Cellular Stress Research Articles

Full-length direct RNA sequencing uncovers stress granule-dependent RNA decay upon cellular stress.

Cells react to stress by triggering response pathways, leading to extensive alterations in the transcriptome to restore cellular homeostasis. The role of RNA metabolism in shaping the cellular response to stress is vital, yet the global changes in RNA stability under these conditions remain unclear. In this work, we employ direct RNA sequencing with nanopores, enhanced by 5' end adapter ligation, to comprehensively interrogate the human transcriptome at single-molecule and -nucleotide resolution. By developing a statistical framework to identify robust RNA length variations in nanopore data, we find that cellular stress induces prevalent 5' end RNA decay that is coupled to translation and ribosome occupancy. Unlike typical RNA decay models in normal conditions, we show that stress-induced RNA decay is dependent on XRN1 but does not depend on deadenylation or decapping. We observed that RNAs undergoing decay are predominantly enriched in the stress granule transcriptome while inhibition of stress granule formation via genetic ablation of G3BP1 and G3BP2 rescues RNA length. Our findings reveal RNA decay as a key component of RNA metabolism upon cellular stress that is dependent on stress granule formation.

Biomimetic astrocyte cell membrane-fused nanovesicles for protecting neurovascular units in hypoxic ischemic encephalopathy

Hypoxic ischemic encephalopathy (HIE) refers to neonatal hypoxic brain injury caused by severe asphyxia during the perinatal period. With a high incidence rate and poor prognosis, HIE accounts for 2.4% of the global disease burden, imposing a heavy burden on families and society. Current clinical treatment for HIE primarily focuses on symptomatic management and supportive care. Therefore, the developments of effective treatment strategies and new drug formulations are critical for improving the prognosis of HIE patients. In order to protect the compromised neurovascular units after HIE, we prepared membrane-fused nanovesicles for delivering rapamycin and si EDN1 (TRCAM@RAPA@si EDN1). Due to the homotypic targeting feature of membrane-fused nanovesicles, we employed astrocyte membranes as synthetic materials to improve the targeting of astrocytes in brain while reducing the clearance of nanovesicles by circulatory system. Additionally, the surface of cell membrane was modified with CXCR3 receptors, enhancing the homing of nanovesicles to infarcted lesions. Lipid vesicles were modified with TK and RVG29 transmembrane peptides, enabling responsive release of internal drugs and blood-brain barrier penetration. Internally loaded rapamycin could promote protective autophagy in astrocytes, improve cellular oxidative stress, while si EDN1 could reduce the expression level of endothelin gene, thereby reducing secondary damage to neurovascular units.Graphical

Metabolic labeling of RNA using ribonucleoside analogs enables the evaluation of RNA synthesis and degradation rates.

Long noncoding RNAs (lncRNAs) are transcripts exceeding 200 nucleotides that do not encode proteins. Despite lacking protein-coding capabilities, lncRNAs play crucial roles in cellular processes, including gene-expression modulation and structural maintenance. The study of lncRNAs has evolved significantly since 2009, with advancements in analytical methodologies providing new insights into their functions and dynamics. Key developments include BRIC-Seq, SLAM-Seq, TUC-Seq, TimeLapse-seq, and Dyrec-Seq. These methodologies have enabled researchers to investigate lncRNA behavior under various conditions, including cellular stress responses and complex biologic systems. Future challenges include developing comprehensive techniques for identifying lncRNA-interacting proteins and advancing in vivo methodologies using model organisms. As the field progresses, integrating these technologies will enhance our understanding of lncRNA biology, potentially leading to novel therapeutic strategies and deeper insights into gene-regulation mechanisms.

The UFMylation pathway is impaired in Alzheimer’s disease

BackgroundAlzheimer’s disease (AD) is characterized by the presence of neurofibrillary tangles made of hyperphosphorylated tau and senile plaques composed of beta-amyloid. These pathognomonic deposits have been implicated in the pathogenesis, although the molecular mechanisms and consequences remain undetermined. UFM1 is an important, but understudied ubiquitin-like protein that is covalently attached to substrates. UFMylation has recently been identified as major modifier of tau aggregation upon seeding in experimental models. However, potential alterations of the UFM1 pathway in human AD brain have not been investigated yet.MethodsHere we used frontal and temporal cortex samples from individuals with or without AD to measure the protein levels of the UFMylation pathway in human brain. We used multivariable regression analyses followed by Bonferroni correction for multiple testing to analyze associations of the UFMylation pathway with neuropathological characteristics, primary biochemical measurements of tau and additional biochemical markers from the same cases. We further studied associations of the UFMylation cascade with cellular stress pathways using Spearman correlations with bulk RNAseq expression data and functionally validated these interactions using gene-edited neurons that were generated by CRISPR-Cas9.ResultsCompared to controls, human AD brain had increased protein levels of UFM1. Our data further indicates that this increase mainly reflects conjugated UFM1 indicating hyperUFMylation in AD. UFMylation was strongly correlated with pathological tau in both AD-affected brain regions. In addition, we found that the levels of conjugated UFM1 were negatively correlated with soluble levels of the deUFMylation enzyme UFSP2. Functional analysis of UFM1 and/or UFSP2 knockout neurons revealed that the DNA damage response as well as the unfolded protein response are perturbed by changes in neuronal UFM1 signaling.ConclusionsThere are marked changes in the UFMylation pathway in human AD brain. These changes are significantly associated with pathological tau, supporting the idea that the UFMylation cascade might indeed act as a modifier of tau pathology in human brain. Our study further nominates UFSP2 as an attractive target to reduce the hyperUFMylation observed in AD brain but also underscores the critical need to identify risks and benefits of manipulating the UFMylation pathway as potential therapeutic avenue for AD.

DNMT1 prolonged absence is a tunable cellular stress that triggers cell proliferation arrest to protect from major DNA methylation loss

Methylation of cytosine in CpG dinucleotides is an epigenetic modification carried out by DNA-methyltransferases (DNMTs) that contributes to chromatin condensation and structure and, thus, to gene transcription regulation and chromosome stability. DNMT1 maintains the DNA methylation pattern of the genome at each cell cycle by copying it to the newly synthesized DNA strand during the S-phase. DNMT1 pharmacological inhibition as well as genetic knockout and knockdown, leads to passive DNA methylation loss. However, these strategies have been associated with different cell fates, even in the same cell background, suggesting that they can question the interpretation of the obtained results. Using a cell system in which endogenous DNMT1 is fused with an inducible degron and can be rapidly degraded, we found that in non-tumoral RPE-1 cells, DNMT1 loss progressively induced cell proliferation slowing-down and cell cycle arrest at the G1/S transition. The latter is due to p21 activation, which is partly mediated by p53 and leads to a global reduction in DNA methylation. DNMT1 restoration rescues cell proliferation, indicating that its deregulation is sensed as tunable cellular stress.

Fluoxetine inhibited RANKL-induced osteoclastic differentiation in vitro

Abstract Selective serotonin reuptake inhibitor correlates with decreased bone mineral density and impedes orthodontic tooth movement. The present study aimed to examine the effects of fluoxetine on osteoclast differentiation and function. Human peripheral blood mononuclear cells (hPBMCs) and murine RAW264.7 cells were cultured with RANKL to stimulate osteoclast differentiation. The resulting multinucleated cells displayed characteristics of mature osteoclasts. Fluoxetine at 0.01–1 μM did not impact cellular viability or oxidative stress. However, 10 μM fluoxetine significantly reduced clonal growth, cell viability, and increased cytotoxicity and lipid peroxidation in RAW 264.7 cells. Further, application of 0.1 μM fluoxetine potently suppressed osteoclast differentiation of both RAW264.7 and hPBMCs, with reduced osteoclast numbers and downregulation of osteoclastic genes matrix metalloproteinase-9, cathepsin K, and integrin β3 at mRNA and protein levels. Fluoxetine also disrupted F-actin ring formation essential for osteoclast resorptive function. Mechanistically, fluoxetine inhibited NF-kB signaling by reducing phosphorylation of pathway members IκBα and p65, preventing IκBα degradation and blocking p65 nuclear translocation. In conclusion, this study demonstrates fluoxetine suppressing osteoclast differentiation in association with disrupting NF-kB activation, providing insight into orthodontic treatment planning for patients taking fluoxetine.

Intertwining of Cellular Osmotic Stress Handling Mechanisms and Heavy Metal Accumulation

Intertwining of Cellular Osmotic Stress Handling Mechanisms and Heavy Metal Accumulation

Sulfur Amino Acid Restriction Mitigates High-Fat Diet-Induced Molecular Alterations in Cardiac Remodeling Primarily via FGF21-Independent Mechanisms

Background/Objectives: Dietary sulfur amino acid restriction (SAAR) elicits various health benefits, some mediated by fibroblast growth factor 21 (FGF21). However, research on SAAR’s effects on the heart is limited and presents mixed findings. This study aimed to evaluate SAAR-induced molecular alterations associated with cardiac remodeling and their dependence on FGF21. Methods: Male C57BL/6J wild-type and FGF21 knockout mice were randomized into four dietary regimens, including normal fat and high-fat diets (HFDs) with and without SAAR, over five weeks. Results: SAAR significantly reduced body weight and visceral adiposity while increasing serum FGF21 levels. In the heart, SAAR-induced molecular metabolic alterations are indicative of enhanced lipid utilization, glucose uptake, and mitochondrial biogenesis. SAAR also elicited opposing effects on the cardiac gene expression of FGF21 and adiponectin. Regarding cellular stress responses, SAAR mitigated the HFD-induced increase in the cardiac expression of genes involved in oxidative stress, inflammation, and apoptosis, while upregulating antioxidative genes. Structurally, SAAR did not induce alterations indicative of cardiac hypertrophy and it counteracted HFD-induced fibrotic gene expression. Overall, most alterations induced by SAAR were FGF21-independent, except for those related to lipid utilization and glucose uptake. Conclusions: Altogether, SAAR promotes cardiac alterations indicative of physiological rather than pathological remodeling, primarily through FGF21-independent mechanisms.

In Silico Enabled Discovery of KAI-11101, a Preclinical DLK Inhibitor for the Treatment of Neurodegenerative Disease and Neuronal Injury.

Dual leucine zipper kinase (DLK), expressed primarily in neuronal cells, is a regulator of neuronal degeneration in response to cellular stress from chronic disease or neuronal injury. This makes it an attractive target for the treatment of neurodegenerative diseases such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis, and neuronal injury, such as chemotherapy-induced peripheral neuropathy. Here, we describe the discovery of a potent, selective, brain-penetrant DLK inhibitor, KAI-11101 (59). Throughout the program's progression, medicinal chemistry challenges such as potency, hERG inhibition, CNS penetration, CYP3A time-dependent inhibition, and kinase selectivity were overcome through the implementation of cutting-edge in silico tools. KAI-11101 displayed an excellent in vitro safety profile and showed neuroprotective properties in an ex vivo axon fragmentation assay as well as dose-dependent activity in a mouse PD model.

AET-net: A framework for subtype classification based on the multi-omics data of breast cancer

Breast cancer (BC) is one of the most prevalent cancers worldwide and remains a significant global public health challenge. The biomechanical characteristics of tumor microenvironments provide critical insights into cellular interactions and mechanical stress responses that potentially influence cancer progression. The integration and analysis of multi-omics data for BC subtype classification present substantial challenges, including high-dimensional data complexity and difficulties in integrating heterogeneous omics data characteristics. To address these challenges, we propose an Autoencoder and Transformer integrated neural network (AET-net) classification framework. The experimental results demonstrate that our model achieves significant performance improvements in predicting BC subtypes based on integrated multi-omics datasets, with an Accuracy of 0.912 and an AUC of 0.9862. These results not only validate the high accuracy of our model in BC subtype classification, providing a valuable tool for diagnostic decision support, but also demonstrate the potential of integrated multi-omics data analysis in enhancing the precision and efficiency of BC subtype identification.

Endothelial Cell Senescence Effect on the Blood-Brain Barrier in Stroke and Cognitive Impairment.

Age is an important risk factor of stroke, cognitive decline, and dementia. Senescent endothelial cells (ECs) accumulate with advancing age through exposure to cellular stress, such as that exerted by hypertension and diabetes. These senescent ECs have altered characteristics, such as altered tight junction proteins, use of a more indiscriminate transcellular transport system, increased inflammation, and increased immune cell interactions. ECs are the main component of the blood-brain barrier (BBB), separating the brain from systemic circulation. As senescent ECs accumulate in the BBB, their altered functioning results in the disruption of the barrier. They have inadequate barrier-forming properties, disrupted extracellular matrix, and increased transcytosis, resulting in an overly permeable barrier. This disruption of the BBB can have important effects in stroke and cognitive impairment, as presented in this review. Besides increasing the permeability of the BBB, senescent ECs can also impair angiogenesis and vascular remodeling, which in ischemic stroke may increase risk of hemorrhagic transformation and worsen outcomes. Senescent ECs may also contribute to microvascular dysfunction, with disruption of cerebral perfusion and autoregulation. These may contribute to vascular cognitive impairment along with increased permeability. With an aging population, there is growing interest in targeting senescence. Several ongoing trials have been evaluating whether senolytics can slow aging, improve vascular health, and reduce the risk of stroke and cognitive decline.

HBV Remodels PP2A Complexes to Rewire Kinase Signaling in Hepatocellular Carcinoma.

Hepatitis B virus (HBV) infections promote liver cancer initiation by inducing inflammation and cellular stress. Despite the primarily indirect effect on oncogenesis, HBV is associated with a recurrent genomic phenotype in HCC, suggesting that it impacts the biology of established HCC. Characterization of the interaction of HBV with host proteins and the mechanistic contributions of HBV to HCC initiation and maintenance could provide insights into HCC biology and uncover therapeutic vulnerabilities. Here, we used affinity purification mass spectrometry to comprehensively map a network of 145 physical interactions between HBV and human proteins in hepatocellular carcinoma (HCC). A subset of the host factors targeted by HBV proteins were preferentially mutated in non-HBV-associated HCC, suggesting that their interaction with HBV influences HCC biology. HBV interacted with proteins involved in mRNA splicing, mitogenic signaling, and DNA repair, with the latter set interacting with the HBV oncoprotein X (HBx). HBx remodeled the PP2A phosphatase complex by excluding striatin regulatory subunits from the PP2A holoenzyme, and the HBx effects on PP2A caused Hippo kinase activation. In parallel, HBx activated mTOR complex 2 (mTORC2), which can prevent YAP degradation. mTORC2-mediated upregulation of YAP was observed in human HCC specimens and mouse HCC models and could be targeted with mTOR kinase inhibitors. Thus, HBV interaction with host proteins rewires HCC signaling rather than directly activating mitogenic pathways, provide an alternative paradigm for the cellular effects of a tumor promoting virus.

Bioinformatic Analysis of Actin-Binding Proteins in the Nucleolus During Heat Shock

Background/Objectives: Actin plays a crucial role not only in the cytoplasm, but also in the nucleus, influencing various cellular behaviors, including cell migration and gene expression. Recent studies reveal that nuclear actin dynamics is altered by cellular stresses, such as DNA damage; however, the effect of heat shock on nuclear actin dynamics, particularly in the nucleolus, remains unclear. This study aims to elucidate the contribution of nucleolar actin to cellular responses under heat shock conditions. Methods: Nuclear actin dynamics in response to heat shock were investigated using nAC-GFP, a GFP-tagged actin chromobody, to visualize nuclear actin in HeLa cells. Bioinformatic analyses were also performed. Results: Heat shock induced the reversible assembly of nAC-GFP in the nucleolus, with disassembly occurring upon recovery in a heat shock protein (Hsp) 70-dependent manner. Because the nucleolus, formed via liquid–liquid phase separation (LLPS), sequesters misfolded proteins under heat shock to prevent irreversible aggregation, we hypothesized that nucleolar actin-binding proteins might also be sequestered in a similar manner. Using several databases, we identified 47 actin-binding proteins localized in the nucleolus and determined the proportion of intrinsically disordered regions (IDRs) known to promote LLPS. Our analysis revealed that many of these 47 proteins exhibited high levels of IDRs. Conclusions: The findings from our bioinformatics analysis and further cellular studies may help elucidate new roles for actin in the heat shock response.

Single-cell RNA sequencing in stroke and traumatic brain injury: Current achievements, challenges, and future perspectives on transcriptomic profiling.

Single-cell RNA sequencing (scRNA-seq) is a high-throughput transcriptomic approach with the power to identify rare cells, discover new cellular subclusters, and describe novel genes. scRNA-seq can simultaneously reveal dynamic shifts in cellular phenotypes and heterogeneities in cellular subtypes. Since the publication of the first protocol on scRNA-seq in 2009, this evolving technology has continued to improve, through the use of cell-specific barcodes, adoption of droplet-based systems, and development of advanced computational methods. Despite induction of the cellular stress response during the tissue dissociation process, scRNA-seq remains a popular technology, and commercially available scRNA-seq methods have been applied to the brain. Recent advances in spatial transcriptomics now allow the researcher to capture the positional context of transcriptional activity, strengthening our knowledge of cellular organization and cell-cell interactions in spatially intact tissues. A combination of spatial transcriptomic data with proteomic, metabolomic, or chromatin accessibility data is a promising direction for future research. Herein, we provide an overview of the workflow, data analyses methods, and pros and cons of scRNA-seq technology. We also summarize the latest achievements of scRNA-seq in stroke and acute traumatic brain injury, and describe future applications of scRNA-seq and spatial transcriptomics.

The Influence of Extra-Ribosomal Functions of Eukaryotic Ribosomal Proteins on Viral Infection

The eukaryotic ribosome is a large ribonucleoprotein complex consisting of four types of ribosomal RNA (rRNA) and approximately 80 ribosomal proteins (RPs), forming the 40S and 60S subunits. In all living cells, its primary function is to produce proteins by converting messenger RNA (mRNA) into polypeptides. In addition to their canonical role in protein synthesis, RPs are crucial in controlling vital cellular processes such as cell cycle progression, cellular proliferation, differentiation, DNA damage repair, genome structure maintenance, and the cellular stress response. Viruses, as obligate intracellular parasites, depend completely on the machinery of the host cell for their replication and survival. During viral infection, RPs have been demonstrated to perform a variety of extra-ribosomal activities, which are especially important in viral disease processes. These functions cover a wide range of activities, ranging from controlling inflammatory responses and antiviral immunity to promoting viral replication and increasing viral pathogenicity. Deciphering the regulatory mechanisms used by RPs in response to viral infections has greatly expanded our understanding of their functions outside of the ribosome. Furthermore, these findings highlight the promising role of RPs as targets for the advancement of antiviral therapies and the development of novel antiviral approaches. This review comprehensively examines the many functions of RPs outside of the ribosome during viral infections and provides a foundation for future research on the host–virus interaction.

Equivocating and Deliberating on the Probability of COVID-19 Infection Serving as a Risk Factor for Lung Cancer and Common Molecular Pathways Serving as a Link

The COVID-19 infection caused by SARS-CoV-2 in late 2019 posed unprecedented global health challenges of massive proportions. The persistent effects of COVID-19 have become a subject of significant concern amongst the medical and scientific community. This article aims to explore the probability of a link between the COVID-19 infection and the risk of lung cancer development. First, this article reports that SARS-CoV-2 induces severe inflammatory response and cellular stress, potentially leading to tumorigenesis through common pathways between SARS-CoV-2 infection and cancer. These pathways include the JAK/STAT3 pathway which is activated after the initiation of cytokine storm following SARS-CoV-2 infection. This pathway is involved in cellular proliferation, differentiation, and immune homeostasis. The JAK/STAT3 pathway is also hyperactivated in lung cancer which serves as a link thereof. It predisposes patients to lung cancer through myriad molecular mechanisms such as DNA damage, genomic instability, and cell cycle dysregulation. Another probable pathway to tumorigenesis is based on the possibility of an oncogenic nature of SARS-CoV-2 through hijacking the p53 protein, leading to cell oxidative stress and interfering with the DNA repair mechanisms. Finally, this article highlights the overexpression of the SLC22A18 gene in lung cancer. This gene can be overexpressed by the ZEB1 transcription factor, which was found to be highly expressed during COVID-19 infection.

Senescence and Stress Signaling Pathways in Corneal Cells After Nitrogen Mustard Injury.

Mustard gas keratopathy (MGK), a complication of exposure to sulfur mustard, is a blinding ocular surface disease involving key cellular pathways, including apoptosis, oxidative stress, and inflammation. Recent studies indicate that cellular senescence contributes to the pathophysiology of mustard gas toxicity. This study aimed to assess senescence and stress-related pathways-particularly mitogen-activated protein kinase (MAPK) signaling-in nitrogen mustard (NM)-induced corneal injury. In vitro, primary human corneal epithelial (P-HCECs), primary human corneal mesenchymal stromal cells (hcMSCs), and human corneal-limbal epithelial cell (HCLE) lines were exposed to varying concentrations of NM. The results demonstrated a dose-dependent increase in cellular senescence, characterized by reduced Ki67 expression, elevated p16, and p21 mRNA levels, as well as activation of the MAPK pathway activation. Treatment with a selective p38-MAPK inhibitor significantly reduced senescence markers and improved cell proliferation following exposure to NM. Overall, these studies indicate that NM exposure triggers cellular senescence and stress-related MAPK signaling, while p38-MAPK inhibition mitigates these effects, suggesting a potential therapeutic strategy.

ADAR1 expression in different cancer cell lines and its change under heat shock.

Adenosine deaminase acting on RNA 1 (ADAR1) plays an essential role in the development of malignancies by modifying the expression of different oncogenes. ADAR1 presents three distinct activities: adenosine-to-inosine RNA editing, modulating IFN pathways, and response to cellular stress factors. Following stressors such as heat shock, ADAR1p110 isoform relocates from the nucleus to the cytoplasm, where it suppresses RNA degradation which leads to the arrest of apoptosis and cell survival. In this study, we assessed the expression of ADAR1 across different cancer cell lines. We revealed that the presence of ADAR1 varies between cells of different origins and that a high transcript level does not reflect protein abundance. Additionally, we subjected cells to a heat shock in order to evaluate how cellular stress factors affect the expression of ADAR1. Our results indicate that ADAR1 transcript and protein levels are relatively stable and do not change under heat shock in examined cell lines. This research lays a groundwork for future directions on ADAR1-related studies suggesting in which types of cancer ADAR1 may be a promising target for novel therapeutic approaches.

Mitochondrial and ER contact sites related to physiology and diseases

Mitochondria and the endoplasmic reticulum (ER) are indispensable organelles in eukaryotic cells, playing pivotal roles in energy production, calcium homeostasis, and lipid synthesis. The specialized contact sites between these two organelles, known as mitochondria-associated ER membranes (MERCs), facilitate fundamental cellular functions such as lipid metabolism, and apoptosis. Disruption of MERCs is implicated in various diseases, including neurodegenerative disorders, cardiovascular diseases, diabetes. This review delves into the dynamics of mitochondria, the importance of MERCs, and their implications in disease pathogenesis. We have explored the processes of mitochondrial fusion and fission, regulated by proteins such as mitofusins and dynamin-related protein 1, and their impact on mitochondrial quality control and cellular stress responses. Furthermore, we investigate the role of the ER in mitochondrial dynamics, with a focus on how ER-mitochondria interactions influence cellular homeostasis. By unraveling the complex mechanisms governing MERCs, we can identify potential therapeutic targets for diseases associated with MERC dysfunction, thereby offering new avenues for treatment.

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 of mCrip2 impaired myogenesis, likely due to Cu accumulation in cells. CUT&RUN and transcriptome analyses revealed its association with gene promoters, including MyoD1 and metallothioneins, 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.