Biology subjects

Sinning, I.

Publications and source records attributed to Sinning, I..

10 recordsLinked to original sources

bioRxiv2025

Secretion and signaling properties of Wnt11 and Wnt3 are determined by their N-terminus

Wnt proteins are evolutionarily conserved signaling molecules that control cell-cell communication in health and disease. While different Wnt proteins share a requirement for palmitoleoylation and the cargo receptor Evi/Wls for their secretion, they interact with diverse signaling receptors and co-receptors to elicit distinct outcomes in signal-receiving cells. The molecular mechanisms underlying this contrast between conserved post-translational modification and secretion and diverse receptor interactions remain poorly understood. Here, we demonstrate that Wnt11 maintains partial secretion capability in the absence of Evi/Wls. Unexpectedly, the secreted protein is also non-palmitoleoylated, yet retains its ability to activate downstream signaling in receiving cells through the Frizzled 6 receptor. We identify the N-terminus as the mediator of this unusual behavior, as Wnt3, which strictly requires Evi/Wls, becomes Evi/Wls-independent when its N-terminus is replaced with that of Wnt11. In different cell lines and in Xenopus laevis embryos, the chimeric Wnt11Nterm-Wnt3 protein recapitulates known Wnt11 phenotypes, demonstrating that the N-terminal portion of Wnt11 is sufficient to determine signaling specificity. Our findings reveal novel determinants of Wnt protein specificity and provide mechanistic insights into the molecular basis of Wnt secretion and downstream signaling.

cell biology↗

bioRxiv2025

DNA-encoded Library Screening Uncovers Potent DNMT2 Inhibitors Targeting a Cryptic Allosteric Binding Site

The human RNA methyltransferase DNMT2 is thought to be involved in various pathophysiological processes, yet, a major challenge in drug targeting DNMT2 is given by the fact that current SAH-derived inhibitors have poor target selectivity and limited cellular permeability. In this study, we have performed a DNA-encoded library (DEL) screening on DNMT2 yielding five non-SAH-like hit structures, three of which feature a peptidomimetic scaffold. All DEL hits could be validated by orthogonal biophysical and biochemical assays for DNMT2 binding. At the same time, the lead structure did not interact with related methyltransferases from the DNMT and NSUN families highlighting an unmatched DNMT2-targeting selectivity profile. Subsequent crystallographic studies revealed the unique ligand binding mode including an active site loop rearrangement and the formation of a cryptic allosteric binding pocket able to modulate the enzymatic activity by non-covalent DNMT2 dimerization. Based on the crystallographic results, we performed a structure-activity relationship study around the inhibitor lead structure resulting in an optimized DNMT2 inhibitor (KD=3.04 {micro}M), which was able to reduce m5C levels in MOLM-13 tRNA. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/632061v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1bf92b3org.highwire.dtl.DTLVardef@38498borg.highwire.dtl.DTLVardef@1645740org.highwire.dtl.DTLVardef@9c61d5_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Structures of aberrant spliceosome intermediates on their way to disassembly

Intron removal during pre-mRNA splicing is of extraordinary complexity and its disruption causes a vast number of genetic diseases in humans1. While key steps of the canonical spliceosome cycle have been revealed by combined structure-function analyses2,3, structural information on an aberrant spliceosome committed to premature disassembly is not available. Here, we report two cryo-EM structures of post-Bact spliceosome intermediates from S. pombe primed for disassembly. We identify the DEAH-box helicase - G patch protein pair (Gih35-Gpl1, homologous to human DHX35-GPATCH1) and show how it maintains catalytic dormancy. In both structures, Gpl1 recognizes a remodeled active site introduced by an over-stabilization of the U5 loop I interaction with the 5 exon leading to a single nucleotide insertion at the 5splice site. Remodeling is communicated to the spliceosome surface and the Ntr1 complex that mediates disassembly is recruited. Our data pave the way for a targeted analysis of splicing quality control.

biochemistry↗

bioRxiv2024

Multi-protein assemblies orchestrate co-translational enzymatic processing on the human ribosome

Most nascent chains undergo rapid co-translational enzymatic processing as soon as their N-terminus becomes accessible at the ribosomal polypeptide tunnel exit (PTE). In eukaryotes, N-terminal methionine excision (NME) by Methionine Aminopeptidases (MAP1 and MAP2), and N-terminal acetylation (NTA) by N-Acetyl-Transferase A (NatA), is the most common set of subsequent modifications carried out on the ribosome. How these two enzymatic processes are coordinated in quick succession in the context of a rapidly translating ribosome has remained elusive. Here, we report that human NatA occupies a non-intrusive distal binding site on the ribosome which does not interfere with most other ribosome associated factors (RAFs). In this position, NatA can partake in a coordinated and dynamic assembly with MAP1 through a complex scaffolding function of the abundant Nascent Polypeptide Associated Complex (NAC). Alternatively, MAP2 can co-occupy the PTE with NatA in preparation for successive NME and NTA. In contrast to MAP1, MAP2 completely covers the PTE and is thus incompatible with NAC and MAP1 recruitment. Both assemblies can compile on the human ribosome independent of nascent chain substrates. Together, our structures provide the structural framework for the rapid coordinated orchestration of NME and NTA in protein biogenesis.

biochemistry↗

bioRxiv2023

The Listeria monocytogenes persistence factor ClpL is a potent stand-alone disaggregase

Heat stress can cause cell death by triggering the aggregation of essential proteins. In bacteria, aggregated proteins are rescued by the canonical Hsp70/AAA+ (ClpB) bi-chaperone disaggregase. Man-made, severe stress conditions applied during e.g. food-processing represent a novel threat for bacteria by exceeding the capacity of the Hsp70/ClpB system. Here, we report on the potent autonomous AAA+ disaggregase ClpL from Listeria monocytogenes that provides enhanced heat resistance to the food-borne pathogen enabling persistence in adverse environments. ClpL shows increased thermal stability and enhanced disaggregation power compared to Hsp70/ClpB, enabling it to withstand severe heat stress and to solubilize tight aggregates. ClpL binds to protein aggregates via aromatic residues present in its N-terminal domain (NTD) that adopts a partially folded and dynamic conformation. Target specificity is achieved by simultaneous interactions of multiple NTDs with the aggregate surface. ClpL shows remarkable structural plasticity by forming diverse higher assembly states through interacting ClpL rings. NTDs become largely sequestered upon ClpL ring interactions. Stabilizing ring assemblies by engineered disulfide bonds strongly reduces disaggregation activity, suggesting that they represent storage states.

biochemistry↗

bioRxiv2022

Trim-Away degrades lysine-less substrates without requiring ligase autoubiquitination

TRIM proteins are the largest family of E3 ligases in mammals. They include the intracellular antibody receptor TRIM21, which is responsible for mediating targeted protein degradation during Trim-Away. Despite their importance, the ubiquitination mechanism of TRIM ligases has remained elusive. Here we show that while TRIM21 activation results in ubiquitination of both ligase and substrate, autoubiquitination is regulatory and not required for substrate degradation. Substrate binding stimulates N-terminal RING autoubiquitination by the E2 Ube2W, but when inhibited by N-terminal acetylation this prevents neither substrate ubiquitination nor degradation and has no impact on TRIM21 antiviral activity. Instead, uncoupling ligase and substrate degradation prevents ligase recycling and extends functional persistence in cells. Substrate ubiquitination mediates degradation but Trim-Away efficiently degrades lysine-less substrates, suggesting a non-canonical ubiquitination mechanism explains its broad substrate specificity.

molecular biology↗

bioRxiv2021

DEAD-box RNA Helicases Act as Nucleotide Exchange Factors for Casein Kinase 2

DDX RNA helicases promote RNA processing but DDX3X is also known to activate casein kinase 1 {varepsilon} (CK1{varepsilon}). Here we show that not only is protein kinase stimulation a latent property of other DDX proteins towards CK1{varepsilon}, but that this extends to casein kinase 2 (CK22) as well. CK22 enzymatic activity is stimulated by a variety of DDX proteins and we identify DDX1/24/41/54 as physiological activators required for full kinase activity in vitro and in Xenopus embryos. Mutational analysis of DDX3X reveals that CK1 and CK2 kinase stimulation engages its RNA binding-but not catalytic motifs. Mathematical modelling of enzyme kinetics and stopped-flow spectroscopy converge that DDX proteins function as nucleotide exchange factor towards CK22 that reduce unproductive reaction intermediates and substrate inhibition. Our study reveals protein kinase stimulation by nucleotide exchange as a new principle in kinase regulation and an evolved function of DDX proteins.

biochemistry↗

bioRxiv2021

The inner nuclear membrane protein Lem2 coordinates RNA degradation at the nuclear periphery

Transcriptionally silent chromatin often localizes to the nuclear periphery. However, whether the nuclear envelope (NE) is a site for post-transcriptional gene repression is unknown. Here we demonstrate that S. pombe Lem2, an NE protein, regulates nuclear exosome-mediated RNA degradation. Lem2 deletion causes accumulation of non-coding RNAs and meiotic transcripts. Indeed, an engineered exosome substrate RNA shows Lem2-dependent localization to the nuclear periphery. Lem2 does not directly bind RNA, but instead physically interacts with the exosome-targeting MTREC complex and promotes RNA recruitment. The Lem2-assisted pathway acts independently of nuclear bodies where exosome factors assemble, revealing that multiple spatially distinct degradation pathways exist. The Lem2 pathway is environmentally responsive: nutrient availability modulates Lem2 regulation of meiotic transcripts. Our data indicate that Lem2 recruits exosome co-factors to the nuclear periphery to coordinate RNA surveillance and regulates transcripts during the mitosis-to-meiosis switch.

molecular biology↗

bioRxiv2020

tructural basis for the complex DNA binding behavior of the plant stem cell regulator WUSCHEL

Stem cells are one of the foundational evolutionary novelties that allowed the independent emergence of multicellularity in the plant and animal lineages. In plants, the homeodomain (HD) transcription factor WUSCHEL (WUS) is essential for the maintenance of stem cells in the shoot apical meristem. WUS has been reported to bind to diverse DNA motifs and to act as transcriptional activator and repressor. However, the mechanisms underlying this remarkable behavior have remained unclear. Here, we quantitatively delineate WUS binding to three divergent DNA motifs and resolve the relevant structural underpinnings. We show that WUS exhibits a strong binding preference for TGAA repeat sequences, while retaining the ability to weakly bind to TAAT elements. This behavior is attributable the formation of dimers through interactions of specific residues in the HD that stabilize WUS DNA interaction. Our results provide a mechanistic basis for dissecting WUS dependent regulatory networks in plant stem cell control.

plant biology↗

bioRxiv2019

Complexin suppresses spontaneous exocytosis by capturing the membrane-proximal regions of VAMP2 and SNAP25

The neuronal protein complexin contains multiple domains that exert both clamping and facilitatory functions to tune spontaneous and action potential triggered synaptic release. We address the clamping mechanism and show that the accessory helix of complexin arrests the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that forms the core machinery of intracellular membrane fusion. In a reconstituted fusion assay, site- and stage-specific photo-cross-linking reveals that prior to fusion the complexin accessory helix laterally binds the membrane-proximal C-terminal ends of SNAP25 and VAMP2. Corresponding complexin interface mutants selectively increase spontaneous release of neurotransmitter in living neurons, implying that the accessory helix suppresses final zippering/assembly of the SNARE four-helix bundle by restraining VAMP2 and SNAP25.

neuroscience↗