CMPK1 Human

What is the function of CMPK1 in human cells?

CMPK1 functions primarily as an enzyme that catalyzes the phosphorylation of pyrimidine nucleoside monophosphates, particularly UMP and CMP, using ATP as a phosphate donor. This reaction produces the corresponding nucleoside diphosphates, which are essential intermediates in nucleic acid synthesis pathways. The enzyme shows preference for UMP and CMP as phosphate acceptors but also displays broad nucleoside diphosphate kinase activity . CMPK1 plays a crucial role in de novo pyrimidine nucleotide biosynthesis, which is essential for DNA and RNA synthesis .

Additionally, CMPK1 has significant importance in the activation of pyrimidine analogs used as chemotherapeutic agents against human cancers and pathogenic viruses . This dual role in both normal metabolism and drug activation makes CMPK1 particularly relevant for understanding both physiological processes and therapeutic resistance mechanisms.

What are the common methods to detect CMPK1 expression in human tissues?

Several methodological approaches can be employed to detect CMPK1 expression in human tissues, each with specific advantages depending on research objectives:

Immunohistochemistry (IHC) using specific antibodies against CMPK1 is widely used for tissue microarray (TMA) analysis, allowing visualization of both nuclear and cytoplasmic localization of the protein . For protein quantification, Western blotting (WB) using validated antibodies such as rabbit polyclonal anti-CMPK1 can detect the approximately 28kDa protein in cell or tissue lysates .

For transcriptional analysis, quantitative real-time PCR (qRT-PCR) measures CMPK1 mRNA levels, while RNA sequencing provides a comprehensive view of expression in relation to other genes. Mass spectrometry (MS) offers high-resolution detection and quantification of CMPK1 protein, though correlation analysis has shown that MS-derived intensity may not directly correspond to IHC measurements of subcellular CMPK1 pools .

Increasingly, integrated approaches combining transcriptomics and proteomics, as demonstrated in type 2 diabetes research, provide multi-level characterization of CMPK1 expression patterns and associated molecular networks .

How is CMPK1 regulated in normal cellular processes?

The regulation of CMPK1 in normal cellular processes involves multiple mechanisms that ensure appropriate enzymatic activity according to cellular needs. At the transcriptional level, CMPK1 expression appears to be influenced by cell cycle progression, with evidence suggesting connections to DNA replication pathways .

Post-translational regulation represents another important mechanism, with particular attention to subcellular localization. CMPK1 has been observed to localize predominantly in the cytoplasm but can also enter the nucleus in certain cellular contexts, such as in HeLa S3 cancer cells . This translocation between cellular compartments may serve as a regulatory switch for its function in different cellular processes.

Gene Set Enrichment Analysis (GSEA) has revealed that CMPK1 expression correlates with specific cellular pathways, including extracellular matrix organization (positive correlation) and DNA replication/cell cycle (negative correlation) . These associations suggest that CMPK1 regulation may be integrated with broader cellular programs related to proliferation and tissue remodeling.

What are the known subcellular localizations of CMPK1 and their biological significance?

CMPK1 demonstrates distinct subcellular localization patterns with significant biological implications. The enzyme predominantly localizes in the cytoplasm but can also enter the nucleus in specific contexts . This dual localization pattern has emerged as particularly important in cancer biology.

Nuclear CMPK1 (nCMPK1) expression has been associated with poor prognosis in triple-negative breast cancer (TNBC) patients, suggesting a function beyond its canonical enzymatic role in nucleotide metabolism . Gene Set Enrichment Analysis has revealed that nuclear CMPK1 expression correlates positively with extracellular matrix (ECM) organization pathways and negatively with DNA replication/cell cycle pathways in TNBC .

This suggests that the nuclear localization of CMPK1 may be involved in regulating gene expression programs related to tumor microenvironment remodeling and cell proliferation. Notably, correlation analysis found no significant relationship between cytoplasmic or nuclear CMPK1 staining intensities and total CMPK1 levels measured by mass spectrometry, indicating that subcellular distribution rather than total expression may be the critical factor in determining CMPK1's biological impact .

How does nuclear vs. cytoplasmic CMPK1 expression correlate with cancer prognosis?

Nuclear CMPK1 (nCMPK1) expression has emerged as a significant prognostic indicator in triple-negative breast cancer (TNBC), distinct from its cytoplasmic counterpart. Research utilizing immunohistochemistry on tissue microarrays has developed a histo-score system combining both intensity and quantity metrics (on a scale of 0-20) to evaluate CMPK1 subcellular expression .

According to multivariate Cox regression analyses stratified by hospital of origin, patients with higher nCMPK1 histo-scores (16-20) demonstrated significantly worse metastasis-free survival compared to those with low scores (0-5), with a hazard ratio (HR) of 2.66 (95% CI: 1.39 to 5.08, P=0.003) . The table below summarizes these findings:

Interestingly, cytoplasmic CMPK1 (cCMPK1) expression did not show the same prognostic significance. Gene Set Enrichment Analysis further established that nCMPK1 expression positively correlates with extracellular matrix organization pathways and negatively with DNA replication/cell cycle genes, providing mechanistic insights into how nuclear localization of CMPK1 might influence tumor progression and metastatic potential .

What experimental models are most suitable for studying CMPK1's role in pyrimidine metabolism?

For investigating CMPK1's role in pyrimidine metabolism, researchers should employ a multi-tiered experimental approach combining in vitro enzymatic assays, cell-based models, and clinical samples.

In vitro systems using recombinant CMPK1 protein produced in E. coli allow for precise kinetic analyses of enzymatic activity with various substrates . These assays can be designed to measure the phosphorylation rates of different pyrimidine monophosphates (UMP, CMP, dCMP) and assess the effects of potential inhibitors or activators on enzyme function.

Cell culture models with modulated CMPK1 expression (overexpression, knockdown, or knockout) are valuable for studying the metabolic consequences of altered CMPK1 activity in a cellular context. Cancer cell lines with different levels of intrinsic chemoresistance, such as the HCT-8 colorectal cancer line mentioned in literature, offer systems to investigate CMPK1's role in drug metabolism and resistance mechanisms .

For translational relevance, patient-derived xenografts and primary cell cultures from tumor samples can provide more physiologically relevant contexts. Isotope-labeling approaches using 13C or 15N-labeled pyrimidine precursors, combined with metabolomic analysis, enable tracking of nucleotide flux through the CMPK1-catalyzed step in the pyrimidine synthesis pathway.

How can researchers effectively analyze the impact of CMPK1 polymorphisms on drug response?

Analyzing the impact of CMPK1 polymorphisms on drug response requires a comprehensive approach integrating genomic, biochemical, and clinical methodologies. Researchers should begin with identification of clinically relevant CMPK1 genetic variants through next-generation sequencing of patient cohorts, focusing on those receiving nucleoside analog-based therapies (such as gemcitabine or 5-fluorouracil).

Research has shown that CMPK1 polymorphisms have prognostic significance in non-small cell lung cancer and pancreatic cancer patients treated with gemcitabine-based chemotherapy . For functional characterization, site-directed mutagenesis should be used to create recombinant CMPK1 proteins carrying the identified polymorphisms, followed by in vitro enzyme assays to assess how these variants affect the kinetic parameters for phosphorylation of both natural substrates and drug metabolites.

Cell-based assays using isogenic cell lines expressing different CMPK1 variants (created through CRISPR/Cas9 gene editing) can determine how these polymorphisms affect drug activation, cytotoxicity, and resistance development. For clinical correlation, retrospective and prospective studies should analyze associations between CMPK1 genotypes and patient outcomes using multivariate models that account for other known predictive factors.

Additionally, researchers should consider how CMPK1 variants might interact with other enzymes in nucleoside metabolic pathways, as polymorphisms in multiple genes may collectively influence drug response through epistatic effects.

What are the challenges in targeting CMPK1 for therapeutic intervention?

Targeting CMPK1 for therapeutic intervention presents several significant challenges that researchers must address. First, CMPK1's essential role in normal nucleotide metabolism means that complete inhibition may cause significant toxicity in healthy tissues. Developing compounds with sufficient selectivity between cancer cells and normal cells requires understanding the differential dependence on CMPK1 activity across tissue types.

Second, CMPK1's dual subcellular localization (cytoplasmic and nuclear) complicates targeting strategies, as compounds may need to reach both compartments to be fully effective. Based on research findings, nuclear CMPK1 correlates with poor prognosis in triple-negative breast cancer, suggesting this pool might be particularly important to target .

Third, CMPK1 plays a role in activating several anticancer nucleoside analogs, so inhibiting it could potentially antagonize existing chemotherapies—researchers must carefully consider this dual role when designing therapeutic strategies . Fourth, resistance mechanisms may emerge through compensatory upregulation of alternative kinases or nucleotide salvage pathways.

To overcome these challenges, researchers should: (1) develop targeted approaches that exploit differences in CMPK1 regulation between normal and cancer cells; (2) consider dual-targeting strategies that combine CMPK1 modulation with inhibition of complementary pathways; (3) explore the therapeutic window where CMPK1 modulation might enhance rather than antagonize nucleoside analog therapies; and (4) investigate tissue-specific delivery systems to improve targeting and reduce systemic toxicity.

How is CMPK1 involved in triple-negative breast cancer progression?

CMPK1 involvement in triple-negative breast cancer (TNBC) progression is primarily associated with its nuclear localization, which serves as an independent poor prognostic marker. Nuclear CMPK1 (nCMPK1) expression correlates with decreased metastasis-free survival in TNBC patients .

Mechanistically, Gene Set Enrichment Analysis (GSEA) revealed that higher nCMPK1 expression positively correlates with extracellular matrix (ECM) organization pathways and negatively with DNA replication/cell cycle pathways . This suggests that nuclear CMPK1 may contribute to TNBC progression through ECM remodeling, potentially enhancing invasive and metastatic capabilities of tumor cells.

The "core matrisome" and "ECM glycoproteins" gene sets showed the most significant positive correlation with nCMPK1 expression, indicating a specific relationship with stromal components of the tumor microenvironment . Interestingly, cytoplasmic CMPK1 did not show the same prognostic significance, highlighting the importance of subcellular localization in determining CMPK1's role in cancer progression.

The study utilized a histo-score system combining both intensity and quantity of CMPK1 nuclear staining, with patients having high scores (16-20) showing significantly worse outcomes (HR=2.66, 95% CI: 1.39 to 5.08, P=0.003) even after adjusting for other clinical factors . These findings suggest that nuclear CMPK1 may have functions beyond its canonical enzymatic role in nucleotide metabolism, potentially affecting transcriptional programs related to tumor-stroma interactions.

What mechanisms connect CMPK1 expression to type 2 diabetes pathophysiology?

The connection between CMPK1 expression and type 2 diabetes mellitus (T2DM) pathophysiology represents an emerging area of research. Recent integrated transcriptomic and proteomic analyses have identified CMPK1 as significantly upregulated in the liver tissues of T2DM patients, particularly in the context of obesity .

The protein-protein interaction (PPI) analysis indicated that CMPK1 was the core protein among six biomarkers that showed differential expression in both proteome and transcriptome datasets with consistent directional changes . Mechanistically, correlation analyses revealed significant associations between CMPK1 expression and key molecules in T2DM-related pathways at both protein and transcriptome levels, suggesting CMPK1 may be integrated into diabetes-specific molecular networks .

While the exact mechanisms remain to be fully elucidated, several potential pathways can be hypothesized: (1) CMPK1's role in nucleotide metabolism may impact energy homeostasis in liver cells, potentially affecting gluconeogenesis or glycogen synthesis; (2) altered pyrimidine nucleotide pools might influence insulin signaling pathways or glucose transporter expression; (3) CMPK1 might participate in metabolic inflammation processes characteristic of T2DM.

The significant upregulation of CMPK1 in diabetic liver tissue was validated through immunohistochemistry, confirming its differential expression at the protein level . These findings collectively suggest that CMPK1 could serve as a novel biomarker for screening and diagnosing T2DM in patients with obesity and potentially represent a new therapeutic target for obesity-related metabolic diseases.

How does CMPK1 contribute to resistance to nucleoside analog chemotherapies?

CMPK1 plays a crucial role in the activation of nucleoside analogs used as chemotherapeutic agents, and alterations in its expression or activity can significantly impact drug efficacy and resistance development. Decreased expression of CMPK1 mRNA has been associated with 5-fluorouracil resistance in the HCT-8 colorectal cancer cell line . This suggests that insufficient activation of the prodrug to its active metabolites may be one mechanism of resistance.

The activation pathway requires CMPK1 to phosphorylate the monophosphate forms of nucleoside analogs to their diphosphate states, a critical step in generating the active triphosphate forms that interfere with DNA synthesis . Additionally, certain genetic polymorphisms of CMPK1 have been reported as prognostic markers for non-small cell lung cancer and pancreatic cancer patients treated with gemcitabine-based chemotherapy, indicating that variations in enzyme structure or activity can affect treatment outcomes .

Resistance mechanisms may include: (1) downregulation of CMPK1 expression through transcriptional repression or epigenetic silencing; (2) mutations affecting the binding affinity for drug metabolites; (3) alterations in CMPK1 subcellular localization, potentially reducing drug activation efficiency; and (4) compensatory upregulation of alternative kinases or nucleotide salvage pathways.

To overcome resistance, researchers might consider combination strategies that target multiple enzymes in the activation pathway, development of nucleoside analogs that bypass the CMPK1-dependent step, or approaches to upregulate or restore CMPK1 expression in resistant cells.

What are the most effective techniques to study CMPK1 in patient-derived samples?

Studying CMPK1 in patient-derived samples requires a multi-modal approach that combines protein and gene expression analysis with functional assessment. Based on the methodologies described in the literature, several techniques have proven effective.

Immunohistochemistry (IHC) on tissue microarrays (TMAs) allows for visualization and quantification of CMPK1 in different subcellular compartments while preserving tissue architecture . A standardized histo-score system combining intensity (scale 0-4) and quantity (percentage of positive cells) metrics provides a reproducible measure of CMPK1 expression across patient cohorts .

For quantitative protein analysis, mass spectrometry (MS) offers high-resolution detection of CMPK1 and can be integrated with proteomic profiling to identify associated protein networks . RNA-sequencing or targeted qRT-PCR provides complementary data on CMPK1 transcript levels and potential splice variants.

For functional analyses, ex vivo culture of patient-derived samples with nucleoside analogs can assess CMPK1-dependent drug activation capacity. Additionally, integrated multi-omics approaches combining transcriptomics and proteomics have successfully identified CMPK1 as a biomarker in type 2 diabetes, demonstrating the power of this integrated strategy .

For genetic analysis, targeted sequencing of the CMPK1 gene can identify polymorphisms that may affect enzyme function or drug responses. When analyzing results, it's essential to consider potential confounding factors such as tissue heterogeneity, prior treatments, and comorbidities.

What are the recommended protocols for recombinant CMPK1 protein production?

The production of recombinant human CMPK1 protein requires careful optimization of expression systems and purification protocols to obtain functional protein suitable for enzymatic and structural studies. Escherichia coli has been successfully employed as an expression host for human CMPK1 .

The recommended protocol involves constructing an expression vector containing the CMPK1 coding sequence (228 amino acids) fused to a 20-amino acid His-tag at the N-terminus to facilitate purification . After transformation into an appropriate E. coli strain, protein expression should be induced under optimized conditions (temperature, inducer concentration, duration).

Purification typically employs a two-step process: (1) initial capture using nickel-affinity chromatography to bind the His-tagged protein, followed by (2) size-exclusion or ion-exchange chromatography for further purification. The final purified protein should be formulated in a stabilizing buffer such as: 20mM Tris-HCl buffer (pH 8.0) containing 20% glycerol (as a cryoprotectant), 1mM DTT (to maintain reduced cysteines), and 100mM NaCl (for ionic strength) .

Quality control should include SDS-PAGE to verify purity (>90% as indicated in the reference), mass spectrometry to confirm protein identity, and enzymatic activity assays to ensure functionality . For long-term storage, it is recommended to store the protein at -20°C with the addition of a carrier protein (0.1% HSA or BSA) to enhance stability, while avoiding multiple freeze-thaw cycles. For short-term use (2-4 weeks), the protein can be stored at 4°C .

How can researchers accurately measure CMPK1 enzymatic activity?

Accurately measuring CMPK1 enzymatic activity requires specialized assays that can detect the phosphorylation of pyrimidine nucleoside monophosphates. A coupled spectrophotometric assay represents one of the most reliable methods, where CMPK1 activity is linked to the oxidation of NADH, which can be monitored by decreasing absorbance at 340 nm.

In this system, the ADP produced by CMPK1 during phosphorylation of UMP or CMP is coupled to pyruvate kinase and lactate dehydrogenase reactions, allowing real-time monitoring of activity. Alternatively, radiometric assays using [γ-32P]ATP or [3H]-labeled substrates provide highly sensitive measurements of phosphoryl transfer, particularly useful when working with low enzyme concentrations or when assessing inhibitors.

For high-throughput screening applications, luminescence-based assays that detect ATP consumption or ADP production can be employed using commercially available kits. HPLC or capillary electrophoresis methods offer the advantage of directly quantifying both substrates and products, providing detailed kinetic parameters.

When conducting these assays, researchers should carefully control reaction conditions, including pH (typically 7.5-8.0), temperature (25-37°C), divalent cation concentration (usually Mg2+), and ionic strength. Enzyme concentration should be optimized to ensure linearity of the reaction rate over the measurement time. Michaelis-Menten kinetic analysis should be performed by varying substrate concentrations (both ATP and pyrimidine nucleoside monophosphates) to determine key parameters such as Km, Vmax, and kcat.

What are the best practices for CMPK1 immunohistochemistry analysis?

Immunohistochemistry (IHC) analysis of CMPK1 requires careful optimization to accurately assess both expression levels and subcellular localization patterns. For tissue preparation, formalin-fixed paraffin-embedded (FFPE) sections are commonly used, with tissue microarrays (TMAs) allowing for high-throughput analysis across multiple samples .

When selecting primary antibodies, validated antibodies with demonstrated specificity for CMPK1 should be used, such as rabbit polyclonal antibody described in the literature, which has been validated for IHC applications . For visualization systems, researchers should employ sensitive detection methods such as polymer-based detection systems with appropriate chromogens (typically DAB) that provide good signal-to-noise ratio.

Importantly, proper assessment of CMPK1 requires evaluation of both nuclear and cytoplasmic staining patterns separately, as these have distinct biological and clinical implications . A standardized scoring system should be implemented, such as the "histo-score" approach described in the literature, which combines both intensity (scale 0-4) and quantity (percentage of positive cells) metrics to create a composite score (range 0-20) .

Appropriate controls must be included in each staining run: positive controls (tissues known to express CMPK1), negative controls (omission of primary antibody), and isotype controls. To ensure reproducibility, at least two independent observers should score the samples blind to clinical data, with discrepancies resolved by consensus or a third observer.

For statistical analysis, appropriate tests for correlation with clinical parameters and survival outcomes should be included, with multivariate analyses to identify independent prognostic factors, as demonstrated in studies of CMPK1 in triple-negative breast cancer .

How can integrated transcriptomics and proteomics be applied to study CMPK1 function?

Integrated transcriptomics and proteomics approaches provide powerful tools for comprehensively characterizing CMPK1 function across multiple molecular levels. As demonstrated in recent research, this integrated strategy successfully identified CMPK1 as a biomarker in type 2 diabetes mellitus, highlighting its utility .

For transcriptomic analysis, RNA sequencing (RNA-seq) or microarray technology can be used to measure CMPK1 mRNA expression levels and identify co-expressed genes that may function in the same pathways. For proteomics, mass spectrometry-based approaches such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) enable quantification of CMPK1 protein levels and post-translational modifications that may affect its function.

Integration of these datasets requires sophisticated bioinformatic approaches: correlation analysis between CMPK1 transcript and protein levels can reveal post-transcriptional regulatory mechanisms; differential expression analysis across experimental conditions (such as disease vs. healthy states) can identify context-specific regulation of CMPK1; and network analysis approaches such as weighted gene co-expression network analysis (WGCNA) or protein-protein interaction (PPI) mapping can position CMPK1 within broader functional networks .

Gene Set Enrichment Analysis (GSEA), as used in triple-negative breast cancer research, provides a powerful method to identify biological pathways and processes associated with CMPK1 expression . For validation of findings from these high-throughput approaches, targeted techniques should be employed, including qRT-PCR for mRNA expression, western blotting or immunohistochemistry for protein levels and localization, and functional assays to assess enzymatic activity.

The integration of multi-omics data with clinical information enables the identification of CMPK1's role in disease processes and its potential utility as a biomarker or therapeutic target.