Biology subjects

St-Germain, J.

Publications and source records attributed to St-Germain, J..

13 recordsLinked to original sources

bioRxiv2026

Quaternary structure and activity of glutamate dehydrogenase are regulated by reversible S-palmitoylation and mitochondrial acyl-protein thioesterases.

Glutamate dehydrogenase (GDH) is a key mitochondrial enzyme that catalyzes the reversible oxidative deamination of glutamate to -ketoglutarate, thereby linking amino acid and carbohydrate metabolism. GDH forms catalytically active hexamers and is regulated by various allosteric modulators, including ADP and GTP. Here, we demonstrate that GDH undergoes auto-palmitoylation in the presence of palmitoyl-CoA, leading to a dose-dependent inhibition of enzymatic activity. Using acyl-PEG exchange assays and mass spectrometry, we show that GDH monomers are predominantly mono-palmitoylated, with modification detected at multiple cysteine residues, including Cys55, Cys115, and Cys197, among the six cysteines in the mature enzyme. Blue Native PAGE analysis revealed that palmitoylation disrupts the native hexameric assembly of mammalian GDH, which is organized as a dimer-of-trimers, promoting dissociation into dimers. Importantly, this modification is reversible, as incubation with mitochondrial acyl-protein thioesterases 1 (APT1) and, to a lesser extent, /{beta} hydrolase domain 10 (ABHD10) restores both the hexameric structure and enzymatic activity. The modified Cys55 residues are positioned near the trimer interface, providing a mechanism by which palmitoylation could prevent hexamer formation, whereas Cys115 and 197 may destabilize individual trimers. These findings establish S-palmitoylation as a novel regulatory mechanism for GDH, linking mitochondrial lipid metabolism to the reversible control of a central metabolic enzyme.

biochemistry↗

bioRxiv2026

Sec23b regulates cell migration by orchestrating collagen I secretion and processing.

Cell migration is critically important for development and for homeostatic processes such as wound healing, while its aberrant regulation may contribute to the development of pathological conditions and diseases. Efficient cell migration requires the coordination of many structural and signalling proteins (e.g. Rho GTPases) that have key functions in controlling actin cytoskeleton organization and dynamics. Given that the actin cytoskeleton has a central role in orchestrating the complex series of events underlying cell migration, characterizing the actin-associated proteome can illuminate important molecular players that could be novel therapeutic targets for diseases associated with dysregulated cell migration. Here, we report a proteomic-based assay that uses a doxycycline-inducible proximity ligation assay to label actin-associated proteins (actin cytoskeletome) during the early stages of cell migration. We found that an unexpected actin-associated protein, Sec23b, influences the rate of cell migration by orchestrating the secretion and post-secretion maturation of Collagen I.

cell biology↗

bioRxiv2025

Characterization of PROTAC specificity and endogenous protein interactomes using ProtacID

Here we describe ProtacID, a flexible BioID (proximity-dependent biotinylation)-based approach to identify PROTAC-proximal proteins in living cells. ProtacID analysis of VHL- and CRBN-recruiting PROTACs targeting a number of different proteins (localized to chromatin or cellular membranes, and tested across six different human cell lines) demonstrates how this technique can be used to validate PROTAC degradation targets and identify non-productive (i.e. non-degraded) PROTAC-interacting proteins, addressing a critical need in the field of PROTAC development. We also demonstrate that ProtacID can be used to characterize native, endogenous multiprotein complexes without the use of antibodies, or modification of the protein of interest with epitope tags or biotin ligase tagging.

molecular biology↗

bioRxiv2024

Cryo-EM of AKAP350 reveals fibrillar clusters and a potential association with DNA

Protein kinase A (PKA) is a promiscuous serine/threonine kinase that phosphorylates a broad-spectrum of effectors involved in vital processes such as glucose, glycogen, and lipid metabolism. Its activity is thus tightly controlled by a family of eukaryotic scaffolding proteins known as the A-kinase anchoring proteins (AKAPs) that confine PKA signaling to specific compartments in the cell. AKAP350 (the protein encoded by AKAP9) is a massive scaffolding protein that anchors PKA to the Golgi apparatus and the centrosome where it nucleates macromolecular signaling hubs that control microtubule nucleation and dynamics. Here, we have expressed and purified full-length AKAP350 from HEK293F cells in a functional conformation. Electron cryo-microscopy (cryo-EM) of the purified protein revealed polydisperse particles forming fibrillar clusters around 50 nm in diameter, and long, thin filaments that reconstructed into double-stranded DNA. Tomographic reconstruction of a tilt series of the purified protein by electron cryo-tomography (cryo-ET) further elucidated these fibrillar clusters as 3D bundles of entangled filaments. Mass spectrometry and DNA sequencing confirmed the co-purification of DNA and DNA binding proteins such as nuclear factor 1 B (NFIB) and nucleolin (NCL). Pulldown of NFIB and NCL, but not of CEP290, CDK5RAP2, and CEP170 was diminished in the presence of DNase-I, suggesting that AKAP350 interaction with these two proteins is mediated by DNA. Overall, this study has achieved a quality purification of AKAP350 from which a previously uncharacterized interaction landscape with DNA and DNA binding proteins was discovered.

biochemistry↗

bioRxiv2024

Adenomatous polyposis coli (APC) regulates internalization and signaling of the chemorepellent receptor, Roundabout (ROBO) 1

The SLIT-ROBO signaling pathway regulates axon guidance and cell migration, and ROBO1 is a receptor for SLIT ligands. ROBO1 undergoes constitutive endocytosis which is enhanced upon SLIT2 binding, but the molecular mechanisms and functional consequences of this process are not well understood. Using pharmacologic inhibitors and molecular techniques, we found that clathrin-mediated endocytosis is necessary for SLIT2-induced inhibition of cell spreading. To explore the underlying mechanisms, we performed BioID to identify ROBO1-interacting proteins whose association with the cytoplasmic domain of ROBO1 is differentially regulated by SLIT2. We discovered that adenomatous polyposis coli (APC), a multifunctional tumor suppressor, constitutively interacts with ROBO1 but dissociates upon binding of SLIT2 and that this dissociation is necessary for clathrin-mediated endocytosis of ROBO1 and subsequent effects on cell morphology. These findings provide new insights into the functional mechanisms by which SLIT2 binding to ROBO1 effects changes in actin cytoskeletal architecture.

cell biology↗

bioRxiv2023

Nucleolar Pol II interactome reveals TBPL1, PAF1, and Pol I at intergenic rDNA drive rRNA biogenesis

Nucleolar ribosomal DNA (rDNA) repeats control ribosome manufacturing. rDNA harbors a ribosomal RNA (rRNA) gene and an intergenic spacer (IGS). RNA polymerase (Pol) I transcribes rRNA genes yielding the rRNA components of ribosomes. Pol II at the IGS induces rRNA production by preventing Pol I from excessively synthesizing IGS non-coding RNAs (ncRNAs) that can disrupt nucleoli. At the IGS, Pol II regulatory processes and whether Pol I function can be beneficial remain unknown. Here, we identify IGS Pol II regulators, uncovering nucleolar optimization via IGS Pol I. Compartment-enriched proximity-dependent biotin identification (compBioID) showed enrichment of the TATA-less promoter-binding TBPL1 and transcription regulator PAF1 with IGS Pol II. TBPL1 localizes to TCT motifs, driving Pol II and Pol I and maintaining its baseline ncRNA levels. PAF1 promotes Pol II elongation, preventing unscheduled R-loops that hyper-restrain IGS Pol I and its ncRNAs. PAF1 or TBPL1 deficiency disrupts nucleolar organization and rRNA biogenesis. In PAF1-deficient cells, repressing unscheduled IGS R-loops rescues nucleolar organization and rRNA production. Depleting IGS Pol I-dependent ncRNAs is sufficient to compromise nucleoli. We present the interactome of nucleolar Pol II and show its control by TBPL1 and PAF1 ensures IGS Pol I ncRNAs maintaining nucleolar structure and operation.

molecular biology↗

bioRxiv2023

Recruitment of FBXO22 for Targeted Degradation of NSD2

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here, we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain of function mutation p.E1099K, resulting in growth suppression, apoptosis, and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 in a covalent and reversible manner to recruit the SCFFBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCFFBXO22. Overall, we present a highly potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a novel FBXO22-dependent TPD strategy.

biochemistry↗

bioRxiv2023

A neuron-specific microexon ablates the novel DNA-binding function of a histone H3K4me0 reader PHF21A

How cell-type-specific chromatin landscapes emerge and progress during metazoan ontogenesis remains an important question. Transcription factors are expressed in a cell-type-specific manner and recruit chromatin-regulatory machinery to specific genomic loci. In contrast, chromatin-regulatory proteins are expressed broadly and are assumed to exert the same intrinsic function across cell types. However, human genetics studies have revealed an unexpected vulnerability of neurodevelopment to chromatin factor mutations with unknown mechanisms. Here, we report that 14 chromatin regulators undergo evolutionary-conserved neuron-specific splicing events involving microexons. Of the 14 chromatin regulators, two are integral components of a histone H3K4 demethylase complex; the catalytic subunit LSD1 and an H3K4me0-reader protein PHF21A adopt neuron-specific forms. We found that canonical PHF21A (PHF21A-c) binds to DNA by AT-hook motif, and the neuronal counterpart PHF21A-n lacks this DNA-binding function yet maintains H3K4me0 recognition intact. In-vitro reconstitution of the canonical and neuronal PHF21A-LSD1 complexes identified the neuronal complex as a hypomorphic H3K4 demethylating machinery with reduced nucleosome engagement. Furthermore, an autism-associated PHF21A missense mutation, 1285 G>A, at the last nucleotide of the common exon immediately upstream of the neuronal microexon led to impaired splicing of PHF21A-n. Thus, ubiquitous chromatin regulatory complexes exert unique intrinsic functions in neurons via alternative splicing of their subunits and potentially contribute to faithful human brain development.

molecular biology↗

bioRxiv2023

Targeting Acute Myeloid Leukemia Stem Cells Through Perturbation of Mitochondrial Calcium

We previously reported that acute myeloid leukemia stem cells (LSCs) are uniquely reliant on oxidative phosphorylation (OXPHOS) for survival. Moreover, maintenance of OXPHOS is dependent on BCL2, creating a therapeutic opportunity to target LSCs using the BCL2 inhibitor drug venetoclax. While venetoclax-based regimens have indeed shown promising clinical activity, the emergence of drug resistance is prevalent. Thus, in the present study, we investigated how mitochondrial properties may influence mechanisms that dictate venetoclax responsiveness. Our data show that utilization of mitochondrial calcium is fundamentally different between drug responsive and non-responsive LSCs. By comparison, venetoclax-resistant LSCs demonstrate a more active metabolic (i.e., OXPHOS) status with relatively high steady-state levels of calcium. Consequently, we tested genetic and pharmacological approaches to target the mitochondrial calcium uniporter, MCU. We demonstrate that inhibition of calcium uptake sharply reduces OXPHOS and leads to eradication of venetoclax-resistant LSCs. These findings demonstrate a central role for calcium signaling in the biology of LSCs and provide a therapeutic avenue for clinical management of venetoclax resistance. SignificanceWe identify increased utilization of mitochondrial calcium as distinct metabolic requirement of venetoclax-resistant LSCs and demonstrate the potential of targeting mitochondrial calcium uptake as a therapeutic strategy.

cancer biology↗

bioRxiv2023

PRMT1 is a critical dependency in clear cell renal cell carcinoma through its role in post-transcriptional regulation of DNA damage response genes

Biallelic inactivation of the Von Hippel-Lindau (VHL) tumor suppressor gene occurs in almost all cases of clear cell renal cell carcinoma (ccRCC) and leads to disrupted oxygen sensing and the upregulation of hypoxia-related genetic programs. Although the loss of VHL appears to be a necessary oncogenic driver event in the majority of ccRCC instances, it is not always a sufficient one. Large genomics studies have revealed that co-deletions of VHL with genes involved in chromatin regulation are common and important co-drivers of tumorigenesis. Several conserved evolutionary subtypes have been described clinically and the majority implicate disruptions in epigenetic regulators. It is now clear that impairments in cellular epigenetic mechanisms are important co-drivers of disease and signal a potential vulnerability in the epigenetic network of ccRCC cells relative to their normal counterparts. Using a clinically relevant panel of ccRCC models, we herein sought to exploit this potential vulnerability by screening a library of small molecule inhibitors that target a spectrum of epigenetic regulators. We identified MS023, an inhibitor of type I protein arginine methyltransferases (PRMTs) as an agent with antitumor activity. Using orthogonal genetic technologies, we further validated PRMT1 as the specific critical dependency for cancer growth. Mechanistically, our transcriptomic and functional analyses suggest that MS023 treatment results in substantial impairments to cell cycle and DNA damage repair (DDR) pathways, while spawning an accumulation of DNA damage over time. Our PRMT1 specific proteomics analysis revealed an interactome rich in RNA binding proteins including the specific regulator of DDR mRNA metabolism: the BCLAF1/THRAP3 complex. Further investigation suggests that MS023 treatment may result in impairments to DDR specific mRNA activities including nucleocytoplasmic transport and RNA splicing. Together, our data supports PRMT1 as a compelling target in ccRCC and informs a potential mechanism-based strategy for translational development.

cancer biology↗

bioRxiv2022

Functional characterization of a PHF8 processed pseudogene in the mouse genome

The majority of pseudogenes are generated when an RNA transcript is reverse-transcribed and integrated into the genome at a new location. Pseudogenes are often considered as an imperfect and silent copy of a functional gene because of the accumulation of numerous mutations in their sequence. Here we report the presence of PHF8-ps, a PHF8 retrotransposed pseudogene in the mouse genome, which has no disruptions in its coding sequence. We showed that this pseudogene was specifically transcribed in testis and can produce a Phf8-ps protein in vivo. As Phf8-ps protein has a well-conserved JmjC domain, we characterized its enzymatic activity and showed that Phf8-ps did not have the intrinsic capability to demethylate H3K9me2 in vitro compared to the parental Phf8 protein. Surprisingly, Phf8-ps did not localize in the nucleus like Phf8 but rather was mostly located at the cytoplasm. Finally, our proteomic analysis of Phf8-ps associated proteins revealed that Phf8-ps interacted with mitochondrial proteins but also with prefoldin subunits (PFDN proteins) that deliver unfolded proteins to the cytosolic chaperonin complex implicated the folding of cytosolic proteins. Together, our findings highlighted Phf8-ps as a new pseudogene-derived protein with distinct molecular functions from Phf8.

biochemistry↗

bioRxiv2020

A SARS-CoV-2 - host proximity interactome

Viral replication is dependent on interactions between viral polypeptides and host proteins. Identifying virus-host protein interactions can thus uncover unique opportunities for interfering with the virus life cycle via novel drug compounds or drug repurposing. Importantly, many viral-host protein interactions take place at intracellular membranes and poorly soluble organelles, which are difficult to profile using classical biochemical purification approaches. Applying proximity-dependent biotinylation (BioID) with the fast-acting miniTurbo enzyme to 27 SARS-CoV-2 proteins in a lung adenocarcinoma cell line (A549), we detected 7810 proximity interactions (7382 of which are new for SARS-CoV-2) with 2242 host proteins (results available at covid19interactome.org). These results complement and dramatically expand upon recent affinity purification-based studies identifying stable host-virus protein complexes, and offer an unparalleled view of membrane-associated processes critical for viral production. Host cell organellar markers were also subjected to BioID in parallel, allowing us to propose modes of action for several viral proteins in the context of host proteome remodelling. In summary, our dataset identifies numerous high confidence proximity partners for SARS-CoV-2 viral proteins, and describes potential mechanisms for their effects on specific host cell functions.

systems biology↗

bioRxiv2020

Nucleotide binding, evolutionary insights and interaction partners of the pseudokinase Unc-51-like kinase 4

Unc-51-like kinase 4 (ULK4) is a pseudokinase that has been linked to the development of several diseases. Even though sequence motifs required for ATP binding in kinases are lacking, ULK4 still tightly binds ATP and the presence of the cofactor is required for structural stability of ULK4. Here we present a high-resolution structure of a ULK4-ATP{gamma}S complex revealing a highly unusual ATP binding mode in which the lack of the canonical VAIK motif lysine is compensated by K39, located N-terminal to C. Evolutionary analysis suggests that degradation of active site motifs in metazoan ULK4 has co-occurred with an ULK4 specific activation loop, which stabilizes the C-helix. In addition, cellular interaction studies using BioID and biochemical validation data revealed high confidence interactors of the pseudokinase and armadillo repeat domains. Many of the identified ULK4 interaction partners were centrosomal and tubulin associated proteins and several active kinases suggesting new roles for ULK4. HighlightsStructure of the ULK4 ATP complex reveals a unique ATP binding mode. Disease associated mutations modulate ATP binding and ULK4 stability Degradation of active site motifs co-occurred in evolution with an ULK4 specific activation loop BioID suggests a role of ULK4 regulating centrosomal and cytoskeletal functions

biochemistry↗