CIB1 Human

What is the basic structure of human CIB1?

Human CIB1 is a 22 kDa protein containing four EF-hand domains, with calcium-binding capability in the third and fourth EF-hands. Structurally, it shares approximately 57% sequence similarity with calcineurin B and 56% similarity with calmodulin . The protein spans 191 amino acids (Gly2-Leu191) and can undergo N-myristoylation, which contributes to its membrane association properties . This post-translational modification is significant for its localization and function in various cellular compartments. The calcium-binding feature is central to its role as a calcium sensor and regulatory protein in multiple signaling pathways .

What are the primary cellular functions of CIB1?

CIB1 participates in multiple cellular processes, making it a multifunctional signaling protein. Key functions include:

• Cell migration regulation: CIB1 both stimulates and inhibits cell migration, depending on cell type and context

• Cell adhesion modulation: Particularly through its interaction with integrins

• Proliferation control: Affects cell cycle progression in various cell types

• Apoptosis regulation: Functions as a calcium-sensitive negative regulator of ASK1-mediated apoptotic signaling

• Calcium signaling: Interacts with calcium-dependent pathways and proteins like IP3R and calcineurin

These diverse functions highlight CIB1's role as a critical node in cellular signaling networks, functioning as both a calcium sensor and a protein-protein interaction hub .

How does CIB1 distribution vary across human tissues?

CIB1 is ubiquitously expressed across human tissues, though expression levels can vary. It has been detected in kidney tissue , breast cancer tissue , testes , and various other tissues. In cellular contexts, CIB1 can localize to both membrane and cytosolic compartments, with its N-myristoylation influencing this distribution. In cancer tissues, such as breast cancer, CIB1 shows specific localization to the cytoplasm of cancer cells as demonstrated by immunohistochemical analyses . The widespread expression pattern of CIB1 correlates with its diverse functional roles across different tissue and cell types .

What are the most effective antibodies and detection methods for CIB1 research?

For CIB1 detection, researchers have several validated options:

Antibodies:

• Polyclonal antibodies raised against human CIB1 (Gly2-Leu191) have shown efficacy in multiple applications

• Monoclonal antibodies like clone 791119 have demonstrated specificity in both Western blot and immunohistochemistry applications

Detection Methods:

• Western Blotting: Effective at 1:50-400 dilution for polyclonal antibodies and around 2 μg/mL for monoclonal antibodies

• Immunohistochemistry: Works well in both paraffin-embedded (1:10-100 dilution) and frozen sections (1:50-500 dilution)

• Immunocytochemistry: Effective at 1:50-500 dilution in formalin-fixed cells

• ELISA: Functional at 1:100-200 dilution

For optimal results, heat-induced epitope retrieval is recommended when working with paraffin-embedded tissues. Visualization can be achieved using HRP-DAB staining systems, with counterstaining using hematoxylin to provide cellular context .

How can researchers effectively study CIB1-protein interactions?

To study CIB1-protein interactions, researchers should consider multiple complementary approaches:

In vitro techniques:

Cellular approaches:

• Proximity ligation assays: To visualize interactions in situ

• Fluorescence resonance energy transfer (FRET): To study dynamic interactions in living cells

When studying CIB1-protein interactions, it's critical to account for calcium's influence, as CIB1's binding properties can be calcium-dependent. Additionally, the N-myristoylation state of CIB1 may affect its interaction profile and should be considered in experimental design .

What are the key considerations for CIB1 knockdown or knockout experiments?

When designing CIB1 depletion experiments, researchers should consider:

Experimental approaches:

• RNA interference: Has been successfully used in various cell types including rat embryo fibroblasts, human cervical adenocarcinoma cells (HeLa S3), and endothelial cells

• CRISPR-Cas9: For permanent gene editing in cellular models

• Mouse models: CIB1-/- mice have been developed and characterized, revealing phenotypes including male infertility

Critical considerations:

• Cell type specificity: CIB1 depletion has drastically different effects depending on cell type. For example, CIB1 loss reduces migration in endothelial cells but increases migration in fibroblasts and HeLa cells

• Functional redundancy: Consider potential compensation by other calcium-binding proteins

• Male fertility impacts: CIB1-/- male mice are sterile, displaying testicular abnormalities including lower mass, histological irregularities, and lack of detectable spermatids

Researchers should include appropriate controls and validate knockdown/knockout efficiency at both mRNA and protein levels. Cell-type specific phenotypes should be thoroughly characterized to account for the context-dependent functions of CIB1 .

How does CIB1 exert opposing effects on cell migration in different cell types?

CIB1's paradoxical effects on cell migration represent a complex regulatory mechanism that varies by cellular context:

Stimulatory effects observed in:

• Chinese hamster ovary cells

• Human T47D breast cancer cells

• HUVECs (human umbilical vein endothelial cells)

Inhibitory effects observed in:

• Rat embryo fibroblasts

• NIH3T3 cells

Mechanistic explanations for this paradox:

• Cell-type specific binding partner expression: Different cell types express unique repertoires of integrins and other CIB1 binding partners, leading to divergent downstream signaling

• Differential pathway activation:

  • In cells where CIB1 inhibits migration, it activates PAK1 and the downstream LIMK/cofilin pathway, which depolymerizes actin filaments

  • In cells where CIB1 promotes migration, it works through PKC and ERK signaling

• Extracellular matrix interactions: The concentration and type of extracellular matrices used in migration studies influence how CIB1 regulates this process

This context-dependent regulation highlights the importance of careful experimental design when studying CIB1 effects on migration, with special attention to cell type, matrix composition, and signaling pathway analysis .

What molecular mechanisms mediate CIB1's regulation of integrins and cell adhesion?

CIB1 was originally identified as a binding partner for the platelet integrin αIIbβ3, but its regulatory role in integrin function extends beyond this initial discovery:

Key molecular mechanisms:

• Direct binding to integrin cytoplasmic domains: CIB1 interacts with membrane-embedded residues of integrins, suggesting a complex structural interaction that may influence integrin activation states

• Influence on integrin conformational changes: CIB1 has been shown to regulate αIIbβ3 activation, potentially by affecting the conformational changes necessary for inside-out signaling

• Impact on adhesion-dependent signaling: Through interactions with multiple binding partners in adhesion complexes, CIB1 modulates downstream signaling cascades that control adhesion strength and dynamics

• Membrane trafficking regulation: CIB1's calcium-binding properties and myristoylation may influence the trafficking of integrins to and from the cell surface

The structure-function relationship of CIB1-integrin interactions remains incompletely characterized. A significant research gap exists in understanding how these interactions occur in the context of a lipid bilayer and how calcium binding to CIB1 might modulate these interactions . Future biophysical and structural studies could provide valuable insights into these mechanisms .

What is the role of CIB1 in cancer progression and metastasis?

CIB1 has emerged as a significant factor in cancer biology, with multiple mechanisms contributing to tumor progression:

Cancer-promoting mechanisms of CIB1:

• Cell cycle and proliferation enhancement: CIB1 promotes cancer cell proliferation through interaction with cell cycle regulatory pathways

• Apoptosis inhibition: CIB1 negatively regulates stress-activated MAPK signaling pathways by targeting ASK1 (Apoptosis Signal-Regulating Kinase 1), interfering with TRAF2 recruitment to ASK1, and inhibiting ASK1 autophosphorylation on Thr-838 . This action blocks ASK1 activation and mitigates apoptotic cell death, as demonstrated in breast cancer MCF7 cells treated with TNF-α

• Migration facilitation: In certain cancer cell types like T47D breast cancer cells, CIB1 stimulates cell migration, potentially contributing to metastatic capability

• Angiogenesis promotion: CIB1 has been implicated in stress-induced angiogenesis, which supports tumor growth

Clinical evidence:

• CIB1 has been detected in breast cancer tissue through immunohistochemical staining, with specific localization to the cytoplasm of cancer cells

• CIB1 has been implicated in tumor growth in multiple studies

These findings suggest CIB1 as a potential therapeutic target or biomarker in cancer, though further clinical studies are needed to fully establish its utility in this context .

How is CIB1 involved in male infertility mechanisms?

CIB1 plays a critical role in male fertility through its effects on spermatogenesis:

Experimental evidence from animal models:

• CIB1-/- male mice are completely sterile

• Testes of CIB1-/- mice exhibit several abnormalities:

  • Lower testicular mass

  • Histological irregularities

  • Absence of detectable spermatids

  • Increased CDC2/CDK1 expression

  • Elevated cell death compared to wild-type littermates

Clinical correlation in humans:

• Men with oligoasthenozoospermia (low sperm count and motility) demonstrate lower CIB1 mRNA and protein levels compared to men with healthy sperm

Potential mechanisms:

• While the precise molecular mechanisms remain unclear, CIB1's functions in cell proliferation and survival appear critical for proper spermatogenesis

• CIB1 may be particularly important during meiosis, though this aspect has not been deeply explored

The essential nature of CIB1 in male fertility presents an important consideration for potential therapeutic strategies targeting CIB1 in other diseases like cancer. Any CIB1-targeting therapy would need to carefully evaluate potential impacts on male reproductive health .

What is known about CIB1's calcium-sensitive modulation of stress responses and relation to neurological disorders?

CIB1 functions as a calcium-sensitive modulator of stress responses, with particular relevance to neuronal systems:

Molecular mechanisms:

• ASK1 pathway inhibition: CIB1 binds to ASK1 and prevents its activation by interfering with TRAF2 recruitment and blocking autophosphorylation on Thr-838

• Calcium-dependent regulation: Calcium influx, such as that induced by membrane depolarization in neurons, can reverse CIB1's inhibitory effect on ASK1 activation

• Neuroprotection vs. neurotoxicity: In dopaminergic neurons, CIB1 mitigates apoptotic cell death induced by 6-hydroxydopamine (6-OHDA), but this protection is reversed by calcium influx

Connection to neurological disorders:

• CIB1 has been linked to Alzheimer's disease (AD) , though detailed mechanisms remain to be elucidated

• The calcium-sensitive nature of CIB1's function may be particularly relevant in neurological conditions where calcium homeostasis is disrupted

• CIB1's role in neural development, along with its functions in taste or gustation, suggests broader neurological significance

The conditional nature of CIB1's protective effects—dependent on calcium levels—highlights the complexity of targeting this protein in neurological disorders. Future research should focus on understanding the precise conditions under which CIB1 exerts protective versus potentially harmful effects in different neuronal populations .

How do calcium binding and myristoylation mechanistically influence CIB1 function?

The interplay between calcium binding and N-myristoylation represents a sophisticated regulatory mechanism for CIB1:

Calcium binding effects:

• CIB1 contains four EF-hand domains, with the third and fourth being functional calcium-binding sites

• Calcium binding likely induces conformational changes that affect CIB1's interaction with binding partners

• These structural changes may determine binding partner selectivity under different calcium concentration conditions

Myristoylation regulation:

• CIB1 undergoes N-myristoylation, which facilitates membrane association

• A major unresolved question is whether the exposure of the N-myristoyl moiety can be allosterically modulated by calcium binding, creating a "myristoyl switch" mechanism

• This switch could dynamically regulate CIB1's subcellular localization in response to calcium fluctuations

Integrated regulatory model:

• Intracellular calcium levels may simultaneously affect CIB1's conformation and localization

• This dual regulation could enable CIB1 to coordinate different signaling responses based on calcium concentration and subcellular compartment

• The absence of structural data showing CIB1 in complex with binding partners limits our understanding of how these mechanisms operate in concert

Resolving these questions requires advanced biophysical approaches, including structural studies of CIB1 with its binding partners in membrane contexts and real-time imaging of CIB1 dynamics in response to calcium fluctuations .

How does CIB1 prioritize interactions with multiple binding partners within the cellular context?

CIB1 interfaces with numerous binding partners, raising questions about interaction selectivity and prioritization:

Known binding partners include:

• Integrins (e.g., αIIbβ3)

• ASK1 (Apoptosis Signal-Regulating Kinase 1)

• PAK1 (p21-activated kinase 1)

• IP3R (inositol 1,4,5-trisphosphate receptor)

• Calcineurin

• TAS1R2 (taste 1 receptor member 2)

Potential selectivity mechanisms:

• Calcium-dependent affinity modulation: Different calcium concentrations may favor specific interactions through conformational changes in CIB1

• Subcellular compartmentalization: Myristoylation-dependent localization could determine which binding partners CIB1 encounters

• Competitive binding: Partners may compete for overlapping binding sites on CIB1

• Temporal regulation: Cell cycle phase or stress conditions might influence partner prioritization

• Concentration-dependent effects: Local concentrations of binding partners could drive preferential interactions

Research challenges:

• Few biophysical characterizations with non-integrin binding partners have been conducted

• The structural basis for CIB1's partner selectivity remains largely unknown

• Comprehensive interaction networks and binding hierarchies have not been established

Advanced proteomics approaches, including proximity labeling techniques and quantitative interaction studies under varying calcium conditions, could help elucidate how CIB1 navigates its complex interaction network in different cellular contexts .

What explains the contradictory data on CIB1's role in different cellular processes and how can researchers address these discrepancies?

The paradoxical effects of CIB1 across different experimental systems present significant research challenges:

Observed contradictions:

• CIB1 both promotes and inhibits cell migration depending on cell type

• CIB1 deletion/depletion reduces migration in some cells (endothelial cells, megakaryocytes) but increases it in others (fibroblasts, HeLa cells)

• Similar paradoxical effects have been observed with binding partners like PAK1

Potential explanations for discrepancies:

• Cell type-specific protein expression profiles: The unique repertoire of integrins and other binding partners in different cells may determine CIB1's functional outcomes

• Experimental condition variations: Different extracellular matrices, growth factor concentrations, and culture conditions may influence results

• Incomplete knockdown vs. knockout effects: Different methodologies for CIB1 depletion may lead to different compensatory mechanisms

• Context-dependent calcium signaling: Variations in calcium handling between cell types could affect CIB1 function

Methodological approaches to resolve contradictions:

• Standardized experimental systems: Using consistent matrices, culture conditions, and analyses across cell types

• Multi-parameter analyses: Simultaneously evaluating multiple CIB1 functions within the same experimental system

• Temporal studies: Examining short-term versus long-term effects of CIB1 modulation

• Systems biology approaches: Comprehensive network analyses to identify cell-specific differences in CIB1-interacting pathways

• Conditional and inducible models: Temporally controlled CIB1 depletion to distinguish direct effects from adaptive responses

These approaches could help reconcile conflicting data and establish a more coherent understanding of CIB1's contextual functions .

What are the most promising therapeutic applications targeting CIB1 in human diseases?

Based on current knowledge, several therapeutic opportunities targeting CIB1 show potential:

Cancer therapeutics:

• Targeting CIB1 could sensitize cancer cells to apoptotic stimuli by relieving inhibition of ASK1-mediated apoptosis

• CIB1 inhibition might reduce tumor angiogenesis, limiting tumor growth

• Combined approaches targeting CIB1 and its key binding partners could provide synergistic anti-tumor effects

Neurological disorders:

• Modulating CIB1's calcium-sensitive regulation of ASK1 could provide neuroprotection in conditions involving oxidative stress

• CIB1-targeted approaches might have relevance for Alzheimer's disease based on reported associations

Cardiovascular applications:

• Given CIB1's implication in cardiac hypertrophy, targeted therapies might address certain cardiovascular conditions

Challenges and considerations:

• Male fertility impacts present a significant concern for systemic CIB1-targeting therapies

• The context-dependent functions of CIB1 necessitate careful therapeutic design to avoid unintended consequences

• Tissue-specific delivery approaches would be advantageous to limit off-target effects

Future development of CIB1-targeting therapeutics should focus on selective modulation of specific CIB1 interactions rather than complete inhibition, potentially allowing for therapeutic intervention without compromising essential functions .

What are the critical unresolved questions in CIB1 structure-function relationships?

Several fundamental questions about CIB1 structure-function relationships remain unanswered:

Structural gaps:

• Ligand-bound structures: No structure of CIB1 in complex with binding partners that explicitly shows both binding partners has been solved

• Membrane interaction dynamics: How CIB1 interacts with supposedly membrane-embedded integrin residues remains poorly understood

• Myristoyl switch mechanism: The structural nature of the N-myristoyl moiety and whether its exposure can be allosterically modulated requires investigation

Functional questions:

• Activation/inhibition mechanisms: How CIB1, which is non-enzymatic, activates or inhibits its binding partners at the molecular level

• Calcium-dependent binding partner selection: The precise role of intracellular calcium in determining CIB1's binding partner preferences

• Regulatory mechanisms of CIB1 itself: How CIB1 expression, localization, and activity are controlled within cells

Technical approaches needed:

• Structural biology studies of CIB1 with binding partners in lipid bilayer contexts

• Advanced biophysical characterization of CIB1 interactions under varying calcium concentrations

• Single-molecule studies to understand the dynamics of CIB1-partner interactions

• Proteomic and systems biology approaches to comprehensively map CIB1's interaction network

Resolving these questions would significantly advance our understanding of CIB1's role in human health and disease and potentially identify novel therapeutic opportunities .

How can researchers reconcile CIB1's multiple functions into a unified biological model?

Integrating CIB1's diverse functions into a coherent biological framework requires both conceptual and experimental advances:

Conceptual framework elements:

• CIB1 as a calcium-responsive signaling node: Position CIB1 as a calcium sensor that coordinates multiple signaling pathways based on calcium levels, cellular context, and binding partner availability

• Context-dependent signaling outcomes: Recognize that the same molecular mechanism can produce different cellular outcomes depending on the specific cellular environment and signaling network state

• Temporal signaling dynamics: Consider how CIB1's functions may change over time during processes like development, cell cycle progression, or stress responses

Experimental approaches for integration:

• Multi-omics systems biology: Combine proteomics, transcriptomics, and metabolomics to map CIB1-dependent networks across different cell types

• Mathematical modeling: Develop computational models that can predict CIB1's functional outcomes based on multiple parameters (calcium levels, binding partner concentrations, etc.)

• Comparative cellular studies: Perform parallel analyses across multiple cell types using standardized conditions to identify conserved versus context-specific functions

• In vivo conditional models: Develop sophisticated animal models with tissue-specific and temporally controlled CIB1 modulation

By combining these approaches, researchers could develop a comprehensive model of CIB1 biology that accounts for its seemingly contradictory functions across different contexts, ultimately providing a more nuanced understanding of this multifunctional protein and its roles in human health and disease .