CINP Human

What is CINP in the context of human molecular biology?

CINP (CDK2-interacting protein) functions as a critical genome maintenance protein within the DNA damage response (DDR) pathway. Methodologically, CINP should be approached as a checkpoint regulator that interacts with ATR-interacting protein (ATRIP) to maintain genomic stability. Research approaches should consider CINP's role in cell cycle regulation, particularly during replication stress conditions. When designing experiments, researchers should account for CINP's involvement in preventing the formation of replication-associated double-strand breaks (DSBs) through its interaction with the ATR-ATRIP complex .

To effectively study CINP's function, researchers typically employ techniques such as:

• RNAi silencing to observe phenotypic consequences

• Coimmunoprecipitation to verify protein interactions

• Immunofluorescence to track CINP localization

• Functional assays measuring DNA damage responses

How does CINP contribute to genome maintenance?

CINP maintains genomic integrity through several mechanisms that require specific methodological approaches to study. When CINP is silenced, cells exhibit increased γH2AX foci formation (a marker of DNA damage), phosphorylation of KAP1, and heightened sensitivity to hydroxyurea (HU) treatment . These phenotypes indicate CINP's critical role in preventing spontaneous DNA damage and responding to replication stress.

Methodologically, researchers can assess CINP's genome maintenance function through:

• Measuring micronuclei formation frequency following CINP depletion (higher frequencies indicate genomic instability)

• Quantifying γH2AX foci that colocalize with DNA repair proteins such as Mre11

• Evaluating sensitivity to replication stress-inducing agents through viability assays

• Assessing checkpoint integrity through cell cycle progression analysis

Experimental validation requires complementation studies with RNAi-resistant CINP cDNA to confirm that observed phenotypes are not off-target effects .

What is the Collegium Internationale Neuro-psychopharmacologicum (CINP)?

The Collegium Internationale Neuro-psychopharmacologicum (CINP) is an international organization dedicated to advancing worldwide neuroscience and education for brain and mental health . Methodologically, researchers engaging with this organization should understand its multifaceted approach to neuropsychopharmacology research dissemination through:

• Publishing the International Journal of Neuropsychopharmacology (IJNP), which serves as a major forum for rapid publication of high-quality research in both basic and clinical neuropsychopharmacology

• Supporting early-career researchers through fellowship programs

• Organizing international congresses and colloquia for knowledge exchange

• Promoting research that advances understanding of neuropsychopharmacological agents and provides insights into the biological basis of neuropsychiatric disorders

When considering CINP as a publication venue or collaboration platform, researchers should note that IJNP prioritizes original research that is of wide interest and clearly advances the field across the spectrum of biological and psychological fields of inquiry .

How does CINP interact with ATRIP to regulate ATR-dependent signaling?

CINP interacts specifically with the N-terminal half of the ATRIP coiled-coil domain, with the minimum binding region spanning amino acids 118-156 . Methodologically, this interaction can be investigated through:

• Yeast two-hybrid screens (which identified CINP as an ATRIP-interacting protein)

• Coimmunoprecipitation experiments with tagged proteins

• Domain deletion/mutation analysis to map interaction surfaces

• Functional assays measuring ATR-dependent phosphorylation events

The following table summarizes experimental approaches for studying CINP-ATRIP interactions:

The ATRIP coiled-coil domain is critical for ATRIP dimerization, stable ATR binding, accumulation at DNA lesions, and ATR-dependent checkpoint signaling . Consequently, CINP appears to regulate ATR-dependent signaling through its interaction with this essential domain.

What are the consequences of CINP silencing on cellular checkpoint integrity?

CINP silencing significantly impairs several aspects of cellular checkpoint integrity, requiring methodological approaches that can accurately measure these effects. Experimental evidence indicates that CINP depletion reduces:

• CHK1 phosphorylation following ionizing radiation (IR) and ultraviolet (UV) radiation exposure

• G2 checkpoint integrity

• Resistance to replication stress induced by hydroxyurea (HU)

Methodologically, researchers should employ a combination of techniques to comprehensively assess checkpoint integrity following CINP depletion:

• Western blotting to quantify phosphorylation of checkpoint proteins (e.g., CHK1)

• Flow cytometry to measure cell cycle distribution and checkpoint activation

• Clonogenic survival assays to determine sensitivity to genotoxic agents

• Immunofluorescence microscopy to visualize DNA damage marker accumulation

When interpreting results, it's important to note that while CINP silencing impairs CHK1 phosphorylation, the effect is not as severe as ATR silencing, suggesting CINP modulates rather than is essential for ATR activation .

How do mutations in CINP contribute to genomic instability?

Mutations or depletion of CINP contribute to genomic instability through multiple mechanisms, requiring specialized methodological approaches for investigation. When CINP is silenced, cells exhibit:

• Increased formation of γH2AX foci (indicating DNA double-strand breaks)

• Colocalization of these foci with DNA repair proteins like Mre11 (confirming they represent actual DNA damage sites)

• Enhanced frequency of micronuclei (a hallmark of genomic instability)

• Heightened sensitivity to replication stress-inducing agents

To methodically study CINP's role in genomic stability, researchers should:

• Perform sequencing of CINP in cells exhibiting chromosomal instability phenotypes

• Introduce structure-based mutations to disrupt specific protein interactions

• Utilize time-lapse microscopy to track chromosomal aberrations during mitosis

• Measure micronuclei formation as a quantitative readout of genomic instability

The experimental approach should include complementation studies with wild-type CINP to confirm that genomic instability phenotypes result specifically from CINP dysfunction rather than off-target effects .

What are the optimal CRISPR guide RNA designs for CINP knockout studies?

The optimal design of CRISPR guide RNAs (gRNAs) for CINP knockout studies requires careful consideration of specificity, efficiency, and experimental validation. Based on the Feng Zhang laboratory's design criteria from the Broad Institute, effective gRNAs for targeting the human CINP gene should:

• Uniquely target the CINP gene with minimal risk of off-target Cas9 binding elsewhere in the genome

• Be carefully selected to target functional domains or early exons to ensure complete knockout

• Be validated through sequence verification before use in genome editing experiments

Methodologically, researchers should:

• Order at least two gRNA constructs per gene to increase success probability

• Verify gRNA sequences against the target gene sequence, especially when targeting specific splice variants or exons

• Utilize vectors that include selection markers for efficient cell line generation

• Validate knockout through Western blotting, RT-PCR, and functional assays

When implementing CRISPR-based CINP knockout strategies, researchers should design experiments that include appropriate controls, such as non-targeting gRNAs and rescue experiments with CINP cDNA resistant to the gRNA targeting.

What techniques are most reliable for quantifying CINP expression levels?

Reliable quantification of CINP expression requires a combination of complementary techniques to ensure accuracy and specificity. Methodologically, researchers should consider:

• Quantitative RT-PCR for mRNA expression analysis

  • Design primers spanning exon-exon junctions to avoid genomic DNA amplification

  • Include multiple reference genes for normalization

  • Validate primer efficiency and specificity

• Western blotting for protein level quantification

  • Use validated antibodies with known specificity

  • Include appropriate loading controls

  • Employ quantitative analysis software for band intensity measurement

• Immunofluorescence for spatial distribution analysis

  • Include appropriate controls for antibody specificity

  • Use confocal microscopy for precise subcellular localization

  • Quantify signal intensity across multiple cells and experiments

• Proteomic approaches for global analysis

  • Mass spectrometry-based quantification

  • SILAC labeling for comparative analysis

  • Co-immunoprecipitation followed by mass spectrometry for interaction partners

Each technique offers distinct advantages, and researchers should select methods based on their specific experimental questions while being aware of the limitations of each approach.

How can researchers measure the impact of CINP on G2 checkpoint integrity?

Measuring CINP's impact on G2 checkpoint integrity requires methodological approaches that can precisely assess cell cycle progression and DNA damage responses. Based on research findings, effective methods include:

• Flow cytometry analysis of cell cycle distribution following DNA damage

  • Treatment with ionizing radiation or other genotoxic agents

  • Staining with propidium iodide for DNA content

  • Quantification of G2/M phase population

• Phospho-histone H3 (pH3) immunostaining to identify mitotic cells

  • Combined with DNA damage markers (e.g., γH2AX)

  • Quantification of mitotic entry despite DNA damage (checkpoint failure)

• Time-lapse microscopy of live cells

  • Expression of fluorescently tagged cell cycle markers

  • Tracking individual cells progressing through G2 to mitosis

  • Measuring timing of G2 arrest and resolution

• Western blot analysis of checkpoint signaling

  • Phosphorylation of CHK1 (reduced following CINP silencing)

  • ATR-dependent substrate phosphorylation

  • Cell cycle regulator status (e.g., CDC25, Cyclin B1)

When designing experiments to assess G2 checkpoint integrity, researchers should include appropriate positive controls (e.g., ATR inhibitors) and perform time-course analyses to capture the dynamic nature of checkpoint activation and resolution .

What research areas are prioritized by the CINP Research Fellowship Program?

The CINP Research Fellowship Program for 2025 prioritizes specific research areas that reflect cutting-edge developments in neuropsychopharmacology. Methodologically, researchers applying to this program should focus on:

• Digital innovations in translational research and clinical studies

• Cutting-edge methodologies and tools that enhance research

• Quality improvement (QI) approaches in neuropsychopharmacology research

The fellowship program places particular emphasis on collaborative research projects, with the expected outcome being a joint research initiative among participants. Methodologically, successful applications should demonstrate:

• Clear integration of digital technologies in neuropsychopharmacological research

• Innovative approaches to translating basic findings to clinical applications

• Implementation of quality improvement frameworks in research design

• Potential for international collaboration and knowledge exchange

Researchers should note that while the program includes online teaching modules with top researchers, the in-person colloquium in Melbourne, Australia (scheduled for June 15, 2025) is no longer compulsory, accommodating those unable to travel .

How can early-career researchers effectively apply for CINP research support?

Early-career researchers seeking CINP research support should adopt methodological approaches that align with the organization's mission and priorities. Based on the available information, effective application strategies include:

• Focusing research proposals on the specified theme for the current cycle (for 2025: digital innovations in translational research and clinical studies)

• Demonstrating a clear connection to neuropsychopharmacology, particularly research that:

  • Advances understanding of existing and new neuropsychopharmacological agents

  • Provides insights into the biological basis of neuropsychiatric disorders

  • Utilizes innovative methodologies from the spectrum of biological and psychological fields

• Preparing for participation in both:

  • Online teaching modules with field-leading researchers

  • The optional in-person colloquium before the 36th CINP World Congress

• Developing collaborative project plans that can be initiated through the fellowship program

Successful applicants who participate in the in-person colloquium will have their registration fee for the 36th CINP World Congress waived, and all participants who complete the full program will receive 'CINP Research Fellow' certificates .

What are the key requirements for publishing in the International Journal of Neuropsychopharmacology?

The International Journal of Neuropsychopharmacology (IJNP), as the official scientific journal of CINP, has specific methodological requirements for publication. Researchers should note:

• Publication focus and scope:

  • Research advancing understanding of neuropsychopharmacological agents (mode of action and clinical application)

  • Studies providing insights into the biological basis of neuropsychiatric disorders and their treatment

  • Work spanning the full spectrum of biological and psychological fields

  • Research utilizing classical and novel techniques in neuropsychopharmacology, neuroimaging, genetics, psychoneuroendocrinology, and neuropsychology

• Ethical considerations:

  • Human subjects must provide written informed consent

  • For minors or incapacitated individuals, consent must be obtained from parents or guardians

  • Research must adhere to publication and research ethics standards

• Publication process:

  • IJNP operates single-anonymized peer review (author identities known to editors and reviewers)

  • Upon acceptance, the Accepted Manuscript version is published within one week

  • The journal publishes 12 issues per year and follows a fully open access model with applicable charges

Researchers should prioritize original research that is of wide interest and clearly advances the field to maximize chances of acceptance in this prestigious neuropsychopharmacology journal.

How can researchers differentiate between direct and indirect effects of CINP modulation?

Differentiating between direct and indirect effects of CINP modulation presents a significant methodological challenge requiring sophisticated experimental approaches. Based on research findings, recommended strategies include:

• Rescue experiments with structure-based mutants

  • Wild-type CINP for complete rescue

  • Domain-specific mutants to identify essential functional regions

  • Phospho-site mutants to assess regulation mechanisms

• Temporal analysis of effects following CINP depletion

  • Acute vs. chronic depletion using inducible systems

  • Time-course experiments to determine primary vs. secondary effects

  • Correlation between CINP protein levels and phenotype onset

• Separation of function studies

  • Targeted disruption of specific protein interactions (e.g., CINP-ATRIP)

  • Domain deletion constructs that maintain some functions while eliminating others

  • Point mutations that affect specific activities

• Systems biology approaches

  • Transcriptomic analysis following CINP modulation

  • Proteomic profiling of signaling pathway alterations

  • Network analysis to identify direct targets vs. downstream effects

When interpreting results, researchers should consider that some phenotypes observed following CINP silencing (like γH2AX foci formation) have been directly validated through complementation with RNAi-resistant CINP cDNA, confirming they are not off-target effects .

What experimental models are most suitable for studying CINP function?

Selecting appropriate experimental models for studying CINP function requires careful consideration of the biological context and research questions. Based on published research, suitable models include:

• Human cell line systems

  • HeLa cells (validated for KAP1 phosphorylation assessment)

  • U2OS cells (validated for γH2AX foci formation)

  • Multiple cell types to confirm that phenotypes are not cell-type specific

• CRISPR-engineered model systems

  • CINP knockout cell lines using validated gRNA designs

  • Knock-in models with tagged CINP for localization and interaction studies

  • Heterozygous models to study gene dosage effects

• Inducible systems for temporal control

  • Tetracycline-inducible shRNA for controlled CINP depletion

  • Degron-tagged CINP for rapid protein degradation

  • Chemical-genetic approaches for specific inhibition

• In vivo models (where appropriate)

  • Assessment of phenotypes in different tissues and developmental stages

  • Tissue-specific conditional knockout models

  • Correlation with human disease phenotypes

When designing experiments, researchers should include methodological controls:

• Multiple siRNA/shRNA constructs to minimize off-target effects

• Rescue experiments with RNAi-resistant cDNA

• Appropriate positive controls (e.g., ATR silencing) for comparative analysis