Biology subjects

Rudolph, J.

Publications and source records attributed to Rudolph, J..

18 recordsLinked to original sources

bioRxiv2026

Peroxisome quality control is gated by a dynamic ATF6α-ABCD3 tether

The endoplasmic reticulum (ER) coordinates protein folding and lipid biosynthesis, while peroxisomes govern fatty-acid catabolism and redox balance. However, little is known about the mechanisms by which these organelles communicate. Here we show that activating transcription factor 6 (ATF6), a cytoprotective ER-resident sensor of the unfolded protein response (UPR), forms a molecular tether that zippers the ER and peroxisomes together at a 23-nm intermembrane distance. ATF6 achieves this by inserting a short segment of its unstructured N-terminal cytosolic tail into the solvated transmembrane cavity of the peroxisomal ABC-type lipid transporter ABCD3, trapping it in an off-state. This interaction is reinforced by Ceapin, a previously identified small molecule molecular-glue ligand, which promotes ER-peroxisome contact formation. The junctions serve as a scaffold that can initiate peroxisome turnover by p62-triggered autophagy (pexophagy), as enhanced lipid processing resulting from organelle zippering leads to peroxisome damage due to excessive reactive oxygen species (ROS) accumulation. These findings define ATF6 as a non-transcriptional regulator of organelle quality control and provide a template for engineering Ceapin analogues against ATF6/ABCD3-dependent colorectal and hepatocellular cancers and related diseases.

cell biology↗

bioRxiv2025

Rational design of potent small molecule SMARCA2/A4 (BRM/BRG1) degraders acting via the recruitment of FBXO22

Target-anchored monovalent degraders are more drug-like than their bivalent counterparts, Proteolysis Targeting Chimeras (PROTACs), while offering greater target specificity control than the E3 ligase-anchored monovalent degraders, also known as molecular glues. However, their discovery has typically been serendipitous, and the rules governing their identification remain unclear. This study focused on the intentional discovery of SMARCA2/A4 monovalent degraders using a library based on SMARCA2/A4 bromodomain-binding ligands. Compound G-6599 emerged as a lead candidate, showing exceptional degradation potency and specificity for SMARCA2/A4. Mechanistic studies revealed that G-6599 operates through the ubiquitin-proteasome pathway and the E3 ligase FBXO22. G-6599 was shown to promote ternary complex formation between SMARCA2 and FBXO22 involving covalent conjugation to a cysteine residue on the latter. Unlike other recently identified FBXO22-dependent degraders, it does not require biotransformation. The selective degradation ability of G-6599, along with its unique mechanism, highlights the therapeutic potential of target-anchored monovalent degraders.

cancer biology↗

bioRxiv2023

A nonenzymatic dependency on inositol-requiring enzyme 1 controls cancer cell cycle progression and tumor growth

Endoplasmic-reticulum resident inositol-requiring enzyme 1 (IRE1) supports protein homeostasis via a cytoplasmic kinase-RNase module. Known cancer dependency on IRE1 entails its enzymatic activation of the transcription factor XBP1s and of RNA decay. We discovered that some cancer cells require IRE1 but not its enzymatic activity. IRE1 knockdown, but not enzymatic inhibition or XBP1 disruption, increased DNA damage and chromosome instability while engaging the TP53 pathway and cyclin-dependent kinase inhibitors and attenuating cell cycle progression. IRE1 depletion downregulated factors involved in chromosome replication and segregation and in chromatin remodeling. Immunoelectron microscopy indicated that endogenous IRE1 can localize to the nuclear envelope. Thus, cancer cells can require IRE1 either enzymatically or nonenzymatically, with significant implications for IRE1s biological role and therapeutic targeting.

cancer biology↗

bioRxiv2023

Targeting the Hippo pathway in cancers via ubiquitination dependent TEAD degradation

The Hippo pathway is among the most frequently altered key signaling pathways in cancer. TEAD1-4 are essential transcription factors and key downstream effectors in the Hippo pathway in human cells. Here, we identified RNF146 as a ubiquitin ligase (E3) of TEADs, which negatively regulates their stability in cells through proteasome-mediated degradation. We show that RNF146-mediated TEAD ubiquitination is dependent on the TEAD PARylation state. We further validated the genetic interaction between RNF146 and the Hippo pathway in cancer cell lines and the model organism Drosophila melanogaster. Despite the RNF146 and proteasome-mediated degradation mechanisms, TEADs are stable proteins with a long half-life in cells. We demonstrate that degradation of TEADs can be greatly enhanced pharmacologically with heterobifunctional chemical inducers of protein degradation (CIDEs). These TEAD-CIDEs can effectively suppress activation of YAP/TAZ target genes in a dose-dependent manner and exhibit significant anti-proliferative effects in YAP/TAZ-dependent tumor cells, thus phenocopying the effect of genetic ablation of TEAD protein. Collectively, this study demonstrates that the ubiquitin-proteasome system plays an important role in regulating TEAD functions and provides a proof-of-concept demonstration that pharmacologically induced TEAD ubiquitination could be leveraged to target YAP/TAZ-driven cancers.

cancer biology↗

bioRxiv2022

Plan versus motion-referenced generalization of fast and slow processes in reach adaptation

Generalization in motor learning refers to the transfer of a learned compensation to other relevant contexts. The generalization function is typically assumed to be of Gaussian shape, centered on the planned motion, although more recent studies associate generalization with the actual motion. Because motor learning is thought to involve multiple adaptive processes with different time constants, we hypothesized that these processes have different time-dependent contributions to the generalization. Guided by a model-based approach, the objective of the present study was to experimentally examine these contributions. We first reformulated a validated two-state adaptation model as a combination of weighted motor primitives, each specified as a Gaussian-shaped tuning function. Adaptation in this model is achieved by updating individual weights of the primitives of the fast and slow adaptive process separately. Depending on whether updating occurred in a plan-referenced or a motion-referenced manner, the model predicted distinct contributions to the overall generalization by the slow and fast process. We tested 23 participants in a reach adaptation task, using a spontaneous recovery paradigm consisting of five successive blocks of a long adaptation phase to a viscous force field, a short adaptation phase with the opposite force, and an error-clamp phase. Generalization was assessed in eleven movement directions relative to the trained target direction. Results of our participant population fell within a continuum of evidence for plan-referenced to evidence for motion-referenced updating. This mixture may reflect the differential weighting of explicit and implicit compensation strategies among participants.

neuroscience↗

bioRxiv2022

Architecture and Assembly Mechanism of Human LACTB

Mitochondria are complex organelles that play a central role in metabolism. Dynamic membrane-associated processes regulate mitochondrial morphology and bioenergetics in response to cellular demand. In tumor cells, metabolic reprogramming requires active mitochondrial metabolism for providing key metabolites and building blocks for tumor growth and rapid proliferation. To counter this, the mitochondrial serine beta-lactamase-like protein (LACTB) alters mitochondrial lipid metabolism and potently inhibits the proliferation of a variety of tumor cells. Mammalian LACTB is localized in the mitochondrial intermembrane space, where it assembles into filaments to regulate the efficiency of essential metabolic processes. However, the structural basis of LACTB polymerization and regulation remains incompletely understood. Here, we describe how human LACTB self-assembles into micron-scale filaments that increase their catalytic activity. The electron cryo-microscopy (cryoEM) structure defines the mechanism of assembly and reveals how highly ordered filament bundles stabilize the active state of the enzyme. We identify and characterize residues that are located at the filament-forming interface, and further show that mutations that disrupt filamentation reduce enzyme activity. Furthermore, our results provide evidence that LACTB filaments can bind lipid membranes. These data reveal the detailed molecular organization and polymerization-based regulation of human LACTB and provide new insights into the mechanism of mitochondrial membrane organization that modulates lipid metabolism.

biophysics↗

bioRxiv2022

Dynamics of endogenous PARP1 and PARP2 during DNA damage revealed by live-cell single-molecule imaging

Summary/AbstractPARP1 contributes to genome architecture and DNA damage repair through its dynamic association with chromatin. PARP1 and PARP2 (PARP1/2) recognize damaged DNA and recruit the DNA repair machinery. Using single molecule microscopy in live cells, we monitored the movement of PARP1/2 on undamaged and damaged chromatin. We identify two classes of freely diffusing PARP1/2 and two classes of bound PARP1/2. The majority (> 60%) of PARP1/2 diffuse freely in both undamaged and damaged nuclei and in the presence of inhibitors of PARP1/2 used for cancer therapy (PARPi). Laser induced DNA damage results in a small fraction of slowly diffusing PARP1 and PARP2 to become transiently bound. Treatment of cells with PARPi in the presence of DNA damage causes subtle changes in the dynamics of bound PARP1/2, in contrast to bulk studies that suggest PARP trapping. Our results imply that next-generation PARPi could specifically target the small fraction of DNA-bound PARP1/2.

biophysics↗

bioRxiv2021

Noncanonical mRNA decay by the endoplasmic-reticulum stress sensor IRE1α promotes cancer-cell survival

Eukaryotic IRE1 mitigates endoplasmic-reticulum (ER) stress by orchestrating the unfolded-protein response (UPR). IRE1 spans the ER membrane, and signals through a cytosolic kinase-endoribonuclease module. The endoribonuclease generates the transcription factor XBP1s by intron excision between similar RNA stem-loop endomotifs, and depletes select cellular mRNAs through regulated IRE1-dependent decay (RIDD). Paradoxically, mammalian RIDD seemingly targets only mRNAs with XBP1-like endomotifs, while in flies RIDD exhibits little sequence restriction. By comparing nascent and total IRE1-controlled mRNAs in human breast cancer cells, we discovered not only canonical endomotif-containing RIDD substrates, but also many targets lacking recognizable motifs--degraded by a process we coin RIDDLE, for RIDD lacking endomotif. IRE1 displayed two basic endoribonuclease modalities: endomotif-specific cleavage, minimally requiring dimers; and endomotif-independent promiscuous processing, requiring phospho-oligomers. An oligomer-deficient mutant that did not support RIDDLE failed to rescue cancer-cell viability. These results link IRE1 oligomers, RIDDLE, and cell survival, advancing mechanistic understanding of the UPR.

cancer biology↗

bioRxiv2021

Automated Modeling of Protein Accumulation at DNA Damage Sites using qFADD.py

Cells are exposed to a plethora of influences that can cause damage to DNA and alter the genome, often with detrimental consequences for health. Cells mitigate this damage through a variety of repair protein pathways, and accurate measurement of the accumulation, action, and dissipation timescales of these repair proteins is required to fully understand the DNA damage response. Recently, we described the Q-FADD (Quantitation of Fluorescence Accumulation after DNA Damage) method, which enhances the analytical power of the widely used laser microirradiation technique. In that study, Q-FADD and its preprocessing operations required licensed software and a significant amount of user overhead to find the model of best fit. Here, we present "qFADD.py", an open-source implementation of the Q-FADD algorithm that is available as both a stand-alone software package and on a publicly accessible webserver (https://qfadd.colorado.edu/). Furthermore, we describe significant improvements to the fitting and preprocessing methods that include corrections for nuclear drift and an automated grid-search for the model of best fit. To improve statistical rigor, the grid-search algorithm also includes automated simulation of replicates. As an example, we discuss the recruitment dynamics of the signaling protein PARP1 to DNA damage sites, and we show how to compare different populations of qFADD.py models. Statement of SignificanceCells are constantly bombarded by factors that can alter or damage their genome, and they have evolved a variety of proteins that can identify and fix this damage. To fully understand how these proteins interact in repair pathways, we need robust methods to quantify the timescales between the initial identification of the DNA damage event and the subsequent protein-protein interactions that lead to repair. Laser microirradiation is a popular method for studying these repair protein cascades in vivo, and methods for quantifying the timescales of recruitment in these experiments have historically been simple to implement but lacking in physical interpretation. Here, we present qFADD.py, the next iteration of the Q-FADD method, which uses Monte Carlo diffusion models to interpret repair protein recruitment timescales to sites of DNA damage. By moving towards automated fitting procedures with minimal bias from the user, qFADD.py provides a statistically robust but low-effort means to analyze laser microirradiation experiments through a biophysical framework.

biophysics↗

bioRxiv2020

Single Cell atlas of uterine myometrium and leiomyomas reveals diverse and novel cell types of non-monoclonal origin.

Uterine leiomyomas are the most common tumors of the female reproductive tract with significant morbidity that includes excessive bleeding, infertility and pregnancy complications. The origin and cellular composition of leiomyomas is controversial, yet very important in better understanding the pathogenesis of these tumors. We applied single-cell RNA sequencing to better understand cellular heterogeneity of uterine leiomyomas and normal myometrium at the molecular level. Our data reveal previously unknown heterogeneity in the smooth muscle cells, fibroblast cells, and endothelial cells of normal myometrium and leiomyomas. We discovered a novel lymphatic endothelial cell population in uterine leiomyomas and that the immune as well as transcriptional profile of leiomyomas is MED12 genotype-dependent. Moreover, we show that leiomyoma cell moiety is not monoclonal in nature. Our work describes unprecedented single cell resolution of normal uterine myometrium and leiomyoma tumors and provides insight into tumor specific hormone responsiveness and extracellular matrix accumulation.

genetics↗

bioRxiv2020

HPF1 and nucleosomes mediate a dramatic switch in activity of PARP1 from polymerase to hydrolase

Poly(ADP-ribose) polymerase 1 (PARP1) is an important player in the response to DNA damage. Recently, histone PARylation factor (HPF1) was shown to be a critical modulator of the activity of PARP1 by facilitating PARylation of histones and redirecting the target amino acid specificity from acidic to serine residues. Here we investigate the mechanism and specific consequences of HPF1-mediated PARylation using nucleosomes as both activators and substrates for PARP1. HPF1 provides that catalytic base Glu284 to substantially redirect PARylation by PARP1 such that the histones in nucleosomes become the primary recipients of PAR chains. Surprisingly, HPF1 partitions most of the reaction product to free ADPR, resulting in much shorter PAR chains compared to reactions in the absence of HPF1. This HPF1-mediated switch from polymerase to hydrolase has important implications for the PARP1-mediated response to DNA damage and raises interesting new questions about the role of intracellular ADPR and depletion of NAD+.

biochemistry↗

bioRxiv2020

Female reproductive tract has low concentration of SARS-CoV2 receptors

There has been significant concern regarding fertility and reproductive outcomes during the SARS-CoV2 pandemic. Recent data suggests a high concentration of SARS-Cov2 receptors, ACE2 or TMPRSS2, in nasal epithelium and cornea, which explains person-to-person transmission. We investigated the prevalence of SARS-CoV2 receptors among reproductive tissues by exploring the single-cell sequencing datasets from uterus, myometrium, ovary, fallopian tube, and breast epithelium. We did not detect significant expression of either ACE2 or TMPRSS2 in the normal human myometrium, uterus, ovaries, fallopian tube, or breast. Furthermore, none of the cell types in the female reproductive organs we investigated, showed the co-expression of ACE2 with proteases, TMPRSS2, Cathepsin B (CTSB), and Cathepsin L (CTSL) known to facilitate the entry of SARS2-CoV2 into the host cell. These results suggest that myometrium, uterus, ovaries, fallopian tube, and breast are unlikely to be susceptible to infection by SARS-CoV2. Our findings suggest that COVID-19 is unlikely to contribute to pregnancy-related adverse outcomes such as preterm birth, transmission of COVID-19 through breast milk, oogenesis and female fertility.

genetics↗

bioRxiv2019

Bridging of nucleosome-proximal DNA double-strand breaks by PARP2 enhances its interaction with HPF1

Poly(ADP-ribose) Polymerase 2 (PARP2) is one of three DNA-dependent PARPs involved in the detection of DNA damage. Upon binding to DNA double-strand breaks, PARP2 uses nicotinamide adenine dinucleotide to synthesize poly(ADP-ribose) (PAR) onto itself and other proteins, including histones. PAR chains in turn promote the DNA damage response by recruiting downstream repair factors. These early steps of DNA damage signaling are relevant for understanding how genome integrity is maintained and how their failure leads to genome instability or cancer. There is no structural information on DNA double-strand break detection in the context of chromatin. Here we present a cryo-EM structure of two nucleosomes bridged by human PARP2 and confirm that PARP2 bridges DNA ends in the context of nucleosomes bearing short linker DNA. We demonstrate that the conformation of PARP2 bound to damaged chromatin provides a binding platform for the regulatory protein Histone PARylation Factor 1 (HPF1), and that the resulting HPF1*PARP2*nucleosome complex is enzymatically active. Our results contribute to a structural view of the early steps of the DNA damage response in chromatin.

biochemistry↗

bioRxiv2019

Memorability of photographs in subjective cognitive decline and mild cognitive impairment: implications for cognitive assessment

INTRODUCTIONImpaired long-term memory is a defining feature of Mild Cognitive Impairment (MCI). We tested whether this impairment is item-specific, limited to some memoranda whereas some remain consistently memorable.\n\nMETHODSWe conducted item-based analyses of long-term visual recognition memory. 394 participants (healthy controls (HC), Subjective Cognitive Decline (SCD), and MCI) in the multicentric DZNE-Longitudinal Cognitive Impairment and Dementia Study (DELCODE) were tested with images from a pool of 835 photographs.\n\nRESULTSWe observed consistent memorability for images in HCs, SCDs, and MCI, predictable by a neural network trained on another healthy sample. Looking at memorability differences between groups, we identified images that could successfully categorize group membership with higher success and a substantial image reduction than the original image set.\n\nDISCUSSIONIndividuals with SCD and MCI show consistent memorability for specific items, while other items show significant diagnosticity. Certain stimulus features could optimize diagnostic assessment, while others could support memory.

neuroscience↗

bioRxiv2019

Activation loop dynamics are controlled by conformation-selective inhibitors of ERK2

Modulating the dynamics of protein kinases expands the inhibitory mechanisms for small molecules. NMR measurements of the MAP kinase, ERK2, have shown that activation by dual-phosphorylation induces global motions involving exchange between two states, \"L\" and \"R\". We show that ERK inhibitors Vertex-11e and SCH772984 exploit the small energetic difference between L and R to shift the equilibrium in opposing directions, while inhibitor GDC-0994 and ATP analogue AMP-PNP retain L{rightleftharpoons}R exchange. An X-ray structure of active 2P-ERK2 complexed with AMP-PNP reveals a shift in the Gly-rich loop along with domain closure to position the nucleotide in a more catalytically productive conformation relative to inactive 0P-ERK2:ATP. X-ray structures of 2P-ERK2 complexed with Vertex-11e or GDC-0994 recapitulate this closure, which is blocked in a complex with a SCH772984 analogue. Thus, the L[->]R shift in 2P-ERK2 is associated with movements needed to form a competent active site. Solution measurements by hydrogen-exchange mass spectrometry (HX-MS) reveal distinct binding modes for Vertex-11e, GDC-0994 and AMP-PNP to active vs inactive ERK2, where the extent of HX protection matches their degree of R-state formation. In addition, Vertex-11e and SCH772984 show opposite effects on HX near the activation loop, suggesting that L{rightleftharpoons}R exchange involves coupling between the activation loop and the active site. Consequently, these inhibitors differentially affect MAP kinase phosphatase activity towards 2P-ERK2. We conclude that global motions in ERK2 promote productive nucleotide binding, and couple with the activation loop to allow control of dephosphorylation by conformation-selective inhibitors.\n\nSIGNIFICANCE STATEMENTProtein kinases in the RAF/MKK/ERK signaling pathway are dysregulated in cancer and are important targets for inhibitor development. Catalytic activation of the MAP kinase, ERK2, induces global motions involving exchange between two conformational states. Using nuclear magnetic resonance (NMR) and hydrogen-exchange mass spectrometry, we show that inhibitors exploit these motions to trap ERK2 in distinct states. Our findings reveal motions of the activation loop coupled to the active site. Inhibitor binding can control these activation loop dynamics to alter its rate of dephosphorylation by MAP kinase phosphatase.

biochemistry↗

bioRxiv2018

Disruption of IRE1α through its Kinase Domain Attenuates Multiple Myeloma

Multiple myeloma (MM) arises from malignant immunoglobulin-secreting plasma cells and remains an incurable, often lethal disease despite recent therapeutic advances. The unfolded-protein response sensor IRE1 supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may coopt the IRE1-XBP1s pathway; however, the validity of IRE1 as a potential MM therapeutic target is controversial. Here we show that genetic disruption of IRE1 or XBP1s, or pharmacologic IRE1 kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice, and augmented efficacy of two well-established frontline antimyeloma agents, bortezomib or lenalidomide. Mechanistically, IRE1 perturbation inhibited expression of key components of the ER-associated degradation machinery, as well as cytokines and chemokines known to promote MM growth. Selective IRE1 kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138- cells from bone marrow of newly diagnosed MM patients or patients whose disease relapsed after 1 - 4 lines of treatment in both US- and EU-based cohorts. IRE1 inhibition preserved survival and glucose-induced insulin secretion by pancreatic microislets. Together, these results establish a strong therapeutic rationale for targeting IRE1 with kinase-based small-molecule inhibitors in MM.\n\nSignificance statementMultiple myeloma (MM) is a lethal malignancy of plasma cells. MM cells have an expanded endoplasmic reticulum (ER) that is constantly under stress due to immunoglobulin hyperproduction. The ER-resident sensor IRE1 mitigates ER stress by expanding the ERs protein-folding capacity while supporting proteasomal degradation of misfolded ER proteins. IRE1 elaborates these functions by deploying its cytoplasmic kinase-RNase module to activate the transcription factor XBP1s. The validity of IRE1 as a potential therapeutic target in MM has been questioned. Using genetic and pharmacologic disruption in vitro and in vivo, we demonstrate that the IRE1-XBP1s pathway plays a critical role in MM growth. We further show that IRE1s kinase domain is an effective and safe potential small-molecule target for MM therapy.

cancer biology↗

bioRxiv2018

Q-FADD: A mechanistic approach for modeling the accumulation of proteins at sites of DNA damage by free diffusion

The repair of DNA damage requires the ordered recruitment of many different proteins that are responsible for signaling and subsequent repair. A powerful tool for studying the orchestrated accumulation of these proteins at damage sites is laser microirradiation in live cells, followed by monitoring of the accumulation of the fluorescently labeled protein in question. Despite the widespread use of this approach, there exists no rigorous method for characterizing this process quantitatively. Here we introduce a free diffusion model that explicitly accounts for the unique topology of individual nuclei and quantitatively describes the accumulation of two test proteins, poly-ADP-ribose polymerases 1 and 2. Application of our model to other proteins will yield novel insights into the timing and mechanism of DNA repair.

biochemistry↗

bioRxiv2018

Poly(ADP-ribose) polymerase 1 Searches DNA via a ‘Monkey Bar’ Mechanism

Poly(ADP-ribose) polymerase 1 (PARP1) is both a first responder to DNA damage and a chromatin architectural protein. How PARP1 rapidly finds DNA damage sites in the context of a nucleus filled with undamaged DNA, to which it also binds, is an unresolved question. Here we show that PARP1 association with DNA is diffusion-limited, and release of PARP1 from DNA is promoted by binding of an additional DNA molecule that facilitates a \"monkey bar\" mechanism, also known as intersegment transfer. The WGR-domain of PARP1 is essential to this mechanism, and a point mutation (W589A) recapitulates the altered kinetics of the domain deletion. Demonstrating the physiological importance of the monkey bar mechanism for PARP1 function, the W589A mutant accumulates at sites of DNA damage more slowly following laser micro-irradiation than wild-type PARP1. Clinically relevant inhibitors of PARP1 did not alter the rate or mechanism of the release of PARP1 from DNA.

biophysics↗