Top-down Proteomics for the Characterization and Quantification of Calreticulin Arginylation DOI Creative Commons
Richard Searfoss, Xingyu Liu, Benjamin A. García

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Aug. 8, 2024

Abstract Arginylation installed by arginyltransferase 1 (ATE1) features an addition of arginine (Arg) to the reactive amino acids ( e . g ., Glu and Asp) at protein N -terminus or side chain. Systemic removal arginylation after ATE1 knockout (KO) in mouse models resulted heart defects leading embryonic lethality. The biological importance has motivated discovery sites on proteins using bottom-up approaches. While proteomics is powerful localizing peptide arginylation, it lacks ability quantify proteoforms level. Here we developed a top-down workflow for characterizing quantifying calreticulin (CALR) arginylation. To generate fully arginylated CALR (R-CALR), have inserted R residue signaling (AA1-17). Upon overexpression KO cells, R-CALR were purified affinity purification analyzed LCMS positive mode. Both showed charge states ranging from 27-68 with 58 as most intense state. Their MS2 spectra electron-activated dissociation (EAD) preferential fragmentation -terminals which yielded sufficient c ions facilitating precise localization sites. calcium-binding domain (CBD) gave minimum characteristic possibly due abundant presence >100 D E residues. Ultraviolet photodissociation (UVPD) compared EAD ETD significantly improved sequence coverage CBD. This method can identify absence, endogenous (low), high levels. our knowledge, work first application post-translational vitro vivo

Language: Английский

Redox regulation: mechanisms, biology and therapeutic targets in diseases DOI Creative Commons
Bowen Li, Hui Ming, Siyuan Qin

et al.

Signal Transduction and Targeted Therapy, Journal Year: 2025, Volume and Issue: 10(1)

Published: March 7, 2025

Redox signaling acts as a critical mediator in the dynamic interactions between organisms and their external environment, profoundly influencing both onset progression of various diseases. Under physiological conditions, oxidative free radicals generated by mitochondrial respiratory chain, endoplasmic reticulum, NADPH oxidases can be effectively neutralized NRF2-mediated antioxidant responses. These responses elevate synthesis superoxide dismutase (SOD), catalase, well key molecules like nicotinamide adenine dinucleotide phosphate (NADPH) glutathione (GSH), thereby maintaining cellular redox homeostasis. Disruption this finely tuned equilibrium is closely linked to pathogenesis wide range Recent advances have broadened our understanding molecular mechanisms underpinning dysregulation, highlighting pivotal roles genomic instability, epigenetic modifications, protein degradation, metabolic reprogramming. findings provide foundation for exploring regulation mechanistic basis improving therapeutic strategies. While antioxidant-based therapies shown early promise conditions where stress plays primary pathological role, efficacy diseases characterized complex, multifactorial etiologies remains controversial. A deeper, context-specific signaling, particularly redox-sensitive proteins, designing targeted aimed at re-establishing balance. Emerging small molecule inhibitors that target specific cysteine residues proteins demonstrated promising preclinical outcomes, setting stage forthcoming clinical trials. In review, we summarize current intricate relationship disease also discuss how these insights leveraged optimize strategies practice.

Language: Английский

Citations

4
N-degron pathways DOI Creative Commons
Alexander Varshavsky

Proceedings of the National Academy of Sciences, Journal Year: 2024, Volume and Issue: 121(39)

Published: Sept. 12, 2024

An N-degron is a degradation signal whose main determinant “destabilizing” N-terminal residue of protein. Specific N-degrons, discovered in 1986, were the first identified signals short-lived intracellular proteins. These N-degrons are recognized by ubiquitin-dependent proteolytic system called Arg/N-degron pathway. Although bacteria lack ubiquitin system, they also have pathways. Studies after 1986 shown that all 20 amino acids genetic code can act, specific sequence contexts, as destabilizing residues. Eukaryotic proteins targeted for conditional or constitutive at least five systems differ both functionally and mechanistically: pathway, Ac/N-degron Pro/N-degron fMet/N-degron newly named, this perspective, GASTC/N-degron pathway (GASTC = Gly, Ala, Ser, Thr, Cys). I discuss these expanded terminology now encompasses entire gamut known

Language: Английский

Citations

13
Targeted Protein Degradation: Principles and Applications of the Proteasome DOI Creative Commons

Yosup Kim,

Eun-Kyung Kim, Yoona Chey

et al.

Cells, Journal Year: 2023, Volume and Issue: 12(14), P. 1846 - 1846

Published: July 13, 2023

The proteasome is a multi-catalytic protease complex that involved in protein quality control via three proteolytic activities (i.e., caspase-, trypsin-, and chymotrypsin-like activities). Most cellular proteins are selectively degraded by the ubiquitination. Moreover, ubiquitin–proteasome system critical process for maintaining homeostasis. Here, we briefly summarize structure of proteasome, its regulatory mechanisms, regulate activity, alterations to activity found diverse diseases, chemoresistant cells, cancer stem cells. Finally, describe potential therapeutic modalities use system.

Language: Английский

Citations

15
N/C-degron pathways and inhibitor development for PROTAC applications DOI
Zhibin Wu, Yunyuan Huang, Ke Liu

et al.

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, Journal Year: 2023, Volume and Issue: 1867(1), P. 194952 - 194952

Published: May 30, 2023

Language: Английский

Citations

3
DOT1L stimulates MYC/Mondo transcription factor activity by promoting its degradation cycle on chromatin DOI Creative Commons

Gian Sepulveda,

Ekaterina Gushchanskaia,

Alexandra Mora-Martin

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Feb. 7, 2024

SUMMARY The proto-oncogene c-MYC is a key representative of the MYC transcription factor network regulating growth and metabolism. MML-1 (Myc- Mondo-like) its homolog in C. elegans . functional molecular cooperation between H3 lysine 79 methyltransferase DOT1L was demonstrated several human cancer types, we have earlier discovered connection DOT-1.1. Here, demonstrate critical role DOT1L/DOT-1.1 c-MYC/MML-1 target genes genome-wide by ensuring removal “spent” factors from chromatin nuclear proteasome. Moreover, uncover previously unrecognized proteolytic activity DOT1L, which may facilitate turnover. This new mechanism regulation lead to development approaches for treatment.

Language: Английский

Citations

0
Opticool: Cutting-edge transgenic optical tools DOI Creative Commons
Kelli D. Fenelon, Julia Krause, Theodora Koromila

et al.

PLoS Genetics, Journal Year: 2024, Volume and Issue: 20(3), P. e1011208 - e1011208

Published: March 22, 2024

Only a few short decades have passed since the sequencing of GFP, yet modern repertoire transgenically encoded optical tools implies an exponential proliferation ever improving constructions to interrogate subcellular environment. A myriad tags for labeling proteins, RNA, or DNA arisen in last decades, facilitating unprecedented visualization components and processes. Development broad array genetically sensors allows real-time, vivo detection molecule levels, pH, forces, enzyme activity, other extracellular phenomena expanding contexts. Optogenetic, optically controlled manipulation systems gained traction biological research community facilitate single-cell, real-time modulation protein function broadening, novel applications. While this field continues explosively expand, references are needed assist scientists seeking use improve these transgenic devices new exciting ways development disease. In review, we endeavor highlight state trajectory tools.

Language: Английский

Citations

0
Top-down Proteomics for the Characterization and Quantification of Calreticulin Arginylation DOI Creative Commons
Richard Searfoss, Xingyu Liu, Benjamin A. García

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Aug. 8, 2024

Abstract Arginylation installed by arginyltransferase 1 (ATE1) features an addition of arginine (Arg) to the reactive amino acids ( e . g ., Glu and Asp) at protein N -terminus or side chain. Systemic removal arginylation after ATE1 knockout (KO) in mouse models resulted heart defects leading embryonic lethality. The biological importance has motivated discovery sites on proteins using bottom-up approaches. While proteomics is powerful localizing peptide arginylation, it lacks ability quantify proteoforms level. Here we developed a top-down workflow for characterizing quantifying calreticulin (CALR) arginylation. To generate fully arginylated CALR (R-CALR), have inserted R residue signaling (AA1-17). Upon overexpression KO cells, R-CALR were purified affinity purification analyzed LCMS positive mode. Both showed charge states ranging from 27-68 with 58 as most intense state. Their MS2 spectra electron-activated dissociation (EAD) preferential fragmentation -terminals which yielded sufficient c ions facilitating precise localization sites. calcium-binding domain (CBD) gave minimum characteristic possibly due abundant presence >100 D E residues. Ultraviolet photodissociation (UVPD) compared EAD ETD significantly improved sequence coverage CBD. This method can identify absence, endogenous (low), high levels. our knowledge, work first application post-translational vitro vivo

Language: Английский

Citations

0