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Secretome translation shaped by Lysosomes and Lunapark-marked ER Junctions

ABSTRACT: Proteomic analysis of membrane fractions from U-2 OS control and Lunapark knockout cells to determine protein abundance per mRNA. Membrane proteins were extracted, digested, and analyzed by DIA LC–MS/MS on a Thermo Orbitrap Ascend Tribrid mass spectrometer with Vanquish Neo LC system

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, U2-os Cell

DISEASE(S): Bone Cancer

SUBMITTER: Heejun Choi

LAB HEAD: Jennifer, Lippincott-Schwartz

PROVIDER: PXD068879 | Pride | 2025-12-29

REPOSITORIES: Pride

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			Action	DRS
	202410_hc_wtko_dia24spd_report.pg_matrix.xlsx	Xlsx
	HC_20241004_ko1_p5zip_8of20_dia24SPD_01.raw	Raw
	HC_20241004_ko1_p5zip_8of20_dia24SPD_02.raw	Raw
	HC_20241004_ko2_1zip_4of20_dia24SPD_01.raw	Raw
	HC_20241004_ko2_1zip_4of20_dia24SPD_02.raw	Raw

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Publications

Secretome translation shaped by lysosomes and lunapark-marked ER junctions.

Choi Heejun H Liao Ya-Cheng YC Yoon Young J YJ Grimm Jonathan J Wang Nan N Lavis Luke D LD Singer Robert H RH Lippincott-Schwartz Jennifer J

Nature 20251105 8095

The endoplasmic reticulum (ER) is a highly interconnected membrane network that serves as a central site for protein synthesis and maturation<sup>1</sup>. A crucial subset of ER-associated transcripts, termed secretome mRNAs, encode secretory, lumenal and integral membrane proteins, representing nearly one-third of human protein-coding genes<sup>1</sup>. Unlike cytosolic mRNAs, secretome mRNAs undergo co-translational translocation, and thus require precise coordination between translation and p ...[more]

PMID: 41193816

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Secretome translation shaped by Lysosomes and Lunapark-marked ER Junctions

Project description:This study investigates how translation of mRNAs encoding membrane and secretory proteins (“secretome mRNAs”) is spatially regulated in human cells. Using live-cell imaging, proteomics, and RNA-sequencing, we show that Lunapark (LNPK)–enriched ER junctions and lysosomes form specialized hubs that enhance secretome mRNA translation. RNA-seq was performed on U-2 OS cells with control and Lunapark knockout conditions to assess transcriptome changes.

2025-09-30 | GSE309254 | GEO

Secretome translation shaped by Lysosomes and Lunapark-marked ER Junctions

Project description:Secretome translation shaped by Lysosomes and Lunapark-marked ER Junctions

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Transcriptome-wide identification of LARP4B binding sites in HEK293 cells

Project description:mRNAs are key molecules in gene expression and subject to diverse regulatory events. Regulation is accomplished by distinct sets of trans-acting factors that interact with mRNAs and form defined mRNA-protein complexes (mRNPs). The resulting “mRNP code” determines the fate of any given mRNA and thus controlling gene expression at the post-transcriptional level. The La-related protein 4B (LARP4B) belongs to an evolutionarily conserved family of RNA binding proteins characterized by the presence of a La-module implicated in direct RNA binding. Biochemical experiments have shown previously direct interactions of LARP4B with factors of the translation machinery. This finding along with the observation of an association with actively translating ribosomes suggested that LARP4B is a factor contributing to the mRNP code. To gain insight into the function of LARP4B in vivo we tested its mRNA association at the transcriptome level and its impact on the proteome. PAR-CLIP analyses allowed us to identify the in vivo RNA targets of LARP4B. We show that LARP4B binds to a distinct set of cellular mRNAs by contacting their 3´UTRs. Biocomputational analysis combined with in vitro binding assays identified the LARP4B binding motif on mRNA targets. The reduction of cellular LARP4B levels leads to a marked destabilization of its mRNA targets and consequently their reduced translation. Our data identify LARP4B as a component of the mRNP code that influences the expression of its mRNA targets by affecting their stability. PAR-CLIP experiments for LARP4B in HEK293cells in two biological replicates

2015-05-26 | E-GEOD-69169 | biostudies-arrayexpress

Development of Trypanosoma congolense IL3000 in the host bloodstream

Project description:Trypanosoma congolense IL3000 parasites were grown in adult MF1 mice with parasites harvested on day 5 post infection ('ascend') or on day 6/7 post infection ('peak').

2018-10-01 | GSE114811 | GEO

Development of Trypanosoma brucei EATRO 1125 in the host bloodstream

Project description:Trypanosoma brucei EATRO 1125 parasites were grown in adult MF1 mice with parasites harvested on day 3 post-infection ('ascend/slender') or on day 6 post-infection ('peak/stumpy').

2018-10-01 | GSE114810 | GEO

AMPK governs lineage specification through Tfeb-dependent regulation of lysosomes

Project description:Faithful execution of developmental programs relies on the acquisition of unique cell identities from pluripotent progenitors, a process governed by combinatorial inputs from numerous signaling cascades that ultimately dictate lineage-specific transcriptional outputs. Despite growing evidence that metabolism is integrated with many molecular networks, how pathways that control energy homeostasis may affect cell fate decisions is largely unknown. Here, we show that AMPK, a central metabolic regulator, plays critical roles in lineage specification. Although AMPK-deficient embryonic stem cells (ESCs) were normal in the pluripotent state, these cells displayed profound defects upon differentiation, failing to generate chimeric embryos and preferentially adopting an ectodermal fate at the expense of the endoderm during embryoid body (EB) formation. AMPK-/- EBs exhibited reduced levels of Tfeb, a master transcriptional regulator of lysosomes, leading to diminished endolysosomal function. Remarkably, genetic loss of Tfeb also yielded endodermal defects, while AMPK-null ESCs over-expressing this transcription factor normalized their differential potential, revealing an intimate connection between Tfeb/lysosomes and germ layer specification. The compromised endolysosomal system resulting from AMPK or Tfeb inactivation blunted Wnt signaling, while up-regulating this pathway restored expression of endodermal markers. Collectively, these results uncover the AMPK pathway as a novel regulator of cell fate determination during differentiation. 2 WT and 2 AMPK DKO ESC lines were differentiated into embryoid bodies (EBs) for various lengths of time (2, 4, 8, and 12 days) in high and low glucose conditions. Both ESC and EB samples were profiled by mRNA-seq to examine how global gene expression changes associated with ESC differentiation are affected by AMPK deletion.

2016-03-09 | E-GEOD-77705 | biostudies-arrayexpress

Ring-shaped Rad51 paralog protein complexes bind Holliday junctions and replication forks as visualized by electron microscopy.

Project description:In mammals, there are five Rad51 paralogs that form two distinct complexes in vivo. One complex is composed of Rad51B-Rad51C-Rad51D-Xrcc2 (BCDX2) and the other Rad51C-Xrcc3 (CX3). We co-expressed and purified human BCDX2 and CX3 protein complexes from insect cells and investigated their binding preferences and structure using transmission electron microscopy (TEM). We visualized the binding of BCDX2 and CX3 to DNA templates containing replication forks and Holliday junctions, intermediates observed during DNA replication and recombination, respectively. We show that both complexes bind with exceptionally high specificity to the DNA junctions with little binding observed elsewhere on the DNAs. Further analysis of the structure of free or DNA-bound BCDX2 and CX3 complexes revealed a multimeric ring structure whose subunits are arranged into a flat disc around a central channel. This work provides the first EM visualization of BCDX2 and CX3 binding to Holliday junctions and forked DNAs and suggests the complexes form ring-shaped structures.

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Proteins enriched on damaged lysosomes in 293T

Project description:Lysosomal membrane permeabilization (LMP) or lysosomal membrane damage is commonly associated with aging and age-related diseases. In searching for cellular mechanisms in response to LMP, we used a proteomic approach to identify proteins enriched on damaged lysosomes. This unbiased approach identified a new phosphoinositide signaling pathway triggered by LMP to mediate rapid lysosomal repair. Specifically, LMP induces fast lysosomal recruitment of PI4K2A which generates high levels of the lipid messenger phosphatidylinositol-4-phosphate (PtdIns4P) on damaged lysosomes. Lysosomal PtdIns4P in turn recruits multiple oxysterol-binding protein (OSBP)-related protein (ORP) family members, including ORP9, ORP10, and ORP11, to orchestrate extensive membrane contact sites (MCSs) between damaged lysosomes and the endoplasmic reticulum (ER). The ORPs subsequently catalyze robust ER-to-lysosomal transport of phosphatidylserine (PS), which is critical for rapid lysosomal repair. The lipid transporter ATG2 is also recruited to damaged lysosomes, activated by PS, and is essential for rapid lysosomal repair. Our findings identify a phosphoinositide-dependent membrane tethering and lipid transport (PITT) pathway essential for the maintenance of lysosomal membrane integrity, with important implications for a wide range of diseases characterized by impaired lysosomal function.

2022-09-07 | PXD028852 | Pride

Multisite electrophysiological recordings by self-assembled loose-patch-like junctions between cultured hippocampal neurons and mushroom-shaped microelectrodes.

Project description:Substrate integrated planar microelectrode arrays is the "gold standard" method for millisecond-resolution, long-term, large-scale, cell-noninvasive electrophysiological recordings from mammalian neuronal networks. Nevertheless, these devices suffer from drawbacks that are solved by spike-detecting, spike-sorting and signal-averaging techniques which rely on estimated parameters that require user supervision to correct errors, merge clusters and remove outliers. Here we show that primary rat hippocampal neurons grown on micrometer sized gold mushroom-shaped microelectrodes (gMμE) functionalized simply by poly-ethylene-imine/laminin undergo self-assembly processes to form loose patch-like hybrid structures. More than 90% of the hybrids formed in this way record monophasic positive action potentials (APs). Of these, 34.5% record APs with amplitudes above 300 μV and up to 5,085 μV. This self-assembled neuron-gMμE configuration improves the recording quality as compared to planar MEA. This study characterizes and analyzes the electrophysiological signaling repertoire generated by the neurons-gMμE configuration, and discusses prospects to further improve the technology.

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The identification of adenylyl cyclase modulators as potential receptors for 6-nitrodopamine in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and their relevance in heart inotropism

Project description:6-Nitrodopamine Q7 Q8 (6-ND Q9 ) has potent positive chronotropic and inotropic effects. At a very low dose, i.e., 10 fM, it causes potentiation of the positive chronotropic effects induced by catecholamines in the rat atria, indicating a distinct mechanism of action. Cyclase-associated proteins (CAP-1 and CAP-2) are potential receptors for 6-ND in human cardiomyocytes. Since cyclic 3′,5′-cyclic adenosine monophosphate (cAMP) plays a fundamental role in the positive inotropic effects of classical catecholamines, it was further investigated whether 6-ND potentiates the positive inotropic effects induced by classical catecholamines in the rat isolated perfused heart. Human-induced pluripotent stem cell (hiPSC)- derived cardiomyocytes were harvested and lysed, and following appropriate separation procedures, membrane proteins were incubated with 6-NDderivatized agarose, centrifuged, and the proteins retained in the agarose eluted with 6-ND (1 mM). The proteins isolated from the chemical pulldown assay were fractionated by SDS-PAGE, the bands were cut and hydrolyzed in situ with trypsin, and then separated and sequenced. A total of 817 proteins were generated, and following screening using UniProt “Retrieve/ID Mapping” function and Gene Ontology cellular component category, 124 proteins were identified as membrane proteins. These experiments identified three proteins that modulate adenylyl cyclase (AC) activity (CAP-1, CAP-2, and STIM1), which are compatible with the pharmacological findings reported for 6-ND in the rat heart. As expected, 6-ND strongly potentiates the inotropic effect induced by noradrenaline in Langendorff’s preparation. In conclusion, 6-ND-induced potentiation of catecholamine-induced chronotropic and inotropic effects is due to the modulation of adenylyl cyclase activity, probably via direct interactions with CAP-1 and CAP-2.

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