scholarly journals Thyroglobulin Interactome Profiling Uncovers Molecular Mechanisms of Thyroid Dyshormonogenesis

2020 ◽  
Author(s):  
Madison T. Wright ◽  
Logan Kouba ◽  
Lars Plate
Keyword(s):  
Molecular Basis ◽  
Therapeutic Strategy ◽  
Molecular Mechanisms ◽  
Protein Quality ◽  
Affinity Purification ◽  
Gene Mutations ◽  
Thyroid Hormone Biosynthesis ◽  
Thyroid Dyshormonogenesis ◽  
Hormone Biosynthesis ◽  
Affinity Purification Mass Spectrometry

ABSTRACTThyroglobulin (Tg) is a secreted iodoglycoprotein serving as the precursor for T3 and T4 hormones. Many characterized Tg gene mutations produce secretion-defective variants resulting in congenital hypothyroidism (CH). Tg processing and secretion is controlled by extensive interactions with chaperone, trafficking, and degradation factors comprising the secretory proteostasis network. While dependencies on individual proteostasis network components are known, the integration of proteostasis pathways mediating Tg protein quality control and the molecular basis of mutant Tg misprocessing remain poorly understood. We employ a multiplexed quantitative affinity purification–mass spectrometry approach to define the Tg proteostasis interactome and changes between WT and several CH-variants. Mutant Tg processing is associated with common imbalances in proteostasis engagement including increased chaperoning, oxidative folding, and routing towards ER-associated degradation components, yet variants are inefficiently degraded. Furthermore, we reveal mutation-specific changes in engagement with N-glycosylation components, suggesting distinct requirements for one Tg variant on dual engagement of both oligosaccharyltransferase complex isoforms for degradation. Modulating dysregulated proteostasis components and pathways may serve as a therapeutic strategy to restore Tg secretion and thyroid hormone biosynthesis.

scholarly journals Thyroglobulin interactome profiling defines altered proteostasis topology associated with thyroid dyshormonogenesis

2020 ◽  
pp. mcp.RA120.002168 ◽  
Author(s):  
Madison T Wright ◽  
Logan Kouba ◽  
Lars Plate
Keyword(s):  
Mass Spectrometry ◽  
Molecular Basis ◽  
Therapeutic Strategy ◽  
Protein Quality ◽  
Affinity Purification ◽  
Gene Mutations ◽  
Thyroid Hormone Biosynthesis ◽  
Thyroid Dyshormonogenesis ◽  
Hormone Biosynthesis ◽  
Affinity Purification Mass Spectrometry

Thyroglobulin (Tg) is a secreted iodoglycoprotein serving as the precursor for T3 and T4 hormones. Many characterized Tg gene mutations produce secretion-defective variants resulting in congenital hypothyroidism (CH). Tg processing and secretion is controlled by extensive interactions with chaperone, trafficking, and degradation factors comprising the secretory proteostasis network. While dependencies on individual proteostasis network components are known, the integration of proteostasis pathways mediating Tg protein quality control and the molecular basis of mutant Tg misprocessing remain poorly understood. We employ a multiplexed quantitative affinity purification–mass spectrometry approach to define the Tg proteostasis interactome and changes between WT and several CH-variants. Mutant Tg processing is associated with common imbalances in proteostasis engagement including increased chaperoning, oxidative folding, and engagement by targeting factors for ER-associated degradation (ERAD). Furthermore, we reveal mutation-specific changes in engagement with N-glycosylation components, suggesting distinct requirements for one Tg variant on dual engagement of both oligosaccharyltransferase complex isoforms for degradation. Modulating dysregulated proteostasis components and pathways may serve as a therapeutic strategy to restore Tg secretion and thyroid hormone biosynthesis.

scholarly journals Identification of intracellular glycosaminoglycan-interacting proteins by affinity purification mass spectrometry

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Henning Großkopf ◽  
Sarah Vogel ◽  
Claudia Damaris Müller ◽  
Sebastian Köhling ◽  
Jan-Niklas Dürig ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Mechanisms ◽  
Protein Localization ◽  
Affinity Purification ◽  
Intracellular Localization ◽  
Enrichment Analysis ◽  
Transport Processes ◽  
Interacting Proteins ◽  
Affinity Purification Mass Spectrometry ◽  
Functional Components

Abstract Glycosaminoglycans (GAGs) are essential functional components of the extracellular matrix (ECM). Artificial GAGs like sulfated hyaluronan (sHA) exhibit pro-osteogenic properties and boost healing processes. Hence, they are of high interest for supporting bone regeneration and wound healing. Although sulfated GAGs (sGAGs) appear intracellularly, the knowledge about intracellular effects and putative interaction partners is scarce. Here we used an affinity-purification mass spectrometry-based (AP-MS) approach to identify novel and particularly intracellular sGAG-interacting proteins in human bone marrow stromal cells (hBMSC). Overall, 477 proteins were found interacting with at least one of four distinct sGAGs. Enrichment analysis for protein localization showed that mainly intracellular and cell-associated interacting proteins were identified. The interaction of sGAG with α2-macroglobulin receptor-associated protein (LRPAP1), exportin-1 (XPO1), and serine protease HTRA1 (HTRA1) was confirmed in reverse assays. Consecutive pathway and cluster analysis led to the identification of biological processes, namely processes involving binding and processing of nucleic acids, LRP1-dependent endocytosis, and exosome formation. Respecting the preferentially intracellular localization of sGAG in vesicle-like structures, also the interaction data indicate sGAG-specific modulation of vesicle-based transport processes. By identifying many sGAG-specific interacting proteins, our data provide a resource for upcoming studies aimed at molecular mechanisms and understanding of sGAG cellular effects.

scholarly journals Chibby cooperates with 14-3-3 to regulate β-catenin subcellular distribution and signaling activity

2008 ◽  
Vol 181 (7) ◽  
pp. 1141-1154 ◽  
Author(s):  
Feng-Qian Li ◽  
Adaobi Mofunanya ◽  
Kimberley Harris ◽  
Ken-Ichi Takemaru
Keyword(s):  
Transcriptional Activation ◽  
Nuclear Export ◽  
Molecular Mechanisms ◽  
Affinity Purification ◽  
Nuclear Accumulation ◽  
Binding Motif ◽  
Transcriptional Coactivator ◽  
Evolutionarily Conserved ◽  
Canonical Wnt ◽  
Affinity Purification Mass Spectrometry

β-Catenin functions in both cell–cell adhesion and as a transcriptional coactivator in the canonical Wnt pathway. Nuclear accumulation of β-catenin is the hallmark of active Wnt signaling and is frequently observed in human cancers. Although β-catenin shuttles in and out of the nucleus, the molecular mechanisms underlying its translocation remain poorly understood. Chibby (Cby) is an evolutionarily conserved molecule that inhibits β-catenin–mediated transcriptional activation. Here, we identified 14-3-3ε and 14-3-3ζ as Cby-binding partners using affinity purification/mass spectrometry. 14-3-3 proteins specifically recognize serine 20 within the 14-3-3–binding motif of Cby when phosphorylated by Akt kinase. Notably, 14-3-3 binding results in sequestration of Cby into the cytoplasm. Moreover, Cby and 14-3-3 form a stable tripartite complex with β-catenin, causing β-catenin to partition into the cytoplasm. Our results therefore suggest a novel paradigm through which Cby acts in concert with 14-3-3 proteins to facilitate nuclear export of β-catenin, thereby antagonizing β-catenin signaling.

scholarly journals Two novel missense mutations in the thyroid peroxidase gene, R665W and G771R, result in a localization defect and cause congenital hypothyroidism

2002 ◽  
pp. 491-498 ◽  
Author(s):  
K Umeki ◽  
T Kotani ◽  
J Kawano ◽  
T Suganuma ◽  
I Yamamoto ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Congenital Hypothyroidism ◽  
Cellular Localization ◽  
Thyroid Peroxidase ◽  
Missense Mutations ◽  
Thyroid Hormone Biosynthesis ◽  
Thyroid Dyshormonogenesis ◽  
Hormone Biosynthesis ◽  
Iodide Organification Defect ◽  
Organification Defect

OBJECTIVE: Thyroid peroxidase (TPO) deficiency is one of the causes of thyroid dyshormonogenesis, because TPO plays a key role in thyroid hormone biosynthesis. To determine the frequency and pattern of TPO abnormalities, we have been screening TPO genes of patients with congenital goitrous hypothyroidism. SUBJECTS AND METHODS: TPO genes of a patient with congenital goitrous hypothyroidism and her parents were directly sequenced, and two novel missense mutations (R665W and G771R) were found. The former was derived from her father and the latter from her mother. R665 and G771 were well conserved in the peroxidase superfamily. When mRNAs containing each of the mutations were transfected into CHO-K1 cells, each cell showed faint TPO enzyme activity. However, immunofluorescence and immunoelectron microscopic analyses revealed that neither of the mutated TPOs reached the plasma membrane. CONCLUSIONS: Two novel missense mutations in the TPO gene were found. TPO proteins encoded by these mutated alleles showed abnormal cellular localization; namely, localization on the plasma membrane was disturbed. The loss of plasma membrane localization in mutated TPOs brought about the iodide organification defect, which was diagnosed as congenital hypothyroidism.

Antennal transcriptome analysis and candidate olfactory genes in Crematogaster rogenhoferi

2021 ◽  
pp. 1-12
Author(s):  
Xiang Zhou ◽  
Jixing Guo ◽  
Mingxia Zhang ◽  
Chunxiu Bai ◽  
Zheng Wang ◽  
...  
Keyword(s):  
Biological Control ◽  
Transcriptome Analysis ◽  
Molecular Basis ◽  
Molecular Mechanisms ◽  
Biological Control Agent ◽  
Control Agent ◽  
Vital Role ◽  
Neuron Membrane ◽  
Ionotropic Receptors ◽  
Soft Scale

Abstract Crematogaster rogenhoferi (Hymenoptera: Formicidae), an omnivorous ant, is one of the dominant predatory natural enemies of a soft scale pest, Parasaissetia nigra Nietner (Homoptera: Coccidae), and can effectively control P. nigra populations in rubber forests. Olfaction plays a vital role in the process of predation. However, the information about the molecular mechanism of olfaction-evoked behaviour in C. rogenhoferi is limited. In this study, we conducted antennal transcriptome analysis to identify candidate olfactory genes. We obtained 53,892 unigenes, 16,185 of which were annotated. Based on annotations, we identified 49 unigenes related to chemoreception, including four odourant-binding proteins, three chemosensory proteins, 37 odourant receptors, two odourant ionotropic receptors and three sensory neuron membrane proteins. This is the first report on the molecular basis of the chemosensory system of C. rogenhoferi. The findings provide a basis for elucidating the molecular mechanisms of the olfactory-related behaviours of C. rogenhoferi, which would facilitate a better application of C. rogenhoferi as a biological control agent.

scholarly journals Loss of histone methyltransferase ASH1L in the developing mouse brain causes autistic-like behaviors

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yuen Gao ◽  
Natalia Duque-Wilckens ◽  
Mohammad B. Aljazi ◽  
Yan Wu ◽  
Adam J. Moeser ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mouse Brain ◽  
Molecular Mechanisms ◽  
Gene Mutations ◽  
Autism Spectrum ◽  
Normal Brain ◽  
Critical Function ◽  
Developmental Defects ◽  
Neurodevelopmental Disease ◽  
Developing Mouse Brain

AbstractAutism spectrum disorder (ASD) is a neurodevelopmental disease associated with various gene mutations. Recent genetic and clinical studies report that mutations of the epigenetic gene ASH1L are highly associated with human ASD and intellectual disability (ID). However, the causality and underlying molecular mechanisms linking ASH1L mutations to genesis of ASD/ID remain undetermined. Here we show loss of ASH1L in the developing mouse brain is sufficient to cause multiple developmental defects, core autistic-like behaviors, and impaired cognitive memory. Gene expression analyses uncover critical roles of ASH1L in regulating gene expression during neural cell development. Thus, our study establishes an ASD/ID mouse model revealing the critical function of an epigenetic factor ASH1L in normal brain development, a causality between Ash1L mutations and ASD/ID-like behaviors in mice, and potential molecular mechanisms linking Ash1L mutations to brain functional abnormalities.

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