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Summary Cu+‐chaperones are a diverse group of proteins that allocate Cu+ions to specific copper proteins, creating different copper pools targeted to specific physiological processes.Symbiotic nitrogen fixation carried out in legume root nodules indirectly requires relatively large amounts of copper, for example for energy delivery via respiration, for which targeted copper deliver systems would be required.MtNCC1 is a nodule‐specific Cu+‐chaperone encoded in theMedicago truncatulagenome, with a N‐terminus Atx1‐like domain that can bind Cu+with picomolar affinities. MtNCC1 is able to interact with nodule‐specific Cu+‐importer MtCOPT1.MtNCC1is expressed primarily from the late infection zone to the early fixation zone and is located in the cytosol, associated with plasma and symbiosome membranes, and within nuclei. Consistent with its key role in nitrogen fixation,ncc1mutants have a severe reduction in nitrogenase activity and a 50% reduction in copper‐dependent cytochromecoxidase activity.A subset of the copper proteome is also affected in thencc1mutant nodules. Many of these proteins can be pulled down when using a Cu+‐loaded N‐terminal MtNCC1 moiety as a bait, indicating a role in nodule copper homeostasis and in copper‐dependent physiological processes. Overall, these data suggest a pleiotropic role of MtNCC1 in copper delivery for symbiotic nitrogen fixation.
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This content will become publicly available on December 5, 2025
Cysteine Rich Intestinal Protein 2 is a copper-responsive regulator of skeletal muscle differentiation and metal homeostasis
Copper (Cu) is essential for respiration, neurotransmitter synthesis, oxidative stress response, and transcription regulation, with imbalances leading to neurological, cognitive, and muscular disorders. Here we show the role of a novel Cu-binding protein (Cu-BP) in mammalian transcriptional regulation, specifically on skeletal muscle differentiation using murine primary myoblasts. Utilizing synchrotron X-ray fluorescence-mass spectrometry, we identified murine cysteine-rich intestinal protein 2 (mCrip2) as a key Cu-BP abundant in both nuclear and cytosolic fractions. mCrip2 binds two to four Cu+ions with high affinity and presents limited redox potential. CRISPR/Cas9-mediated deletion ofmCrip2impaired myogenesis, likely due to Cu accumulation in cells. CUT&RUN and transcriptome analyses revealed its association with gene promoters, includingMyoD1andmetallothioneins, suggesting a novel Cu-responsive regulatory role for mCrip2. Our work describes the significance of mCrip2 in skeletal muscle differentiation and metal homeostasis, expanding understanding of the Cu-network in myoblasts. Copper (Cu) is essential for various cellular processes, including respiration and stress response, but imbalances can cause serious health issues. This study reveals a new Cu-binding protein (Cu-BP) involved in muscle development in primary myoblasts. Using unbiased metalloproteomic techniques and high throughput sequencing, we identified mCrip2 as a key Cu-BP found in cell nuclei and cytoplasm. mCrip2 binds up to four Cu+ions and has a limited redox potential. Deleting mCrip2 using CRISPR/Cas9 disrupted muscle formation due to Cu accumulation. Further analyses showed that mCrip2 regulates the expression of genes like MyoD1, essential for muscle differentiation, and metallothioneins in response to copper supplementation. This research highlights the importance of mCrip2 in muscle development and metal homeostasis, providing new insights into the Cu-network in cells.
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- Award ID(s):
- 2320718
- PAR ID:
- 10634296
- Editor(s):
- Bhagwat, Ashok
- Publisher / Repository:
- PLOS
- Date Published:
- Journal Name:
- PLOS Genetics
- Volume:
- 20
- Issue:
- 12
- ISSN:
- 1553-7404
- Page Range / eLocation ID:
- e1011495
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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