Isoleucine-to-valine substitutions support cellular physiology during isoleucine deprivation.

Aminoacyl-tRNA synthetases (ARSs) couple tRNAs with their corresponding amino acids. While ARSs can bind structurally similar amino acids, extreme specificity is ensured by subsequent editing activity. Yet, we found that upon isoleucine (I) restriction, healthy fibroblasts consistently incorporated valine (V) into proteins at isoleucine codons, resulting from misacylation of tRNAIle with valine by wildtype IARS1.

mTORC1 restricts TFE3 activity by auto-regulating its presence on lysosomes.

To stimulate cell growth, the protein kinase complex mTORC1 requires intracellular amino acids for activation. Amino-acid sufficiency is relayed to mTORC1 by Rag GTPases on lysosomes, where growth factor signaling enhances mTORC1 activity via the GTPase Rheb. In the absence of amino acids, GATOR1 inactivates the Rags, resulting in lysosomal detachment and inactivation of mTORC1.

Aberrant neurodevelopment in human iPS cell-derived models of Alexander disease.

Alexander disease (AxD) is a rare and severe neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP). While the exact disease mechanism remains unknown, previous studies suggest that mutant GFAP influences many cellular processes, including cytoskeleton stability, mechanosensing, metabolism, and proteasome function. While most studies have primarily focused on GFAP-expressing astrocytes, GFAP is also expressed by radial glia and neural progenitor cells, prompting questions about the impact of GFAP mutations on central nervous system (CNS) development.

ALS-associated C21ORF2 variant disrupts DNA damage repair, mitochondrial metabolism, neuronal excitability and NEK1 levels in human motor neurons.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease leading to motor neuron loss. Currently mutations in > 40 genes have been linked to ALS, but the contribution of many genes and genetic mutations to the ALS pathogenic process remains poorly understood. Therefore, we first performed comparative interactome analyses of five recently discovered ALS-associated proteins (C21ORF2, KIF5A, NEK1, TBK1, and TUBA4A) which highlighted many novel binding partners, and both unique and shared interactors.

Deciphering lineage specification during early embryogenesis in mouse gastruloids using multilayered proteomics.

Gastrulation is a critical stage in embryonic development during which the germ layers are established. Advances in sequencing technologies led to the identification of gene regulatory programs that control the emergence of the germ layers and their derivatives. However, proteome-based studies of early mammalian development are scarce.

Galectin-3 and prohibitin 1 are autoantigens in IgG4-related cholangitis without clear-cut protective effects against toxic bile acids.

Background And Aims: IgG4-related cholangitis (IRC) is the hepatobiliary manifestation of IgG4-related disease, a systemic B cell-driven fibro-inflammatory disorder. Four autoantigens have recently been described in IgG4-RD: annexin A11, galectin-3, laminin 511-E8, and prohibitin 1. We have previously reported a protective role of annexin A11 and laminin 511-E8 in human cholangiocytes against toxic bile acids.

A farnesyl-dependent structural role for CENP-E in expansion of the fibrous corona.

Correct chromosome segregation during cell division depends on proper connections between spindle microtubules and kinetochores. During prometaphase, kinetochores are temporarily covered with a dense protein meshwork known as the fibrous corona. Formed by oligomerization of ROD/ZW10/ZWILCH-SPINDLY (RZZ-S) complexes, the fibrous corona promotes spindle assembly, chromosome orientation, and spindle checkpoint signaling.

Pyruvate metabolism controls chromatin remodeling during CD4 T cell activation.

Upon antigen-specific T cell receptor (TCR) engagement, human CD4 T cells proliferate and differentiate, a process associated with rapid transcriptional changes and metabolic reprogramming. Here, we show that the generation of extramitochondrial pyruvate is an important step for acetyl-CoA production and subsequent H3K27ac-mediated remodeling of histone acetylation. Histone modification, transcriptomic, and carbon tracing analyses of pyruvate dehydrogenase (PDH)-deficient T cells show PDH-dependent acetyl-CoA generation as a rate-limiting step during T activation.

Early adipogenesis is repressed through the newly identified FHL2-NFAT5 signaling complex.

The LIM-domain-only protein FHL2 is a modulator of signal transduction and has been shown to direct the differentiation of mesenchymal stem cells towards osteoblast and myocyte phenotypes. We hypothesized that FHL2 may simultaneously interfere with the induction of the adipocyte lineage. Therefore, we investigated the role of FHL2 in adipocyte differentiation.

TRIB3 Modulates PPARγ-Mediated Growth Inhibition by Interfering with the MLL Complex in Breast Cancer Cells.

Aberrant expression or activity of proteins are amongst the best understood mechanisms that can drive cancer initiation and progression, as well as therapy resistance. TRIB3, a member of the Tribbles family of pseudokinases, is often dysregulated in cancer and has been associated with breast cancer initiation and metastasis formation. However, the underlying mechanisms by which TRIB3 contributes to these events are unclear.

FER regulates endosomal recycling and is a predictor for adjuvant taxane benefit in breast cancer.

Elevated expression of non-receptor tyrosine kinase FER is an independent prognosticator that correlates with poor survival of high-grade and basal/triple-negative breast cancer (TNBC) patients. Here, we show that high FER levels are also associated with improved outcomes after adjuvant taxane-based combination chemotherapy in high-risk, HER2-negative patients. In TNBC cells, we observe a causal relation between high FER levels and sensitivity to taxanes.

Comprehensive Profiling of Mammalian Tribbles Interactomes Implicates TRIB3 in Gene Repression.

The three human Tribbles (TRIB) pseudokinases have been implicated in a plethora of signaling and metabolic processes linked to cancer initiation and progression and can potentially be used as biomarkers of disease and prognosis. While their modes of action reported so far center around protein-protein interactions, the comprehensive profiling of TRIB interactomes has not been reported yet. Here, we have developed a robust mass spectrometry (MS)-based proteomics approach to characterize Tribbles' interactomes and report a comprehensive assessment and comparison of the TRIB1, -2 and -3 interactomes, as well as domain-specific interactions for TRIB3.

The Human 2-Cys Peroxiredoxins form Widespread, Cysteine-Dependent- and Isoform-Specific Protein-Protein Interactions.

Redox signaling is controlled by the reversible oxidation of cysteine thiols, a post-translational modification triggered by HO acting as a second messenger. However, HO actually reacts poorly with most cysteine thiols and it is not clear how HO discriminates between cysteines to trigger appropriate signaling cascades in the presence of dedicated HO scavengers like peroxiredoxins (PRDXs). It was recently suggested that peroxiredoxins act as peroxidases and facilitate HO-dependent oxidation of redox-regulated proteins via disulfide exchange reactions.

Cardiomyocytes stimulate angiogenesis after ischemic injury in a ZEB2-dependent manner.

The disruption in blood supply due to myocardial infarction is a critical determinant for infarct size and subsequent deterioration in function. The identification of factors that enhance cardiac repair by the restoration of the vascular network is, therefore, of great significance. Here, we show that the transcription factor Zinc finger E-box-binding homeobox 2 (ZEB2) is increased in stressed cardiomyocytes and induces a cardioprotective cross-talk between cardiomyocytes and endothelial cells to enhance angiogenesis after ischemia.

Proteomic Profiling of Two Distinct Populations of Extracellular Vesicles Isolated from Human Seminal Plasma.

Body fluids contain many populations of extracellular vesicles (EV) that differ in size, cellular origin, molecular composition, and biological activities. EV in seminal plasma are in majority originating from prostate epithelial cells, and hence are also referred to as prostasomes. Nevertheless, EV are also contributed by other accessory sex glands, as well as by the testis and epididymis.

A novel three step protocol to isolate extracellular vesicles from plasma or cell culture medium with both high yield and purity.

Extracellular vesicles (EV) are membrane encapsulated nanoparticles that can function in intercellular communication, and their presence in biofluids can be indicative for (patho)physiological conditions. Studies aiming to resolve functionalities of EV or to discover EV-associated biomarkers for disease in liquid biopsies are hampered by limitations of current protocols to isolate EV from biofluids or cell culture medium. EV isolation is complicated by the >10-fold numerical excess of other types of particles, including lipoproteins and protein complexes.

The Lipid Raft Component Stomatin Interacts with the Na Taurocholate Cotransporting Polypeptide (NTCP) and Modulates Bile Salt Uptake.

The sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the basolateral membrane of hepatocytes, where it mediates the uptake of conjugated bile acids and forms the hepatocyte entry receptor for the hepatitis B and D virus. Here, we aimed to identify novel protein-protein interactions that could play a role in the regulation of NTCP. To this end, NTCP was precipitated from HA-tagged hNTCP-expressing HepG2 cells, and chloride channel CLIC-like 1 (CLCC1) and stomatin were identified as interacting proteins by mass spectrometry.

Diverse Gene Fusions Confer Resistance to EGFR-Targeted Therapy via Differential Modulation of BRAF Activity.

Fusion genes can be oncogenic drivers in a variety of cancer types and represent potential targets for targeted therapy. The gene is frequently involved in oncogenic gene fusions, with fusion frequencies of 0.2%-3% throughout different cancers.

Steroidogenic control of liver metabolism through a nuclear receptor-network.

Objective: Coupling metabolic and reproductive pathways is essential for the survival of species. However, the functions of steroidogenic enzymes expressed in metabolic tissues are largely unknown.

Methods And Results: Here, we show that in the liver, the classical steroidogenic enzyme Cyp17a1 forms an essential nexus for glucose and ketone metabolism during feed-fast cycles.

Cysteine oxidation triggers amyloid fibril formation of the tumor suppressor p16.

The tumor suppressor p16 induces cell cycle arrest and senescence in response to oncogenic transformation and is therefore frequently lost in cancer. p16 is also known to accumulate under conditions of oxidative stress. Thus, we hypothesized it could potentially be regulated by reversible oxidation of cysteines (redox signaling).

De Novo Heterozygous POLR2A Variants Cause a Neurodevelopmental Syndrome with Profound Infantile-Onset Hypotonia.

The RNA polymerase II complex (pol II) is responsible for transcription of all ∼21,000 human protein-encoding genes. Here, we describe sixteen individuals harboring de novo heterozygous variants in POLR2A, encoding RPB1, the largest subunit of pol II. An iterative approach combining structural evaluation and mass spectrometry analyses, the use of S.

Nemo-like Kinase Drives Foxp3 Stability and Is Critical for Maintenance of Immune Tolerance by Regulatory T Cells.

The Foxp3 transcription factor is a crucial determinant of both regulatory T (T) cell development and their functional maintenance. Appropriate modulation of tolerogenic immune responses therefore requires the tight regulation of Foxp3 transcriptional output, and this involves both transcriptional and post-translational regulation. Here, we show that during T cell activation, phosphorylation of Foxp3 in T cells can be regulated by a TGF-β activated kinase 1 (TAK1)-Nemo-like kinase (NLK) signaling pathway.