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Plant Communications Nov 2023Jasmonates (JAs) are plant hormones with crucial roles in development and stress resilience. They activate MYC transcription factors by mediating the proteolysis of MYC...
Jasmonates (JAs) are plant hormones with crucial roles in development and stress resilience. They activate MYC transcription factors by mediating the proteolysis of MYC inhibitors called JAZ proteins. In the absence of JA, JAZ proteins bind and inhibit MYC through the assembly of MYC-JAZ-Novel Interactor of JAZ (NINJA)-TPL repressor complexes. However, JAZ and NINJA are predicted to be largely intrinsically unstructured, which has precluded their experimental structure determination. Through a combination of biochemical, mutational, and biophysical analyses and AlphaFold-derived ColabFold modeling, we characterized JAZ-JAZ and JAZ-NINJA interactions and generated models with detailed, high-confidence domain interfaces. We demonstrate that JAZ, NINJA, and MYC interface domains are dynamic in isolation and become stabilized in a stepwise order upon complex assembly. By contrast, most JAZ and NINJA regions outside of the interfaces remain highly dynamic and cannot be modeled in a single conformation. Our data indicate that the small JAZ Zinc finger expressed in Inflorescence Meristem (ZIM) motif mediates JAZ-JAZ and JAZ-NINJA interactions through separate surfaces, and our data further suggest that NINJA modulates JAZ dimerization. This study advances our understanding of JA signaling by providing insights into the dynamics, interactions, and structure of the JAZ-NINJA core of the JA repressor complex.
Topics: Arabidopsis Proteins; Arabidopsis; Repressor Proteins; Cyclopentanes
PubMed: 37322867
DOI: 10.1016/j.xplc.2023.100639 -
Plant & Cell Physiology Sep 2023Strigolactones (SLs) play fundamental roles in regulating plant architecture, which is a major factor determining crop yield. The perception and signal transduction of...
Strigolactones (SLs) play fundamental roles in regulating plant architecture, which is a major factor determining crop yield. The perception and signal transduction of SLs require the formation of a complex containing the receptor DWARF14 (D14), an F-box protein D3 and a transcriptional regulator D53 in an SL-dependent manner. Structural and biochemical analyses of D14 and its orthologs DAD2 and AtD14, D3 and the complexes of ASK1-D3-AtD14 and D3CTH-D14 have made great contributions to understanding the mechanisms of SL perception. However, structural analyses of D53 and the D53-D3-D14 holo-complex are challenging, and the biochemical mechanism underlying the complex assembly remains poorly understood. Here, we found that apo-D53 was rather flexible and reconstituted the holo-complex containing D53, S-phase kinase-associated protein 1 (SKP1), D3 and D14 with rac-GR24. The cryo-electron microscopy (cryo-EM) structure of SKP1-D3-D14 in the presence of D53 was analyzed and superimposed on the crystal structure of ASK1-D3-AtD14 without D53. No large conformational rearrangement was observed, but a 9Å rotation appeared between D14 and AtD14. Using hydrogen-deuterium exchange monitored by mass spectrometry, we analyzed dynamic motifs of D14, D3 and D53 in the D53-SKP1-D3-D14 complex assembly process and further identified two potential interfaces in D53 that are located in the N and D2 domains, respectively. Together, our results uncovered the dynamic conformational changes and built a model of the holo-complex D53-SKP1-D3-D14, offering valuable information for the biochemical and genetic mechanisms of SL perception and signal transduction.
Topics: Plant Growth Regulators; Cryoelectron Microscopy; F-Box Proteins; Lactones; Signal Transduction
PubMed: 37384578
DOI: 10.1093/pcp/pcad067 -
Journal of Cachexia, Sarcopenia and... Oct 2023Skeletal muscle mass and strength diminish during periods of disuse but recover upon return to weight bearing in healthy adults but are incomplete in old muscle. Efforts...
BACKGROUND
Skeletal muscle mass and strength diminish during periods of disuse but recover upon return to weight bearing in healthy adults but are incomplete in old muscle. Efforts to improve muscle recovery in older individuals commonly aim at increasing myofibrillar protein synthesis via mammalian target of rapamycin (mTOR) stimulation despite evidence demonstrating that old muscle has chronically elevated levels of mammalian target of rapamycin complex 1 (mTORC1) activity. We hypothesized that protein synthesis is higher in old muscle than adult muscle, which contributes to a proteostatic stress that impairs recovery.
METHODS
We unloaded hindlimbs of adult (10-month) and old (28-month) F344BN rats for 14 days to induce atrophy, followed by reloading up to 60 days with deuterium oxide (D O) labelling to study muscle regrowth and proteostasis.
RESULTS
We found that old muscle has limited recovery of muscle mass during reloading despite having higher translational capacity and myofibrillar protein synthesis (0.029 k/day ± 0.002 vs. 0.039 k/day ± 0.002, P < 0.0001) than adult muscle. We showed that collagen protein synthesis was not different (0.005 k (1/day) ± 0.0005 vs. 0.004 k (1/day) ± 0.0005, P = 0.15) in old compared to adult, but old muscle had higher collagen concentration (4.5 μg/mg ± 1.2 vs. 9.8 μg/mg ± 0.96, P < 0.01), implying that collagen breakdown was slower in old muscle than adult muscle. This finding was supported by old muscle having more insoluble collagen (4.0 ± 1.1 vs. 9.2 ± 0.9, P < 0.01) and an accumulation of advanced glycation end products (1.0 ± 0.06 vs. 1.5 ± 0.08, P < 0.001) than adult muscle during reloading. Limited recovery of muscle mass during reloading is in part due to higher protein degradation (0.017 1/t ± 0.002 vs. 0.028 1/t ± 0.004, P < 0.05) and/or compromised proteostasis as evidenced by accumulation of ubiquitinated insoluble proteins (1.02 ± 0.06 vs. 1.22 ± 0.06, P < 0.05). Last, we showed that synthesis of individual proteins related to protein folding/refolding, protein degradation and neural-related biological processes was higher in old muscle during reloading than adult muscle.
CONCLUSIONS
Our data suggest that the failure of old muscle to recover after disuse is not due to limitations in the ability to synthesize myofibrillar proteins but because of other impaired proteostatic mechanisms (e.g., protein folding and degradation). These data provide novel information on individual proteins that accumulate in protein aggregates after disuse and certain biological processes such as protein folding and degradation that likely play a role in impaired recovery. Therefore, interventions to enhance regrowth of old muscle after disuse should be directed towards the identified impaired proteostatic mechanisms and not aimed at increasing protein synthesis.
Topics: Humans; Rats; Animals; Aged; Muscular Atrophy; Aging; Muscle, Skeletal; Muscular Disorders, Atrophic; TOR Serine-Threonine Kinases; Collagen; Mammals
PubMed: 37448295
DOI: 10.1002/jcsm.13285 -
Advances and prospects in deuterium metabolic imaging (DMI): a systematic review of in vivo studies.European Radiology Experimental Jun 2024Deuterium metabolic imaging (DMI) has emerged as a promising non-invasive technique for studying metabolism in vivo. This review aims to summarize the current... (Review)
Review
BACKGROUND
Deuterium metabolic imaging (DMI) has emerged as a promising non-invasive technique for studying metabolism in vivo. This review aims to summarize the current developments and discuss the futures in DMI technique in vivo.
METHODS
A systematic literature review was conducted based on the PRISMA 2020 statement by two authors. Specific technical details and potential applications of DMI in vivo were summarized, including strategies of deuterated metabolites detection, deuterium-labeled tracers and corresponding metabolic pathways in vivo, potential clinical applications, routes of tracer administration, quantitative evaluations of metabolisms, and spatial resolution.
RESULTS
Of the 2,248 articles initially retrieved, 34 were finally included, highlighting 2 strategies for detecting deuterated metabolites: direct and indirect DMI. Various deuterated tracers (e.g., [6,6'-H2]glucose, [2,2,2'-H3]acetate) were utilized in DMI to detect and quantify different metabolic pathways such as glycolysis, tricarboxylic acid cycle, and fatty acid oxidation. The quantifications (e.g., lactate level, lactate/glutamine and glutamate ratio) hold promise for diagnosing malignancies and assessing early anti-tumor treatment responses. Tracers can be administered orally, intravenously, or intraperitoneally, either through bolus administration or continuous infusion. For metabolic quantification, both serial time point methods (including kinetic analysis and calculation of area under the curves) and single time point quantifications are viable. However, insufficient spatial resolution remains a major challenge in DMI (e.g., 3.3-mL spatial resolution with 10-min acquisition at 3 T).
CONCLUSIONS
Enhancing spatial resolution can facilitate the clinical translation of DMI. Furthermore, optimizing tracer synthesis, administration protocols, and quantification methodologies will further enhance their clinical applicability.
RELEVANCE STATEMENT
Deuterium metabolic imaging, a promising non-invasive technique, is systematically discussed in this review for its current progression, limitations, and future directions in studying in vivo energetic metabolism, displaying a relevant clinical potential.
KEY POINTS
• Deuterium metabolic imaging (DMI) shows promise for studying in vivo energetic metabolism. • This review explores DMI's current state, limits, and future research directions comprehensively. • The clinical translation of DMI is mainly impeded by limitations in spatial resolution.
Topics: Humans; Deuterium; Animals
PubMed: 38825658
DOI: 10.1186/s41747-024-00464-y -
Clinical Cancer Research : An Official... Dec 2023An accurate and noninvasive assessment of tumor response following treatment other than traditional anatomical imaging techniques is essential. Deuterium magnetic...
PURPOSE
An accurate and noninvasive assessment of tumor response following treatment other than traditional anatomical imaging techniques is essential. Deuterium magnetic resonance spectroscopic (MRS) imaging has been demonstrated as an alternative for cancer metabolic imaging by high-field MRI using deuterium-labeled molecules. The study aim was to use 2H tissue labeling and deuterium MRI at clinical field strength for tumor visualization and assessment of three anticancer therapies in pancreatic cancer model mice.
EXPERIMENTAL DESIGN
MIA PaCa-2 pancreatic carcinoma and C26 colorectal carcinoma models of BALB/c-nu mice was prepared, and repeated deuterium MRI was performed during the first 10 days of free drinking of 30% D2O to track 2H distribution in tissues. 2H accumulation in the tumor after irradiation, bevacizumab administration, or gemcitabine administration was also measured in MIA PaCa-2-bearing mice. Confirmatory proton MRI, ex vivo metabolic hyperpolarization 13C-MRS, and histopathology were performed.
RESULTS
The mouse's whole-body distribution of 2H was visible 1 day after drinking, and the signal intensity increased daily. Although the tumor size did not change 1 and 3 days after irradiation, the amount of 2H decreased significantly. The 2H image intensity of the tumor also significantly decreased after the administration of bevacizumab or gemcitabine. Metabolic hyperpolarization 13C-MRS, proton MRI, and 2H-NMR spectroscopy confirmed the efficacy of the anticancer treatments.
CONCLUSIONS
Deuterium MRI at 1.5T proved feasible to track 2H distribution throughout mouse tissues during D2O administration and revealed a higher 2H accumulation in the tumor xenografts. This research demonstrated a promising successful method for preliminary assessment of radiotherapy and chemotherapy of cancer.
Topics: Humans; Mice; Animals; Water; Deuterium; Protons; Bevacizumab; Gemcitabine; Magnetic Resonance Imaging; Neoplasms
PubMed: 37732903
DOI: 10.1158/1078-0432.CCR-23-1635 -
Translational Exercise Biomedicine May 2024'OMICs encapsulates study of scaled data acquisition, at the levels of DNA, RNA, protein, and metabolite species. The broad objectives of OMICs in biomedical exercise... (Review)
Review
OBJECTIVES
'OMICs encapsulates study of scaled data acquisition, at the levels of DNA, RNA, protein, and metabolite species. The broad objectives of OMICs in biomedical exercise research are multifarious, but commonly relate to biomarker development and understanding features of exercise adaptation in health, ageing and metabolic diseases.
METHODS
This field is one of exponential technical (i.e., depth of feature coverage) and scientific (i.e., in health, metabolic conditions and ageing, multi-OMICs) progress adopting and approaches.
RESULTS
Key findings in exercise biomedicine have led to the identification of OMIC features linking to heritability or adaptive responses to exercise e.g., the forging of GWAS/proteome/metabolome links to cardiovascular fitness and metabolic health adaptations. The recent addition of stable isotope tracing to proteomics ('dynamic proteomics') and metabolomics ('fluxomics') represents the next phase of state-of-the-art in 'OMICS.
CONCLUSIONS
These methods overcome limitations associated with point-in-time 'OMICs and can be achieved using substrate-specific tracers or deuterium oxide (DO), depending on the question; these methods could help identify how individual protein turnover and metabolite flux may explain exercise responses. We contend application of these methods will shed new light in translational exercise biomedicine.
PubMed: 38660119
DOI: 10.1515/teb-2024-2006 -
Structural Premise of Selective Deubiquitinase USP30 Inhibition by Small-Molecule Benzosulfonamides.Molecular & Cellular Proteomics : MCP Aug 2023Dampening functional levels of the mitochondrial deubiquitylating enzyme Ubiquitin-specific protease 30 (USP30) has been suggested as an effective therapeutic strategy...
Dampening functional levels of the mitochondrial deubiquitylating enzyme Ubiquitin-specific protease 30 (USP30) has been suggested as an effective therapeutic strategy against neurodegenerative disorders such as Parkinson's Disease. USP30 inhibition may counteract the deleterious effects of impaired turnover of damaged mitochondria, which is inherent to both familial and sporadic forms of the disease. Small-molecule inhibitors targeting USP30 are currently in development, but little is known about their precise nature of binding to the protein. We have integrated biochemical and structural approaches to gain novel mechanistic insights into USP30 inhibition by a small-molecule benzosulfonamide-containing compound, USP30. Activity-based protein profiling mass spectrometry confirmed target engagement, high selectivity, and potency of USP30 for USP30 against 49 other deubiquitylating enzymes in a neuroblastoma cell line. In vitro characterization of USP30 enzyme kinetics inferred slow and tight binding behavior, which is comparable with features of covalent modification of USP30. Finally, we blended hydrogen-deuterium exchange mass spectrometry and computational docking to elucidate the molecular architecture and geometry of USP30 complex formation with USP30, identifying structural rearrangements at the cleft of the USP30 thumb and palm subdomains. These studies suggest that USP30 binds to this thumb-palm cleft, which guides the ubiquitin C terminus into the active site, thereby preventing ubiquitin binding and isopeptide bond cleavage, and confirming its importance in the inhibitory process. Our data will pave the way for the design and development of next-generation inhibitors targeting USP30 and associated deubiquitinylases.
Topics: Deubiquitinating Enzymes; Mitochondrial Proteins; Mitophagy; Ubiquitin-Protein Ligases; Ubiquitination; Sulfonamides
PubMed: 37385347
DOI: 10.1016/j.mcpro.2023.100609 -
Frontiers in Oncology 2023Cancer metabolism has emerged as a pivotal area of research recently. The ability to visualize and comprehend the metabolic processes of cancer holds immense clinical... (Review)
Review
Cancer metabolism has emerged as a pivotal area of research recently. The ability to visualize and comprehend the metabolic processes of cancer holds immense clinical value, particularly in the diagnosis of malignant tumors and the assessment of treatment responses. Deuterium Metabolic Imaging (DMI), as a robust, simple, and versatile MR spectroscopic imaging tool, demonstrates promise in tumor diagnosis and treatment efficacy assessment. This review explored the latest developments and applications of DMI in oncology across various tumor metabolic axes, with a specific emphasis on its potential for clinical translation. DMI offers invaluable insights into tumor biology, treatment responses, and prognostic outcomes. Notably, DMI can identify early responses to immunotherapy, a prominent area of current research interest. In conclusion, DMI harbors the potential to evolve into a convenient and efficient imaging technique in clinical practice, thereby advancing precision medicine and improving the diagnosis and evaluation of cancer treatments.
PubMed: 38090478
DOI: 10.3389/fonc.2023.1285209 -
Proceedings of the National Academy of... Jul 2023E3 ubiquitin ligase Mdm2 facilitates β-arrestin ubiquitination, leading to the internalization of G protein-coupled receptors (GPCRs). In this process, β-arrestins...
E3 ubiquitin ligase Mdm2 facilitates β-arrestin ubiquitination, leading to the internalization of G protein-coupled receptors (GPCRs). In this process, β-arrestins bind to Mdm2 and recruit it to the receptor; however, the molecular architecture of the β-arrestin-Mdm2 complex has not been elucidated yet. Here, we identified the β-arrestin-binding region (ABR) on Mdm2 and solved the crystal structure of β-arrestin1 in complex with Mdm2 peptide. The acidic residues of Mdm2 bind to the positively charged concave side of the β-arrestin1 N-domain. The C-tail of β-arrestin1 is still bound to the N-domain, indicating that Mdm2 binds to the inactive state of β-arrestin1, whereas the phosphorylated C-terminal tail of GPCRs binds to activate β-arrestins. The overlapped binding site of Mdm2 and GPCR C-tails on β-arrestin1 suggests that the binding of GPCR C-tails might trigger the release of Mdm2. Moreover, hydrogen/deuterium exchange experiments further show that Mdm2 binding to β-arrestin1 induces the interdomain interface to be more dynamic and uncouples the IP-induced oligomer of β-arrestin1. These results show how the E3 ligase, Mdm2, interacts with β-arrestins to promote the internalization of GPCRs.
Topics: beta-Arrestins; Ubiquitin-Protein Ligases; Arrestins; beta-Arrestin 1; Ubiquitination; Receptors, G-Protein-Coupled; beta-Arrestin 2; Phosphorylation
PubMed: 37399373
DOI: 10.1073/pnas.2301934120 -
Nature Communications Jul 2023Membrane efflux pumps play a major role in bacterial multidrug resistance. The tripartite multidrug efflux pump system from Escherichia coli, AcrAB-TolC, is a target for...
Membrane efflux pumps play a major role in bacterial multidrug resistance. The tripartite multidrug efflux pump system from Escherichia coli, AcrAB-TolC, is a target for inhibition to lessen resistance development and restore antibiotic efficacy, with homologs in other ESKAPE pathogens. Here, we rationalize a mechanism of inhibition against the periplasmic adaptor protein, AcrA, using a combination of hydrogen/deuterium exchange mass spectrometry, cellular efflux assays, and molecular dynamics simulations. We define the structural dynamics of AcrA and find that an inhibitor can inflict long-range stabilisation across all four of its domains, whereas an interacting efflux substrate has minimal effect. Our results support a model where an inhibitor forms a molecular wedge within a cleft between the lipoyl and αβ barrel domains of AcrA, diminishing its conformational transmission of drug-evoked signals from AcrB to TolC. This work provides molecular insights into multidrug adaptor protein function which could be valuable for developing antimicrobial therapeutics.
Topics: Membrane Transport Proteins; Escherichia coli Proteins; Multidrug Resistance-Associated Proteins; Biological Transport; Escherichia coli; Anti-Bacterial Agents; Bacterial Outer Membrane Proteins
PubMed: 37463890
DOI: 10.1038/s41467-023-39615-x