Biology subjects

Kramer, H. B.

Publications and source records attributed to Kramer, H. B..

4 recordsLinked to original sources

bioRxiv2024

Conserved helical motifs in the Ikaros IDR mediate NuRD interaction and transcriptional repression

The transcription factor IKZF1/Ikaros is essential for B cell development, and recurrently mutated in human B-ALL. Ikaros has been ascribed both activating and repressive functions via interactions with coactivator and corepressor complexes, but the relative abundance of Ikaros-associated coregulatory complexes and their contribution to Ikaros-mediated gene regulation are not well understood. To address this issue, we performed an unbiased identification of Ikaros-interacting proteins in pre-B cells, and found that Ikaros interacts overwhelmingly with corepressors and heterochromatin-associated proteins. Time-resolved analysis of transcription and chromatin state identified transcriptional repression as the immediate response to Ikaros induction. Transcriptional repression preceded transcriptional activation by several hours, and was accompanied by a rapid loss of chromatin accessibility and reduced levels of H3K27ac particularly at enhancers. Functional characterisation of intrinsically disordered regions in the Ikaros protein identified highly conserved helical motifs that mediate Ikaros association with the NuRD corepressor complex and contribute to the silencing of target genes in pre-B cells and antiproliferative functions of Ikaros in human B-ALL.

molecular biology↗

bioRxiv2023

Novel protein interaction network of human calcitonin receptor-like receptor revealed by label-free quantitative proteomics

BackgroundG protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) signalling is implicated in skin-related and cardiovascular diseases, migraine and cancer. However, beyond its agonists and receptor activity-modifying proteins (RAMPs), proteins which bind to CLR and define its properties in primary human cells remain insufficiently understood. AimWe aimed to profile the CLR interactome in primary human dermal lymphatic endothelial cells (HDLEC), where this GPCR is expressed. Materials and methodsImmunoprecipitation (IP) of core- and terminally-glycosylated CLR from primary in vitro cultured HDLEC was conducted using rabbit polyclonal anti-human CLR serum (with pre- immune serum serving as a control) and confirmed by immunoblotting. Total HDLEC and co-immunoprecipitated CLR proteomes were analysed by label-free quantitative nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS). Quantitative in-situ proximity ligation assay (PLA) using ZEISS LSM 710 confocal microscope and ZEN Blue 3.0 and Image J software was performed to confirm nLC-MS/MS findings. All experiments were repeated at least three times (biological replicates). For statistical analysis of PLA data, distribution was analysed using Shapiro-Wilk normality test followed by an unpaired t-test or Mann-Whitney test with a p-value of [≤]0.05 interpreted as significant. For MS data of CLR IP samples, statistical analysis was performed using t-test with a permutation-based false discovery rate (FDR)-adjusted p-value of [≤]0.006 interpreted as significant. ResultsA total of 4,902 proteins were identified and quantified by nLC-MS/MS in primary HDLEC and 46 were co-immunoprecipitated with CLR (p<0.006). Direct interaction with the GPCR was confirmed for five of these by PLA (p<0.01). ConclusionsThis is the first study of its kind to identify novel binding partners of CLR expressed in primary human cells. Our integrative quantitative approach, combining immunoprecipitation of core- and terminally-glycosylated CLR, nLC-MS/MS, and PLA, could be applied in a similar fashion to study its interactome in a variety of human cells and tissues, and its contribution to a range of diseases, where the role of this GPCR is implicated.

cell biology↗

bioRxiv2021

Solvent Precipitation SP3 (SP4) enhances recovery for proteomics sample preparation without magnetic beads

Complete, reproducible extraction of protein material is essential for comprehensive and unbiased proteome analyses. A current gold standard is single-pot, solid-phase-enhanced sample preparation (SP3), in which organic solvent and magnetic beads are used to denature and capture proteins, with subsequently washes allowing contaminant removal. However, SP3 is dependent on effective protein immobilisation onto beads, risks losses during wash steps, and experiences a drop-off in protein recovery at higher protein inputs. Magnetic beads may also contaminate samples and instruments, and become costly for larger scale protein preparations. Here, we propose solvent precipitation SP3 (SP4) as an alternative to SP3, omitting magnetic beads and employing brief centrifugation--either with or without low-cost inert glass beads--as the means of aggregated protein capture. SP4 recovered equivalent or greater protein yields for 1-5000 {micro}g preparations and improved reproducibility (median protein R2 0.99 (SP4) vs. 0.97 (SP3)). Deep proteome profiling (n = 9,076) also demonstrated improved recovery by SP4 and a significant enrichment of membrane and low-solubility proteins vs. SP3. The effectiveness of SP4 was verified in three other labs, each confirming equivalent or improved proteome characterisation over SP3. This work suggests that protein precipitation is the primary mechanism of SP3, and reliance on magnetic beads presents protein losses, especially at higher concentrations and amongst hydrophobic proteins. SP4 represents an efficient and effective alternative to SP3, provides the option to omit beads entirely, and offers virtually unlimited scalability of input and volume--all whilst retaining the speed and universality of SP3. BRIEFSolvent precipitation SP3 (SP4) captures aggregated protein for proteomics sample clean-up by omitting magnetic beads, instead employing brief centrifugation--with or without low-cost inert glass beads. SP4 offers improvements to protein yields, higher reproducibility, and greater recovery of membrane proteins, with verifications from three labs. Protein precipitation appears to be the primary mechanism of SP3, with reliance on magnetic beads presenting protein losses, especially at higher concentrations. SP4 presents an effective alternative to SP3 with improved scalability and equal speed and universality. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/461247v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1ba615corg.highwire.dtl.DTLVardef@14df25dorg.highwire.dtl.DTLVardef@62f0c1org.highwire.dtl.DTLVardef@1ac436a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

NGLY1 knockdown or pharmacological inhibition induces cellular autophagy

Pan-caspase inhibitor Z-VAD-fmk acts as an inhibitor of peptide:N-glycanase (NGLY1); an endoglycosidase which cleaves N-linked glycans from glycoproteins exported from the endoplasmic reticulum during ER-associated degradation (ERAD). Pharmacological N-glycanase inhibition by Z-VAD-fmk or siRNA knockdown (KD) induces GFP-LC3 positive puncta in HEK 293 cells. Activation of ER stress markers or reactive oxygen species (ROS) induction are not observed. In NGLY1 inhibition or KD, upregulation of autophagosome formation without impairment of autophagic flux are observed. Enrichment and proteomics analysis of autophagosomes after Z-VAD-fmk treatment or NGLY1 KD reveals comparable autophagosomal protein content. Upregulation of autophagy represents a cellular adaptation to NGLY1 inhibition or KD, and ATG13-deficient mouse embryonic fibroblasts (MEFs) show reduced viability under these conditions. In contrast, treatment with pan-caspase inhibitor, Q-VD-OPh does not induce cellular autophagy. Therefore, experiments with Z-VAD-fmk are complicated by the effects of NGLY1 inhibition and Q-VD-OPh represents an alternative caspase inhibitor free from this limitation.

cell biology↗