Biology subjects

Seeliger, M.

Publications and source records attributed to Seeliger, M..

7 recordsLinked to original sources

bioRxiv2025

Ocular delivery of different VCP inhibitory formulations prevents retinal degeneration in rhodopsin 255 isoleucine deletion mice

Rhodopsin-mediated autosomal dominant retinitis pigmentosa (RHO-adRP) is a progressive inherited retinal degenerative disorder currently lacking effective treatments. A recurrent 3-base pair deletion in the RHO gene, resulting in the loss of isoleucine at codon 255 or 256 (RHO{Delta}I255 or RHO{Delta}I256), has been identified in patients from the United Kingdom, Germany, Belgium, China, and Korea, suggesting a broad geographic distribution. This mutation leads to rhodopsin (RHO) misfolding, its retention in the endoplasmic reticulum (ER), and aggregation with wild-type (WT) RHO, ultimately triggering ER stress and photoreceptor degeneration. These aggregates are primarily cleared via the ER-associated degradation (ERAD) pathway, with valosin-containing protein (VCP) playing a key role in their retrotranslocation and proteasomal degradation. Pharmacological or genetic inhibition of VCP has shown neuroprotective effects in other models of adRP, but the poor aqueous solubility of VCP inhibitors and challenges in retinal drug delivery hinders clinical translation. To overcome these limitations, we evaluated and compared three VCP-targeted therapeutic strategies in Rho{Delta}I255 knock-in mouse retinae: (1) small-molecule inhibitors (ML240, NMS-873) solubilized in DMSO, (2) ML240 encapsulated in monomethoxy-polyethylene glycol (mPEG)-cholane nanoparticles, and (3) small interfering RNA (siRNA) targeting VCP, delivered via magnetic nanoparticles. Neuroprotective effects were assessed in vitro in retinal explants and in vivo following intravitreal injection. Our findings provide the first evidence that VCP inhibition restores RHO trafficking to the outer segments and prevents photoreceptor cell death in the Rho{Delta}I255 model. Among the three approaches, nanocarrier-encapsulated ML240 exhibited superior efficacy, enabling sustained drug delivery and enhanced photoreceptor protection. These results establish a preclinical proof-of-concept for nanocarrier-mediated VCP inhibition as a promising therapeutic strategy for RHO-adRP and potentially other ER-stress-related retinal degenerations. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC="FIGDIR/small/661245v1_ufig1.gif" ALT="Figure 1"> View larger version (73K): org.highwire.dtl.DTLVardef@12f9366org.highwire.dtl.DTLVardef@76437dorg.highwire.dtl.DTLVardef@48dd6corg.highwire.dtl.DTLVardef@1c11812_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Calculating Protein-Ligand Residence Times Through State Predictive Information Bottleneck based Enhanced Sampling

Understanding drug residence times in target proteins is key to improving drug efficacy and understanding target recognition in biochemistry. While drug residence time is just as important as binding affinity, atomiclevel understanding of drug residence times through molecular dynamics (MD) simulations has been difficult primarily due to the extremely long timescales. Recent advances in rare event sampling have allowed us to reach these timescales, yet predicting protein-ligand residence times remains a significant challenge. Here we present a semi-automated protocol to calculate the ligand residence times across 12 orders of magnitudes of timescales. In our proposed framework, we integrate a deep learning-based method, the state predictive information bottleneck (SPIB), to learn an approximate reaction coordinate (RC) and use it to guide the enhanced sampling method metadynamics. We demonstrate the performance of our algorithm by applying it to six different protein-ligand complexes with available benchmark residence times, including the dissociation of the widely studied anti-cancer drug Imatinib (Gleevec) from both wild-type Abl kinase and drug-resistant mutants. We show how our protocol can recover quantitatively accurate residence times, potentially opening avenues for deeper insights into drug development possibilities and ligand recognition mechanisms. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/589710v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@11dc13borg.highwire.dtl.DTLVardef@79103dorg.highwire.dtl.DTLVardef@194b67org.highwire.dtl.DTLVardef@a570e3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

Extended DNA binding interface beyond the canonical SAP domain contributes to SDE2 function at DNA replication forks

Elevated DNA replication stress causes instability of the DNA replication fork and DNA mutations, which underlies tumorigenesis. The DNA replication stress regulator SDE2 binds to TIMELESS (TIM) of the fork protection complex (FPC) and enhances its stability, thereby supporting replisome activity at DNA replication forks. Here, we structurally and functionally characterize a new conserved DNA binding motif related to SAP (SAF-A/B, Acinus, PIAS) in human SDE2 and establish its preference for single-stranded DNA (ssDNA). The nuclear magnetic resonance solution structure of SDE2SAP reveals a helix-extended loop-helix core aligned parallel to each other, consistent with known canonical SAP folds. Notably, its DNA interaction extends beyond the core SAP domain and is augmented by two lysine residues in the C-terminal tail, which is uniquely positioned adjacent to SAP and conserved in the pre-mRNA splicing factor SF3A3. Mutation in the SAP domain with extended C-terminus not only disrupts ssDNA binding but also impairs TIM localization at replication forks, thus inhibiting efficient fork progression. Together, our study establishes SDE2SAP as an essential element for SDE2 to exert its role in preserving replication fork integrity via FPC regulation and highlights the structural diversity of the DNA-protein interactions achieved by a specialized DNA binding motif.

biochemistry↗

bioRxiv2021

Mural cell SRF controls pericyte migration, vessel patterning and blood flow

RationalePericytes (PCs) and vascular smooth muscle cells (vSMCs), collectively known as mural cells (MCs), are recruited through PDGFB-PDGFRB signaling. MCs are essential for vascular integrity, and their loss has been associated with numerous diseases. Most of this knowledge is based on studies in which MCs are insufficiently recruited or fully absent upon inducible ablation. In contrast, little is known about the physiological consequences that result from impairment of specific MC functions. ObjectiveHere, we characterize the role of the transcription factor serum response factor (SRF) in MCs and study its function in developmental and pathological contexts. Methods and ResultsWe generated a mouse model of MC-specific inducible Srf gene deletion and studied its consequences during retinal angiogenesis. By postnatal day (P)6, PCs lacking SRF were morphologically abnormal and failed to properly co-migrate with angiogenic sprouts. As a consequence, PC-deficient vessels at the retinal sprouting front became dilated and leaky. By P12, also the vSMCs had lost SRF, which coincided with the formation of pathological arteriovenous (AV) shunts. Mechanistically, we show that PDGFB-dependent SRF activation is mediated via MRTF co-factors. We further show that MRTF-SRF signaling promotes pathological PC activation during ischemic retinopathy. RNA-sequencing, immunohistology, in vivo live imaging and in vitro experiments demonstrated that SRF regulates expression of contractile SMC proteins essential to maintain the vascular tone. ConclusionsSRF is crucial for distinct functions in PCs and vSMCs. SRF directs PC migration downstream of PDGFRB signaling and mediates pathological PC activation during ischemic retinopathy. In vSMCs, SRF is essential for expression of the contractile machinery, and its deletion triggers formation of AV shunts. These essential roles in physiological and pathological contexts provide a rational for novel therapeutic approaches through targeting SRF activity in MCs.

developmental biology↗

bioRxiv2021

Protein flexibility and dissociation pathway differentiation can explain onset of resistance mutations in kinases

Understanding how point mutations can render a ligand or a drug ineffective against a given biological target is a problem of immense fundamental and practical relevance. Often the efficacy of such resistance mutations can be explained purely on a thermo-dynamic basis wherein the mutated system displays a reduced binding affinity for the ligand. However, the more perplexing and harder to explain situation is when two protein sequences have the same binding affinity for a drug. In this work, we demonstrate how all-atom molecular dynamics simulations, specifically using recent developments grounded in statistical mechanics and information theory, can provide a detailed mechanistic rationale for such variances. We establish the dissociation mechanism for the popular anti-cancer drug Imatinib (Gleevec) against wild-type and N387S mutant of Abl kinase. We show how this single point mutation triggers a non-local response in the proteins flexibility and eventually leads to pathway differentiation during dissociation. This pathway differentiation explains why Gleevec has a long residence time in the wild-type Abl, but for the mutant, by opening up a backdoor pathway for ligand exit, an order of magnitude shorter residence time is obtained. We thus believe that this work marks an efficient and scalable approach to pinpoint the molecular determinants of resistance mutations in biomolecular receptors of pharmacological relevance that are hard to explain using a simple structural perspective and require mechanistic and kinetic insights. Significance statementRelapse in late-stage cancer patients is often correlated with the onset of drug resistance mutations. Some of these mutations are very far from the binding site and thus hard to explain from a purely structural perspective. Here we employ all-atom molecular dynamics simulations aided by ideas from information theory that can reach timescales of seconds with minimal human bias in how the sampling is enhanced. Through these we explain how a single point mutation triggers a non-local response in the protein kinases flexibility and eventually leads to pathway differentiation during dissociation, thereby significantly reducing the residence time of the drug.

biochemistry↗

bioRxiv2020

Voltage-gated calcium channel subunit α2δ-3 shapes light responses of mouse retinal ganglion cells mainly in low and moderate light levels

Little is known about the function of the auxiliary 2{delta} subunits of voltage-gated calcium channels in the retina. We investigated the role of 2{delta}-3 (Cacna2d3) using a mouse in which 2{delta}-3 was knocked out by LacZ insertion. Behavior experiments indicated a normal optokinetic reflex in 2{delta}-3 knockout animals. Strong expression of 2{delta}-3 could be localized to horizontal cells using the LacZ-reporter, but horizontal cell mosaic and currents carried by horizontal cell voltage-gated calcium channels were unchanged by the 2{delta}-3 knockout. In vivo electroretinography revealed unaffected photoreceptor activity and signal transmission to depolarizing bipolar cells. We recorded visual responses of retinal ganglion cells with multi-electrode arrays in scotopic to photopic luminance levels and found subtle changes in 2{delta}-3 knockout retinas. Spontaneous activity in OFF ganglion cells was elevated in all luminance levels. Differential response strength to high- and low-contrast Gaussian white noise was compressed in ON ganglion cells during mesopic ambient luminance and in OFF ganglion cells during scotopic and mesopic ambient luminances. In a subset of ON ganglion cells, we found a sharp increase in baseline spiking after the presentation of drifting gratings in scotopic luminance. This increase happened after gratings of different spatial properties in knockout compared to wild type retinas. In a subset of ON ganglion cells of the 2{delta}-3 knockout, we found altered delays in rebound-like spiking to full-field contrast steps in scotopic luminance. In conclusion, 2{delta}-3 seems to participate in shaping visual responses mostly within brightness regimes when rods or both rods and cones are active.

neuroscience↗

bioRxiv2019

IRE1β negatively regulates IRE1α signaling in response to endoplasmic reticulum stress

IRE1{beta} is an ER stress sensor uniquely expressed in epithelial cells lining mucosal surfaces. Here, we show that intestinal epithelial cells expressing IRE1{beta} have an attenuated response to ER stress. IRE1{beta} assembles with and blocks activation of the closely related and most evolutionarily ancient stress-sensor IRE1 to suppress stress-induced xbp1 splicing, a key mediator of the unfolded protein response. In comparison, IRE1{beta} has weak xbp1 splicing activity, largely explained by a non-conserved amino acid in the kinase domain that impairs its phosphorylation and restricts oligomerization. This enables IRE1{beta} to act as a dominant negative suppressor of IRE1. The inhibitory effect is amplified in cells by disrupting an XBP1-dependent feedback loop regulating stress-induced expression of IRE1. Thus IRE1{beta} functions to negatively regulate IRE1 signaling, perhaps enabling intestinal epithelial cells to manage the response to chronic stress stimuli at the host-environment interface.

cell biology↗