Developmental remodeling shapes neural circuits via activity-dependent pruning of synapses and axons. Regulation of the cytoskeleton is critical for this process, as microtubule loss via enzymatic severing is an early step of pruning across many circuits and species. However, how microtubule-severing enzymes, such as spastin, are activated in specific neuronal compartments remains unknown. Here, we reveal that polyglutamylation, a post-translational tubulin modification enriched in neurons, plays an instructive role in developmental remodeling by tagging microtubules for severing. Motor neuron-specific gene deletion of enzymes that add or remove tubulin polyglutamylation-TTLL glutamylases vs. CCP deglutamylases-accelerates or delays neuromuscular synapse remodeling in a neurotransmission-dependent manner. This mechanism is not specific to peripheral synapses but also operates in central circuits, e.g., the hippocampus. Thus, tubulin polyglutamylation acts as a cytoskeletal rheostat of remodeling that shapes neuronal morphology and connectivity.

• MeSH
• Hippocampus metabolism   cytology   MeSH
• Polyglutamic Acid * metabolism   MeSH
• Microtubules * metabolism   MeSH
• Motor Neurons * metabolism   MeSH
• Mice MeSH
• Neuromuscular Junction metabolism   MeSH
• Synaptic Transmission MeSH
• Neurons * metabolism   MeSH
• Neuronal Plasticity * physiology   MeSH
• Peptide Synthases metabolism   genetics   MeSH
• Protein Processing, Post-Translational MeSH
• Spastin metabolism   MeSH
• Synapses metabolism   MeSH
• Tubulin metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

The circadian clock in choroid plexus (ChP) controls processes involved in its physiological functions, but the signals that synchronize the clock have been sparsely studied. We found that the ChP clock in the fourthventricle (4V) is more robust than that in the lateral ventricle (LV) and investigated whether both clocks use information about mealtime as a signal to synchronize with the current activity state. Exposure of mPer2Luc mice to a 10-day reverse restricted feeding (rRF) protocol, in which food was provided for 6 h during daytime, advanced the phase of the ChP clock in 4V and LV, as evidenced by shifted (1) PER2-driven bioluminescence rhythms of ChP explants ex vivo and (2) daily profiles in clock gene expression in both ChP tissues in vivo. In contrast, clocks in other brain regions (DMH, ARC, LHb) of the same mice did not shift. The 4V ChP responded more strongly than the LV ChP to rRF by modulating the expression of genes to ensure a decrease in resistance to cerebrospinal fluid drainage and increase the secretory capacity of ChP cells. Mechanistically, rRF affects the ChP clock through food-induced increases in insulin, glucose and temperature levels, as in vitro all three signals significantly shifted the clocks in both ChP tissues, similar to rRF. The effect of glucose was partially blocked by OSMI-1, suggesting involvement of O-linked N-acetylglucosamine posttranslational modification. We identified mechanisms that can signal to the brain the time of feeding and the associated activity state via resetting of the ChP clock.

• MeSH
• Circadian Clocks * physiology   genetics   MeSH
• Period Circadian Proteins metabolism   genetics   MeSH
• Circadian Rhythm physiology   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Choroid Plexus * metabolism   physiology   MeSH
• Gene Expression Regulation MeSH
• Feeding Behavior * physiology   MeSH
• Lateral Ventricles metabolism   physiology   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Proteomics is nowadays increasingly becoming part of the routine clinical practice of diagnostic laboratories, especially due to the advent of advanced mass spectrometry techniques. This review focuses on the application of proteomic analysis in the identification of pathological conditions in a hospital setting, with a particular focus on the analysis of protein biomarkers. In particular, the main purpose of the review is to highlight the challenges associated with the identification of specific disease-causing proteins, given their complex nature and the variety of posttranslational modifications (PTMs) they can undergo. PTMs, such as phosphorylation and glycosylation, play critical roles in protein function but can also lead to diseases if dysregulated. Proteomics plays an important role especially in various medical fields ranging from cardiology, internal medicine to hemato-oncology emphasizing the interdisciplinary nature of this field. Traditional methods such as electrophoretic or immunochemical methods have been mainstay in protein detection; however, these techniques are limited in terms of specificity and sensitivity. Examples include the diagnosis of multiple myeloma and the detection of its specific protein or amyloidosis, which relies heavily on these conventional methods, which sometimes lead to false positives or inadequate disease monitoring. Mass spectrometry in this respect emerges as a superior alternative, providing high sensitivity and specificity in the detection and quantification of specific protein sequences. This technique is particularly beneficial for monitoring minimal residual disease (MRD) in the diagnosis of multiple myeloma where traditional methods fall short. Furthermore mass spectrometry can provide precise typing of amyloid proteins, which is crucial for the appropriate treatment of amyloidosis. This review summarizes the opportunities for proteomic determination using mass spectrometry between 2012 and 2024, highlighting the transformative potential of mass spectrometry in clinical proteomics and encouraging its wider use in diagnostic laboratories.

• MeSH
• Amyloidosis * diagnosis   MeSH
• Biomarkers analysis   MeSH
• Mass Spectrometry * methods   MeSH
• Humans MeSH
• Multiple Myeloma * diagnosis   MeSH
• Protein Processing, Post-Translational MeSH
• Proteomics * methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

OBJECTIVE: Thyroid cancer (TC) is the most common endocrine malignancy, with 90%-95% of the cases representing non-medullary thyroid cancer (NMTC). Familial cases account only for a few of all cases and the underlying genetic causes are still poorly understood. METHODS: We whole-genome sequenced affected and unaffected members of an Italian NMTC family and applied our in-house developed Familial Cancer Variant Prioritization Pipeline (FCVPPv2) which prioritized 12 coding variants. We refined this selection using the VarSome American College of Medical Genetics and Genomics (ACMG) implementation, SNAP2 predictions and further in silico scores. RESULTS: We prioritized 4 possibly pathogenic variants in 4 genes including Ret proto-oncogene (RET), polypeptide N-acetylgalactosaminyltransferase 10 (GALNT10), ubinuclein-1 (UBN1), and prostaglandin I2 receptor (PTGIR). The role of RET point mutations in medullary thyroid carcinoma is well established. Similarly, somatic rearrangements of RET are known in papillary TC, a specific histotype of NMTC. In contrast to RET, no germline variants in PTGIR, GALNT10, or UBN1 have been linked to the development of TC to date. However, alterations in these genes have been shown to affect pathways related to cell proliferation, apoptosis, growth, and differentiation, as well as posttranslational modification and gene regulation. A thorough review of the available literature together with computational evidence supported the interpretation of the 4 shortlisted variants as possibly disease-causing in this family. CONCLUSIONS: Our results implicate the first germline variant in RET in a family with NMTC as well as the first germline variants in PTGIR, GALNT10, and UBN1 in TC.

• MeSH
• Adult MeSH
• Genetic Predisposition to Disease * genetics   MeSH
• Middle Aged MeSH
• Humans MeSH
• N-Acetylgalactosaminyltransferases genetics   MeSH
• Thyroid Neoplasms * genetics   MeSH
• Carcinoma, Neuroendocrine * genetics   MeSH
• Polypeptide N-acetylgalactosaminyltransferase MeSH
• Proto-Oncogene Mas MeSH
• Proto-Oncogene Proteins c-ret genetics   MeSH
• Pedigree MeSH
• Whole Genome Sequencing * methods   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

Histones are positively charged proteins found in the chromatin of eukaryotic cells. They regulate gene expression and are required for the organization and packaging of DNA within the nucleus. Histones are extremely conserved, allowing for transcription, replication, and repair. This review delves into their complex structure and function in DNA assembly, their role in nucleosome assembly, and the higher-order chromatin structures they generate. We look at the five different types of histone proteins: H1, H2A, H2B, H3, H4, and their variations. These histones bind with DNA to produce nucleosomes, the basic units of chromatin that are essential for compacting DNA and controlling its accessibility. Their dynamic control of chromatin accessibility has important implications for genomic stability and cellular activities. We elucidate regulatory mechanisms in both normal and pathological situations by investigating their structural features, diverse interaction mechanisms, and chromatin impact. In addition, we discuss the functions of histone post-translational modifications (PTMs) and their significance in various disorders. These alterations, which include methylation, acetylation, phosphorylation, and ubiquitination, are crucial in regulating histone function and chromatin dynamics. We specifically describe and explore the role of changed histones in the evolution of cancer, neurological disorders, sepsis, autoimmune illnesses, and inflammatory conditions. This comprehensive review emphasizes histone's critical role in genomic integrity and their potential as therapeutic targets in various diseases.

• MeSH
• Chromatin metabolism   genetics   chemistry   MeSH
• DNA * metabolism   chemistry   MeSH
• Genome MeSH
• Histones * metabolism   chemistry   genetics   MeSH
• Humans MeSH
• Neoplasms genetics   metabolism   MeSH
• Protein Processing, Post-Translational MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Spermatogenesis starts with the onset of puberty within the seminiferous epithelium of the testes. It is a complex process under intricate control of the endocrine system. Physiological regulations by steroid hormones in general and by estrogens in particular are due to their chemical nature prone to be disrupted by exogenous factors acting as endocrine disruptors (EDs). 17α-Ethynylestradiol (EE2) is an environmental pollutant with a confirmed ED activity and a well-known effect on spermatogenesis and chromatin remodeling in haploid germ cells. The aim of our study was to assess possible effects of two doses (2.5ng/ml; 2.5 μg/ml) of EE2 on both histone-to-protamine exchange and epigenetic profiles during spermatogenesis performing a multi/transgenerational study in mice. Our results demonstrated an impaired histone-to-protamine exchange with a significantly higher histone retention in sperm nuclei of exposed animals, when this process was accompanied by the changes of histone post-translational modifications (PTMs) abundancies with a prominent effect on H3K9Ac and partial changes in protamine 1 promoter methylation status. Furthermore, individual changes in molecular phenotypes were partially transmitted to subsequent generations, when no direct trans-generational effect was observed. Finally, the uncovered specific localization of the histone retention in sperm nuclei and their specific PTMs profile after EE2 exposure may indicate an estrogenic effect on sperm motility and early embryonic development via epigenetic mechanisms.

• MeSH
• Endocrine Disruptors pharmacology   toxicity   MeSH
• Epigenesis, Genetic * drug effects   MeSH
• Ethinyl Estradiol * pharmacology   MeSH
• Histones * metabolism   MeSH
• Mice MeSH
• Protein Processing, Post-Translational drug effects   MeSH
• Protamines * metabolism   genetics   MeSH
• Spermatogenesis * drug effects   genetics   MeSH
• Spermatozoa drug effects   metabolism   MeSH
• Testis * drug effects   metabolism   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

OBJECTIVE: Redox signaling mediated by reversible oxidative cysteine thiol modifications is crucial for driving cellular adaptation to dynamic environmental changes, maintaining homeostasis, and ensuring proper function. This is particularly critical in pancreatic β-cells, which are highly metabolically active and play a specialized role in whole organism glucose homeostasis. Glucose stimulation in β-cells triggers signals leading to insulin secretion, including changes in ATP/ADP ratio and intracellular calcium levels. Additionally, lipid metabolism and reactive oxygen species (ROS) signaling are essential for β-cell function and health. METHODS: We employed IodoTMT isobaric labeling combined with tandem mass spectrometry to elucidate redox signaling pathways in pancreatic β-cells. RESULTS: Glucose stimulation significantly increases ROS levels in β-cells, leading to targeted reversible oxidation of proteins involved in key metabolic pathways such as glycolysis, the tricarboxylic acid (TCA) cycle, pyruvate metabolism, oxidative phosphorylation, protein processing in the endoplasmic reticulum (ER), and insulin secretion. Furthermore, the glucose-induced increase in reversible cysteine oxidation correlates with the presence of other post-translational modifications, including acetylation and phosphorylation. CONCLUSIONS: Proper functioning of pancreatic β-cell metabolism relies on fine-tuned regulation, achieved through a sophisticated system of diverse post-translational modifications that modulate protein functions. Our findings demonstrate that glucose induces the production of ROS in pancreatic β-cells, leading to targeted reversible oxidative modifications of proteins. Furthermore, protein activity is modulated by acetylation and phosphorylation, highlighting the complexity of the regulatory mechanisms in β-cell function.

• MeSH
• Insulin-Secreting Cells * metabolism   drug effects   MeSH
• Adaptation, Physiological physiology   MeSH
• Glucose * metabolism   MeSH
• Humans MeSH
• Mice MeSH
• Oxidation-Reduction * MeSH
• Protein Processing, Post-Translational MeSH
• Reactive Oxygen Species * metabolism   MeSH
• Insulin Secretion drug effects   physiology   MeSH
• Signal Transduction * physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Obesity is a major health burden. Preadipocytes proliferate and differentiate in mature adipocytes in the adipogenic process, which could be a potential therapeutic approach for obesity. Deficiency of SIRT6, a stress-responsive protein deacetylase and mono-ADP ribosyltransferase enzyme, blocks adipogenesis. Mutants of SIRT6 (N308K/A313S) were recently linked to the in the long lifespan Ashkenazi Jews. In this study, we aimed to clarify how these new centenarian-associated SIRT6 genetic variants affect adipogenesis at the transcriptional and epigenetic level. METHODS: We analyzed the role of SIRT6 wild-type (WT) or SIRT6 centenarian-associated mutant (N308K/A313S) overexpression in adipogenesis, by creating stably transduced preadipocyte cell lines using lentivirus on the 3T3-L1 model. Histone post-translational modifications (PTM: acetylation, methylation) and transcriptomic changes were analyzed by mass spectrometry (LC-MS/MS) and RNA-Seq, respectively, in 3T3-L1 adipocytes. In addition, the adipogenic process and related signaling pathways were investigated by bioinformatics and biochemical approaches. RESULTS: Overexpression of centenarian-associated SIRT6 mutant increased adipogenic differentiation to a similar extent compared to the WT form. However, it triggered distinct histone PTM profiles in mature adipocytes, with significantly higher acetylation levels, and activated divergent transcriptional programs, including those dependent on signaling related to the sympathetic innervation and to PI3K pathway. 3T3-L1 mature adipocytes overexpressing SIRT6 N308K/A313S displayed increased insulin sensitivity in a neuropeptide Y (NPY)-dependent manner. CONCLUSIONS: SIRT6 N308K/A313S overexpression in mature adipocytes ameliorated glucose sensitivity and impacted sympathetic innervation signaling. These findings highlight the importance of targeting SIRT6 enzymatic activities to regulate the co-morbidities associated with obesity.

• MeSH
• Adipogenesis * genetics   MeSH
• 3T3-L1 Cells * MeSH
• Epigenesis, Genetic * genetics   MeSH
• Histones metabolism   genetics   MeSH
• Humans MeSH
• Mutation MeSH
• Mice MeSH
• Obesity genetics   metabolism   MeSH
• Protein Processing, Post-Translational genetics   MeSH
• Sirtuins * genetics   metabolism   MeSH
• Adipocytes * metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Sarcopenia is associated with reduced quality of life and premature mortality. The sex disparities in the processes underlying sarcopenia pathogenesis, which include mitochondrial dysfunction, are ill-understood and can be decisive for the optimization of sarcopenia-related interventions. To improve the knowledge regarding the sex differences in skeletal muscle aging, the gastrocnemius muscle of young and old female and male rats was analyzed with a focus on mitochondrial remodeling through the proteome profiling of mitochondria-enriched fractions. To the best of our knowledge, this is the first study analyzing sex differences in skeletal muscle mitochondrial proteome remodeling. Data demonstrated that age induced skeletal muscle atrophy and fibrosis in both sexes. In females, however, this adverse skeletal muscle remodeling was more accentuated than in males and might be attributed to an age-related reduction of 17beta-estradiol signaling through its estrogen receptor alpha located in mitochondria. The females-specific mitochondrial remodeling encompassed increased abundance of proteins involved in fatty acid oxidation, decreased abundance of the complexes subunits, and enhanced proneness to oxidative posttranslational modifications. This conceivable accretion of damaged mitochondria in old females might be ascribed to low levels of Parkin, a key mediator of mitophagy. Despite skeletal muscle atrophy and fibrosis, males maintained their testosterone levels throughout aging, as well as their androgen receptor content, and the age-induced mitochondrial remodeling was limited to increased abundance of pyruvate dehydrogenase E1 component subunit beta and electron transfer flavoprotein subunit beta. Herein, for the first time, it was demonstrated that age affects more severely the skeletal muscle mitochondrial proteome of females, reinforcing the necessity of sex-personalized approaches towards sarcopenia management, and the inevitability of the assessment of mitochondrion-related therapeutics.

• MeSH
• Estradiol metabolism   MeSH
• Fibrosis metabolism   MeSH
• Muscle, Skeletal * metabolism   pathology   MeSH
• Rats MeSH
• Mitochondria metabolism   pathology   MeSH
• Mitophagy MeSH
• Proteome metabolism   MeSH
• Sarcopenia * metabolism   pathology   MeSH
• Sex Factors MeSH
• Aging * metabolism   MeSH
• Muscular Atrophy metabolism   pathology   MeSH
• Mitochondria, Muscle metabolism   pathology   MeSH
• Ubiquitin-Protein Ligases metabolism   genetics   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

CIP/KIP and INK4 families of Cyclin-dependent kinase inhibitors (CKIs) are well-established cell cycle regulatory proteins whose canonical function is binding to Cyclin-CDK complexes and altering their function. Initial experiments showed that these proteins negatively regulate cell cycle progression and thus are tumor suppressors in the context of molecular oncology. However, expanded research into the functions of these proteins showed that most of them have non-canonical functions, both cell cycle-dependent and independent, and can even act as tumor enhancers depending on their posttranslational modifications, subcellular localization, and cell state context. This review aims to provide an overview of canonical as well as non-canonical functions of CIP/KIP and INK4 families of CKIs, discuss the potential avenues to promote their tumor suppressor functions instead of tumor enhancing ones, and how they could be utilized to design improved treatment regimens for cancer patients.

• Publication type
• Journal Article MeSH
• Review MeSH