Biology subjects

Loehr, J.

Publications and source records attributed to Loehr, J..

8 recordsLinked to original sources

bioRxiv2024

The combination of optogenetic-induced protein aggregation and proximity biotinylation assays strongly implicates endolysosomal proteins in the early stages of α-synuclein aggregation

Alpha-synuclein (-syn) aggregation is a defining feature of Parkinsons disease (PD) and related synucleinopathies. Despite significant research efforts focused on understanding -syn aggregation mechanisms, the early stages of this process remain elusive, largely due to limitations in experimental tools that lack the temporal resolution to capture these dynamic events. Here, we introduce UltraID-LIPA, an innovative platform that combines the Light-Inducible Protein Aggregation (LIPA) system with the UltraID proximity-dependent biotinylation assay to identify -syn-interacting proteins and uncover key mechanisms driving its oligomerization. UltraID-LIPA successfully identified 38 -syn-interacting proteins, including both established and novel candidates, highlighting the accuracy and robustness of the approach. Notably, a strong interaction with endolysosomal and membrane-associated proteins was observed, supporting the hypothesis that interactions with membrane-bound organelles are pivotal in the early stages of -syn aggregation. This powerful platform provides new insights into dynamic protein aggregation events, enhancing our understanding of synucleinopathies and other proteinopathies.

neuroscience↗

bioRxiv2024

Coupling proximity biotinylation with genomic targeting to characterize locus-specific changes in chromatin environments

Regulating gene expression involves significant and frequent changes in the chromatin environment at the locus level, especially at regulatory sequences. However, their modulation in response to pharmacological treatments or pathological conditions remain mostly undetermined. Here, we report versatile locus-specific proteomics tools to address this knowledge gap, which combine the targeting ability of the CRISPR/Cas9 system and the protein-labelling capability of the highly reactive biotin ligases TurboID (in CasTurbo) and UltraID (in CasUltra). CasTurbo and CasUltra enabled rapid chromatin protein labelling under mild conditions at repetitive sequences like centromeres and telomeres, as well as non-amplified genes. We applied CasUltra to A375 melanoma cell lines to decipher the protein environment of the MYC promoter and characterize the molecular effects of the bromodomain inhibitor JQ1, which targets bromodomain and extra-terminal (BET) proteins that regulate MYC expression. We quantified the consequences of BET protein displacement from the MYC promoter and found that it was associated with a considerable reorganisation of the chromatin composition. In addition, BET protein retention at the MYC promoter was consistent with a model of increased JQ1 resistance. Thus, through the combination of proximity biotinylation and CRISPR-Cas9-dependent genomic targeting, CasTurbo and CasUltra have successfully demonstrated their utility in profiling the proteome associated with a genomic locus in living cells. In BriefKougnassoukou Tchara et al. report the development and application of CasTurbo and CasUltra, two locus-specific proteomics tools that fuse catalytically dead Cas9 to the engineered biotin ligases TurboID and UltraID. These tools enabled the quantitative mapping of locus-specific chromatin remodelling due to pharmacological inhibition. HighlightsO_LICasTurbo and CasUltra were developed for locus-specific label-free proteomics C_LIO_LICasTurbo mapped the proteins localized to the centromeres and telomeres C_LIO_LIProteins bound to the MYC promoter were quantified in melanoma cells with CasUltra C_LIO_LICasUltra is compatible with investigating pharmacological treatment effects C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=191 SRC="FIGDIR/small/605321v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1f05800org.highwire.dtl.DTLVardef@21754dorg.highwire.dtl.DTLVardef@9c4cc4org.highwire.dtl.DTLVardef@17412a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Systematic profiling of the acetyl lysine machinery reveals a role for MAPKAPK2 in bromodomain inhibitor resistance

Bromodomain (BRD)-containing proteins are chemically tractable multi-domain scaffolding molecules involved in acetyl lysine (Kac) signaling. BRD inhibitors have shown promise in clinical oncology, including melanomas; however, their narrow therapeutic windows and issues with resistance in pre-clinical models highlight the need to better understand the functions of and interconnection between BRD-containing proteins. Here, we use complementary interaction-mapping techniques (affinity purification and proximity-dependent biotinylation) to map the interactions of 39 of the 42 BRD-containing proteins and 110 additional proteins that physically or functionally associate with them. We uncover 3,892 novel interactions and reveal the intricate connectivity of the Kac machinery. Chemical inhibition of multiple BRD classes revealed that inhibiting BETs--but not mSWI/SNF or CREBBP/EP300 proteins--dramatically rewired the interactome. Finally, we identified MAPKAPK2 activity as a critical determinant of BET inhibitor sensitivity in melanoma through its impact on chromatin composition remodeling. In BriefKougnassoukou Tchara et al. generate a static protein interaction map of the human acetyl lysine machinery by coupling two complementary functional proteomics approaches (FLAG affinity purification and proximity-dependent biotinylation) to mass spectrometry. They also investigate network changes upon bromodomain inhibition, and describe a novel resistance mechanism mediated by the p38 stress signaling pathway that causes significant metabolic changes. HighlightsO_LITwo complementary interaction proteomics analyses of the human acetyl lysine machinery were performed. C_LIO_LINovel target- and compound-specific impacts of bromodomain inhibitors were identified. C_LIO_LIMAPKAPK2 was identified as a novel resistance gene to BET bromodomain inhibitors in melanoma. C_LIO_LIBET bromodomain inhibition leads to metabolic adaptation in melanoma. C_LI

cancer biology↗

bioRxiv2024

Novel, postnatal manifestation of an epidermal barrier defect in a mouse model of isolated sulfite oxidase deficiency

Sulfite oxidase deficiency is a rare inborn error in metabolism leading to early childhood death due to rapidly progressing neurodegeneration. A new mouse model of sulfite oxidase deficiency carrying a homozygous deletion in the Suox gene resembles the human pathology in terms of neonatal death and elevation of sulfite and thiosulfate in plasma and urine, respectively. Homozygous Suox-/- mice are initially born healthy, display growth retardation starting at postnatal day 4 and die in average at day 9.6. Here we report that Suox-/- mice develop dry and scaly skin early postnatally, showing that sulfite oxidase is essential to maintain a functional skin barrier after birth. At postnatal day 5 Suox-/- mice develop altered epidermal morphology and dysregulated early and late keratinocyte differentiation accompanied by increased stress response. We propose a sulfite-induced cleavage of disulfide bonds in key epidermal proteins essential for a functional barrier.

biochemistry↗

bioRxiv2021

Recurrent chromosomal translocations in sarcomas create a mega-complex that mislocalizes NuA4/TIP60 to Polycomb target loci

Chromosomal translocations frequently promote carcinogenesis by producing gain-of-function fusion proteins. Recent studies have identified highly recurrent chromosomal translocations in patients with Endometrial Stromal Sarcomas (ESS) and Ossifying FibroMyxoid Tumors (OFMT) leading to an in-frame fusion of PHF1 (PCL1) to six different subunits of the NuA4/TIP60 complex. While NuA4/TIP60 is a co-activator that acetylates chromatin and loads the H2A.Z histone variant, PHF1 is part of the Polycomb repressive complex 2 (PRC2) linked to transcriptional repression of key developmental genes through methylation of histone H3 on lysine 27. In this study, we characterize the fusion protein produced by the EPC1-PHF1 translocation. The chimeric protein assembles a mega-complex harboring both NuA4/TIP60 and PRC2 activities and leads to mislocalization of chromatin marks in the genome, in particular over an entire topologically- associating domain including part of the HOXD cluster. This is linked to aberrant gene expression, most notably increased expression of PRC2 target genes. Furthermore, we show that JAZF1, implicated with a PRC2 component in the most frequent translocation in ESS, JAZF1-SUZ12, is a potent transcription activator that physically associates with NuA4/TIP60, its fusion creating similar outcomes as EPC1-PHF1. Importantly, the specific increased expression of PRC2 targets/HOX genes was also confirmed with ESS patient samples. Altogether, these results indicate that most chromosomal translocations linked to these sarcomas employ the same molecular oncogenic mechanism through a physical merge of NuA4/TIP60 and PRC2 complexes leading to mislocalization of histone marks and aberrant polycomb target gene expression.

molecular biology↗

bioRxiv2019

JMJD6 participates in the maintenance of ribosomal DNA integrity in response to DNA damage

Ribosomal DNA (rDNA) is the most transcribed genomic region and contains hundreds of tandem repeats. Maintaining these rDNA repeats as well as the level of rDNA transcription is essential for cellular homeostasis. DNA damages generated in rDNA need to be efficiently and accurately repaired as rDNA repeats instability has been reported in cancer, aging and neurological diseases. Here, we describe that the histone demethylase JMJD6 is rapidly recruited at nucleolar DNA damage and is crucial for the relocalisation of rDNA in nucleolar caps. Yet, JMJD6 is dispensable for rDNA transcription inhibition. Mass spectrometry study revealed that JMJD6 interacts with the nucleolar protein Treacle and modulates its interaction with NBS1. Moreover, cells deficient for JMJD6 show increased sensitivity to nucleolar DNA damage as well as loss and rearrangements of rDNA repeats upon irradiation. Altogether our data reveals that rDNA transcription inhibition is uncoupled from rDNA relocalisation into nucleolar caps and that JMJD6 is required for rDNA stability upon the rDNA damage response through its role in nucleolar caps formation.\n\nAuthor summaryRibosomal DNA is the most transcribed genomic region composed of repeated sequences. Transcribed rDNA is essential for cellular homeostasis and cell proliferation. Numerous pathologies such as cancer and neurological disorders are described to present defective rDNA repeats maintenance. The mechanisms involved in the control of rDNA integrity involve major DNA repair pathways such as NonHomologous End-Joining and Homologous Recombination. However, how they are controlled and orchestrated is poorly understood. Here, we identified JMJD6 as a new member of the maintenance of rDNA integrity. We observed that JMJD6 controls the recruitment of NBS1 in the nucleolus in order to lead to the proper response to DNA damage at rDNA repeats.\n\nAuthor contributionsYC and DT wrote the manuscript with input from their coauthors. JF, CC, JH, S-KM, JL, J-PL and YC performed the experiments. JC supervised JF and JH during purification and mass spectrometry analysis. YC and DT conceived the project, designed research and coordinated the studies.

cell biology↗

bioRxiv2018

Versatile in vivo genome editing with the prototypical Streptococcus thermophilus CRISPR1-Cas9 system

Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes.

molecular biology↗

bioRxiv2017

A marker-free co-selection strategy for high efficiency human genome engineering

Targeted genome editing using engineered nucleases facilitates the creation of bona fide cellular models for biological research and may be applied to human cell-based therapies. Broadly applicable and versatile methods for increasing the levels of gene editing in cell populations remain highly desirable due to the variable efficiency between distinct genomic loci and cell types. Harnessing the multiplexing capabilities of CRISPR-Cas9 and Cpf1 systems, we designed a simple and robust co-selection strategy for enriching cells harboring either nuclease-driven non-homologous end joining (NHEJ) or homology-directed repair (HDR) events. Selection for dominant alleles of the endogenous sodium-potassium pump (Na+,K+-ATPase) that render cells resistant to ouabain is used to enrich for custom modifications at another unlinked locus of interest, effectively increasing the recovery of engineered cells. The process was readily adaptable to transformed and primary cells, including hematopoietic stem and progenitor cells (HSPCs). The use of universal CRISPR reagents and a commercially available small molecule inhibitor streamlines the incorporation of marker-free genetic changes in human cells.

molecular biology↗