Project description:Endoplasmic reticulum (ER) plasticity and ER-phagy are intertwined processes essential for maintaining ER dynamics. We investigated the interplay between two isoforms of the ER-phagy receptor FAM134B in regulating ER remodeling in differentiating myoblasts. During myogenesis, the canonical FAM134B1 is degraded, while its isoform FAM134B2 is transcriptionally upregulated. The switch, favoring FAM134B2, indicates its significance as a regulator of ER morphology during myogenesis. FAM134B2 partial reticulon homology domain, with its rigid conformational characteristics, enables an efficient ER reshaping. FAM134B2 action increases in the active phase of differentiation leading to ER restructuring via ER-phagy, which then reverts to physiological levels when myotubes are mature and the ER reorganized. Knocking out both FAM134B isoforms in myotubes results in aberrant proteome landscape and the formation of dilated ER structures, both of which are rescued by FAM134B2 re-expression. Our results underscore how the fine tuning of FAM134B isoforms and ER-phagy orchestrate the ER dynamics during myogenesis providing insights into the molecular mechanisms governing ER homeostasis in muscle cells.
Project description:Endoplasmic reticulum (ER) plasticity and ER-phagy are intertwined processes essential for maintaining ER dynamics. We investigated the interplay between two isoforms of the ER-phagy receptor FAM134B in regulating ER remodeling in differentiating myoblasts. During myogenesis, the canonical FAM134B1 is degraded, while its isoform FAM134B2 is transcriptionally upregulated. The switch, favoring FAM134B2, indicates its significance as a regulator of ER morphology during myogenesis. FAM134B2 partial reticulon homology domain, with its rigid conformational characteristics, enables an efficient ER reshaping. FAM134B2 action increases in the active phase of differentiation leading to ER restructuring via ER-phagy, which then reverts to physiological levels when myotubes are mature and the ER reorganized. Knocking out both FAM134B isoforms in myotubes results in aberrant proteome landscape and the formation of dilated ER structures, both of which are rescued by FAM134B2 re-expression. Our results underscore how the fine tuning of FAM134B isoforms and ER-phagy orchestrate the ER dynamics during myogenesis providing insights into the molecular mechanisms governing ER homeostasis in muscle cells.
Project description:Endoplasmic reticulum (ER) remodeling is vital for cellular organization. ER-phagy, a selective autophagy targeting ER, plays an important role in maintaining ER morphology and function. The FAM134 protein family, including FAM134A, FAM134B, and FAM134C, mediates ER-phagy. While FAM134B mutations are linked to hereditary sensory and autonomic neuropathy in humans, the physiological role of the other FAM134 proteins remains unknown. To address this, we investigated the roles of FAM134 proteins using single and combined knockouts (KOs) in mice. Single KO in young mice showed no major phenotypes, however, combined Fam134b and Fam134c deletion (Fam134b/cdKO), but not the combination including Fam134a deletion, led to rapid neuromuscular and somatosensory degeneration, resulting in premature death. Fam134b/cdKO mice show rapid loss of motor and sensory axons in the peripheral nervous system. Long axons from Fam134b/cdKO mice exhibited expanded tubular ER with a transverse ladder-like appearance, whereas no obvious abnormalities were observed in cortical ER. Our study unveils critical roles of FAM134C and FAM134B in the formation of tubular ER network in axons of both motor and sensory neurons.
Project description:Endoplasmic reticulum (ER) plasticity and ER-phagy are intertwined processes essential for maintaining ER dynamics. We investigated the interplay between two isoforms of the ER-phagy receptor FAM134B in regulating ER remodeling in differentiating myoblasts. During myogenesis, the canonical FAM134B1 is degraded, while its isoform FAM134B2 is transcriptionally upregulated. The switch, favoring FAM134B2, is an important regulator of ER morphology during myogenesis. FAM134B2 partial reticulon homology domain, with its rigid conformational characteristics, enables efficient ER reshaping. FAM134B2 action increases in the active phase of differentiation leading to ER restructuring via ER-phagy, which then reverts to physiological levels when myotubes are mature and the ER is reorganized. Knocking out both FAM134B isoforms in myotubes results in an aberrant proteome landscape and the formation of dilated ER structures, both of which are rescued by FAM134B2 re-expression. Our results underscore how the fine-tuning of FAM134B isoforms and ER-phagy orchestrate the ER dynamics during myogenesis providing insights into the molecular mechanisms governing ER homeostasis in muscle cells.
Project description:Barth syndrome (BTHS) is a rare X-linked recessively inherited disorder caused by variants in the TAFAZZIN gene. The pathogenic variants lead to impaired conversion of monolysocardiolipin (MLCL) into mature phospholipid cardiolipin (CL). The accumulation of MLCL and mature CL deficiency is a diagnostic marker for BTHS. The clinical spectrum includes cardiomyopathy, skeletal myopathy, neutropenia, and delays in growth. In severely affected BTHS patients, the cardiac phenotype is early onset, heterogeneous and unpredictable. Ultimately, these patients may require a cardiac transplant early in their life. Unfortunately, the pathophysiological mechanisms of BTHS are poorly understood, and treatment options for BTHS remain symptomatic. In this study, we analysed heart samples from five paediatric male BTHS individuals (5 month-15 years old) and compared them to tissues from 24 non-failing donors (19-71 years old) using a newly developed integrated omics method that combines metabolomics, lipidomics and proteomics using a single sample. This comprehensive analysis confirms expected changes in established diagnostic markers such as CL and MLCL, as well as severe and pleiotropic alterations in mitochondrial phenotype and metabolic output, a substrate shift in energy metabolism, and an elevation of heart-failure markers. It also reveals striking interindividual differences between BTHS individuals. Combined, we describe a powerful analytical tool for the in-depth analysis of metabolic disorders and a solid foundation for the understanding of BTHS disease phenotypes in cardiac tissues.
Project description:Generation of a new library of targeted mass spectrometry assays for accurate protein quantification in triple negative breast cancer (TNBC) tissues. Primary tumor tissue lysates from 105 TNBC patients treated at Masaryk Memorial Cancer Institute (MMCI) in Brno, Czech Republic, were used to generate the spectral library. This project covers raw files from data-dependent acquisition (DDA) – parallel accumulation-serial fragmentation (PASEF) measurements of 12 hydrophilic chromatography (HILIC) fractions of aliquot pool from complete set of 105 samples measured on timsTOF Pro; raw files of 16 individual samples measured in data-independent acquisition (DIA) – PASEF mode and used for hybrid library generation and for demonstrative quantitative DIA data extraction; Pulsar archive generated in Spectronaut 16.0 from 12 DDA-PASEF measurements of HILIC fractions and from 16 data-independent acquisition DIA-PASEF measurements of individual samples. The 16 DIA-PASEF runs of individual samples used for library generation were analyzed using newest versions of Spectronaut (version 18.5) and DIA-NN (version 1.8.1) software tools in library-based setting using the newly generated library as well as in library-free setting showing library-based method to outperform the use of predicted libraries in the terms of identification numbers.