terminal (SPN) domain, followed by a set of ankyrin repeats. Both domains are involved in an intramolecular interaction which is believed to regulate accessibility for additional interaction partners, such as Ras family G-proteins, CaMKII and cytoskeletal proteins. Here we analyse the functional relevance of the SPN-Ank module; we show that binding of active Ras or Rap1a to the SPN domain can differentially regulate the localization of Shank3 in dendrites. In Shank1 and Shank3, the linker between the SPN and Ank domains binds to inactive CaMKII. Due to this interaction, both Shank1 and Shank3 exert a negative effect on CaMKII activity at postsynaptic sites in mice in vivo. The relevance of the SPN-Ank intramolecular interaction was further analysed in primary cultured neurons; here we observed that in the context of full-length Shank3, a closed conformation of the SPN-Ank tandem is necessary for proper clustering of Shank3 on the head of dendritic spines. Shank3 variants carrying Ank repeats which are not associated with the SPN domain lead to the atypical formation of postsynaptic clusters on dendritic shafts, at the expense of dendritic spines. Our data show that the SPN-Ank tandem motif contributes to the regulation of postsynaptic signalling, and is also necessary for proper targeting of Shank3 to postsynaptic sites. Our data also suggest how missense variants found in autistic patients which alter SPN and Ank domains affect the synaptic function of Shank3.

neuroscience↗

bioRxiv2022

Regulation of Liprin-α phase separation by CASK is disrupted by a mutation in its CaM kinase domain

CASK is a unique membrane associated guanylate kinase (MAGUK), due to its Ca2+/calmodulin-dependent kinase (CaMK) domain. We describe four male patients with a severe neurodevelopmental disorder with microcephaly carrying missense variants affecting the CaMK domain. One boy who carried the p.E115K variant and died at an early age showed pontocerebellar hypoplasia (PCH) in addition to microcephaly, thus exhibiting the classical MICPCH phenotype observed in individuals with CASK loss-of-function variants. All four variants selectively weaken the interaction of CASK with Liprin-2, a component of the presynaptic active zone. Liprin- proteins form spherical condensates in a process termed liquid-liquid phase separation (LLPS), which we observe here in Liprin-2 overexpressing HEK293T cells and primary cultured neurons. Condensate formation is reversed by interaction of Liprin-2 with CASK; this is associated with altered phosphorylation of Liprin-2. The p.E115K variant fails to interfere with condensate formation. As the individual carrying this variant had the severe MICPCH disorder, we suggest that regulation of Liprin-2-mediated LLPS is a new functional feature of CASK which must be maintained to prevent PCH.

genetics↗

bioRxiv2021

Mutations affecting the N-terminal domains of SHANK3 point to different pathomechanisms in neurodevelopmental disorders.

Shank proteins are major scaffolds of the postsynaptic density of excitatory synapses. Mutations in SHANK genes are associated with autism and intellectual disability. The relevance of missense mutations for these pathologies is unclear. Several missense mutations in SHANK3 affect the N-terminal region, consisting of the Shank/ProSAP N-terminal (SPN) domain and a set of Ankyrin (Ank) repeats. Here we identify a novel SHANK3 missense mutation (p.L270M) in the Ankyrin repeats in patients with an ADHD-like phenotype. We functionally analysed this and a series of other mutations, using biochemical and biophysical techniques. We observe two major effects: (i) a loss of binding to {delta}-catenin (e.g. in the p.L270M variant), and (ii) interference with the intramolecular interaction between N-terminal SPN domain and the Ank repeats. This also interferes with binding to the -subunit of the calcium-/calmodulin dependent kinase II (CaMKII), and appears to be associated with a more severe neurodevelopmental pathology.

neuroscience↗

bioRxiv2021

Autism associated SHANK3 missense point mutations impact conformational fluctuations and protein turnover at synapses

Members of the SH3- and ankyrin-rich repeat (SHANK) protein family are considered as master scaffolds of the post-synaptic density of glutamatergic synapses. Several missense mutations within the canonical SHANK3 isoform have been proposed as causative for the development of autism spectrum disorders (ASDs). However, there is a surprising paucity of data linking missense mutation-induced changes in protein structure and dynamics to the occurrence of ASD-related synaptic phenotypes. In this work, we focus on two ASD-associated point mutations, both located within the same domain of SHANK3. In a proof-of-principle study we demonstrate that both mutant proteins show indeed distinct changes in secondary and tertiary structure as well as higher conformational fluctuations. Local and surprisingly also distal structural disturbances of protein folding result in altered synaptic targeting and changes of protein turnover at synaptic sites.

neuroscience↗

bioRxiv2020

Conformational dynamics regulate SHANK3 actin and Rap1 binding

Actin-rich cellular protrusions direct versatile biological processes from cancer cell invasion to dendritic spine development. The stability, morphology and specific biological function of these protrusions are regulated by crosstalk between three main signaling axes: integrins, actin regulators and small GTPases. SHANK3 is a multifunctional scaffold protein, interacting with several actin-binding proteins, and a well-established autism risk gene. Recently, SHANK3 was demonstrated to sequester integrin-activating small GTPases Rap1 and R-Ras to inhibit integrin activity via its N-terminal SPN domain. Here, we demonstrate that SHANK3 interacts directly with actin using its SPN domain. Actin binding can be inhibited by an intramolecular closed conformation of SHANK3, where the adjacent ARR domain covers the actin-binding interface of the SPN domain. Actin and Rap1 compete with each other for binding to SHANK3 and loss of SHANK3-actin binding augments inhibition of Rap1-mediated integrin activity. This dynamic crosstalk has functional implications for filopodia formation in cancer cells, dendritic spine morphology in neurons and autism-linked phenotypes in vivo.

cell biology↗