PEF1 Human

What is the PEF1 gene and what protein does it encode?

PEF1 (penta-EF-hand domain containing 1) encodes a calcium-binding protein that belongs to the penta-EF-hand protein family. The encoded protein features five helix-loop-helix motifs that form the calcium-binding PEF-hand region, consistent with other members of this protein family. PEF1 is also known by synonyms including ABP32 and PEF1A, and the encoded protein is identified as PEF1_HUMAN in protein databases. PEF1 has been characterized as having functional associations with over 4,400 biological entities spanning multiple categories, including molecular profiles, organisms, chemicals, and disease states . The gene has been identified with NCBI Gene ID 553115, and alternative splicing results in multiple transcript variants. Notably, a pseudogene has been identified on chromosome 1 .

What are the structural features of the PEF1 protein?

The PEF1 protein contains five conserved EF-hand domains characteristic of calcium-binding proteins in this family. Structural analyses reveal that these domains form helix-loop-helix motifs that create the calcium-binding regions essential for function. Unlike some family members such as ALG-2, PEF1 contains an additional N-terminal extension with predicted disordered regions that share relatively low amino acid sequence identity compared to the highly conserved PEF-hand regions . This structural organization resembles that of peflin, another human PEF-hand protein with which PEF1 shares approximately 34% sequence identity. The conserved calcium-binding domains maintain their structural integrity across fungal and human orthologs, suggesting evolutionary conservation of functional importance .

What is the physiological function of PEF1 in human cells?

PEF1 functions primarily as a calcium-binding protein that forms a heterodimer with the programmed cell death 6 gene product (PDCD6, also known as ALG-2). Through this interaction, PEF1 modulates calcium signaling pathways within cells . Research also indicates that human PEF1, similar to its orthologs in other organisms, plays a critical role in membrane protection during cellular stress, particularly in host-pathogen interactions . This membrane-protective function appears to be conserved across species, as evidenced by studies in fungi where PEF1 orthologs contribute to membrane integrity and repair mechanisms. The protein localizes to areas of membrane reorganization and perturbation, functioning as a "first responder" that stabilizes the plasma membrane at sites requiring immediate repair to maintain cell viability .

How is PEF1 expression regulated in different human tissues?

PEF1 shows variable expression patterns across different human tissues, particularly in brain regions. According to data from the Allen Brain Atlas, PEF1 expression demonstrates tissue-specific regulation in both adult and developing human brain tissues . These expression profiles have been documented through multiple approaches, including microarray analysis and RNA-sequencing technologies. The gene shows differential expression during developmental stages of the human brain, suggesting temporal regulation of its function. PEF1's expression patterns are documented in several datasets including the Allen Brain Atlas Adult Human Brain Tissue Gene Expression Profiles, Allen Brain Atlas Developing Human Brain Tissue Gene Expression Profiles by Microarray, and Allen Brain Atlas Developing Human Brain Tissue Gene Expression Profiles by RNA-seq . This variable expression suggests tissue-specific functions that may correlate with calcium signaling requirements in different cellular contexts.

How does PEF1 interact with PDCD6/ALG-2 in calcium-dependent signaling pathways?

PEF1 forms a functional heterodimer with PDCD6 (also known as ALG-2), which affects calcium-dependent signaling cascades in human cells. The interaction occurs through the calcium-binding EF-hand domains, which undergo conformational changes upon calcium binding. Research suggests that PEF1 shares approximately 33% sequence identity with the human cytosolic calcium sensor protein ALG-2, which initiates plasma membrane repair during mechanical and chemical forms of membrane disruption . This interaction modulates the function of PDCD6/ALG-2 in calcium-dependent membrane repair mechanisms. The specific binding interface between these proteins involves the conserved PEF-hand regions, while the N-terminal extensions may contribute to target recognition and functional specificity. The heterodimer formation appears to be regulated by calcium concentration, suggesting a dynamic response mechanism to cellular calcium fluctuations and providing a regulatory layer to calcium-dependent signaling pathways in response to membrane perturbation .

What role does PEF1 play in membrane integrity and cellular stress responses?

PEF1 serves as a critical component in maintaining membrane integrity during cellular stress. Research on orthologous PEF-hand proteins, including studies in Candida albicans, demonstrates that these proteins localize to sites of membrane perturbation and are essential for membrane repair mechanisms . In human cells, PEF1 functions similarly by responding to membrane disruption events, particularly during host-pathogen interactions. The protein acts as a specialized "first responder" that stabilizes the plasma membrane at sites requiring immediate repair to maintain cell viability . This function extends to both normal membrane reorganization during cellular processes and abnormal damage from external factors. The calcium-binding capability of PEF1 is central to this function, as calcium influx through damaged membranes serves as a signal for recruiting repair machinery. PEF1's role in membrane protection represents an evolutionarily conserved mechanism that contributes to cellular resilience against mechanical stress, chemical perturbation, and pathogen-induced damage .

How do mutations in PEF1 affect calcium homeostasis and related cellular processes?

While the search results don't directly address PEF1 mutations in humans, insights can be drawn from studies of PEF1 deletion in model organisms. In Candida albicans, deletion of PEF1 (pef1Δ) results in compromised plasma membrane integrity, particularly in actively growing regions such as hyphal tips . These mutants show increased permeability to membrane-impermeable dyes like propidium iodide, indicating disrupted membrane integrity. By extension, mutations in human PEF1 would likely affect calcium-dependent membrane repair mechanisms, disrupting calcium homeostasis at sites of membrane damage. Such disruptions could impair cellular responses to mechanical stress and membrane perturbations. Additionally, since PEF1 interacts with PDCD6/ALG-2 in calcium signaling pathways, mutations could potentially disrupt protein-protein interactions essential for proper calcium sensing and downstream signaling cascades. This could have widespread implications for cellular processes dependent on calcium signaling, including vesicular trafficking, cytoskeletal organization, and cell death pathways .

What is the relationship between PEF1 and chromosome cohesion mechanisms?

Research suggests a potential role for a CDK (cyclin-dependent kinase) named Pef1 in chromosome cohesion mechanisms. Studies have demonstrated that inhibition of Pef1 kinase activity enhances cohesin binding to Cohesin Attachment Regions (CARs) in Mis4-deficient cells, improving the establishment of sister chromatid cohesion and chromosome segregation . Specifically, when Pef1-as (an analog-sensitive variant) was inhibited, there was an approximately 4-fold increase in Psm3 K106 acetylation, although the levels remained low compared to wild-type cells . This suggests that Pef1 may regulate cohesin loading or stability through phosphorylation-dependent mechanisms. The research indicates that Pef1 functions in opposition to protein phosphatase 4 in regulating these processes, highlighting a balanced regulatory system for chromosome cohesion. These findings suggest potential research directions for investigating human PEF1's role in cell cycle progression and chromosome maintenance .

What experimental techniques are most effective for studying PEF1 localization and dynamics?

For investigating PEF1 localization and dynamics, fluorescent protein tagging combined with live-cell imaging represents a highly effective approach. Studies in model organisms have successfully employed GFP-tagged Pef1 to track its subcellular localization during various cellular processes . For human PEF1 research, similar approaches using GFP or other fluorescent protein fusions expressed from vectors like pEF1/V5-His can be employed . These tagged constructs allow visualization of protein dynamics in real-time, particularly during membrane stress events or calcium fluctuations.

Complementary techniques include:

• Immunofluorescence microscopy using specific antibodies against PEF1 or its interaction partners

• Fluorescence Recovery After Photobleaching (FRAP) to assess protein mobility and turnover rates

• Calcium imaging using indicators like Fura-2 or GCaMP to correlate calcium fluctuations with PEF1 recruitment

• Super-resolution microscopy techniques such as STORM or PALM for nanoscale visualization of PEF1 clustering

These approaches can be combined with membrane integrity assays using dyes like propidium iodide or FM4-64, which have been successfully employed to correlate PEF1 function with membrane integrity in model organisms .

How can researchers effectively study PEF1 interactions with binding partners?

To study PEF1 interactions with binding partners such as PDCD6/ALG-2, researchers can employ a combination of biochemical, biophysical, and cellular approaches:

Biochemical methods:

• Co-immunoprecipitation with antibodies against PEF1 or suspected binding partners

• Proximity-dependent biotin labeling (BioID or TurboID) to identify proteins in close proximity to PEF1

• Glutathione S-transferase (GST) pulldown assays with recombinant proteins

• Chemical crosslinking followed by mass spectrometry (XL-MS) to map interaction interfaces

Biophysical techniques:

• Surface plasmon resonance (SPR) or bio-layer interferometry (BLI) to measure binding kinetics

• Isothermal titration calorimetry (ITC) to determine thermodynamic parameters of binding

• Förster resonance energy transfer (FRET) to assess protein-protein interactions in live cells

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map binding interfaces

Functional approaches:

• Mutational analysis targeting the EF-hand domains to disrupt calcium binding

• Calcium chelation experiments to assess calcium-dependency of interactions

• Expression of truncated protein variants to identify minimal binding domains

• Generation of analog-sensitive kinase variants (as used with Pef1-as) to study phosphorylation-dependent interactions

These approaches can be effectively combined to build a comprehensive understanding of PEF1's interaction network and the regulatory mechanisms governing these interactions.

What are the best methods for manipulating PEF1 expression in experimental systems?

Researchers interested in manipulating PEF1 expression levels can utilize several complementary approaches:

Overexpression systems:

• Mammalian expression vectors like pEF1/V5-His, which are 6.2 kb vectors designed for high-level, constitutive expression of recombinant proteins in mammalian cells

• Lentiviral or adenoviral delivery systems for efficient transduction in difficult-to-transfect cell types

• Inducible expression systems (e.g., Tet-On/Off) for temporal control of expression levels

Knockdown/knockout approaches:

• CRISPR-Cas9 genome editing for complete knockout of PEF1

• RNA interference (siRNA or shRNA) for transient or stable knockdown

• CRISPRi for inducible transcriptional repression

• Analog-sensitive kinase approaches for functional studies of kinase activity, as demonstrated with Pef1-as

Rescue experiments:

• Expression of wild-type PEF1 in knockout backgrounds

• Introduction of specific mutations to determine structure-function relationships

• Species-specific complementation to assess evolutionary conservation of function

For optimal results, these methods should be validated using quantitative PCR, western blotting, and functional assays to confirm the degree of expression manipulation. When studying calcium-dependent functions, these approaches should be combined with calcium manipulation techniques, such as calcium ionophores or chelators, to assess the relationship between calcium levels and PEF1 function .

What experimental designs are suitable for investigating PEF1's role in membrane protection?

Investigating PEF1's role in membrane protection requires experimental designs that can assess membrane integrity under various conditions. Based on successful approaches with orthologous proteins, the following experimental designs are recommended:

Membrane integrity assays:

• Propidium iodide exclusion assays to assess membrane permeability

• FM4-64 uptake experiments to evaluate membrane internalization rates

• Calcein-AM retention assays to monitor cell viability and membrane integrity

• Lactate dehydrogenase (LDH) release assays to quantify membrane damage

Membrane stress induction methods:

• Treatment with membrane-perturbing agents (e.g., detergents like SDS at sub-lethal concentrations)

• Calcium depletion using chelators like EGTA or BAPTA

• Mechanical stress through hypo-osmotic shock or microfluidic cell stretching

• Pore-forming toxins or antimicrobial peptides to create defined membrane lesions

Real-time imaging approaches:

• Live-cell imaging with fluorescently tagged PEF1 to monitor recruitment to damaged membrane sites

• Dual-color imaging with membrane markers to correlate PEF1 localization with membrane dynamics

• Calcium imaging to correlate calcium influx with PEF1 recruitment and repair processes

Genetic manipulation strategies:

• Comparison of wild-type and PEF1-knockout cells under membrane stress conditions

• Rescue experiments with wild-type or mutant PEF1 in knockout backgrounds

• Overexpression of dominant-negative PEF1 variants with disrupted calcium binding

These experimental designs can be implemented in cell culture systems or relevant model organisms to comprehensively investigate PEF1's role in membrane protection mechanisms. The approaches mirror successful strategies used in fungal systems where PEF1 orthologs have been shown to maintain membrane integrity during stress and normal growth processes .

How is PEF1 implicated in human disease pathologies?

While the provided search results don't explicitly detail PEF1's involvement in specific human diseases, its fundamental role in calcium signaling and membrane protection suggests potential disease implications. Given that PEF1 orthologs in fungal pathogens like Candida albicans contribute to their virulence , human PEF1 may play roles in disease contexts where membrane integrity and calcium homeostasis are disrupted. The protein's expression in various brain tissues according to the Allen Brain Atlas suggests potential neurological relevance. As a calcium-binding protein that interacts with PDCD6/ALG-2, PEF1 may influence cell death pathways implicated in neurodegenerative diseases or cancer. Additionally, its membrane-protective function could be significant in conditions characterized by cellular stress and membrane damage, such as ischemia-reperfusion injury, muscular dystrophies, or disorders involving excitotoxicity. Future research targeting specific disease models may reveal more direct connections between PEF1 dysfunction and human pathologies.

What techniques are available for screening PEF1-targeting compounds for therapeutic development?

To screen compounds targeting PEF1 for potential therapeutic applications, researchers can implement several complementary approaches:

Biochemical screening approaches:

• Fluorescence-based calcium binding assays to identify compounds that modulate PEF1's calcium affinity

• AlphaScreen or FRET-based assays to identify disruptors or enhancers of PEF1-PDCD6 interaction

• Surface plasmon resonance (SPR) to assess compound binding to recombinant PEF1 protein

• Differential scanning fluorimetry (thermal shift assays) to identify stabilizing compounds

Cell-based screening platforms:

• High-content imaging screens using PEF1-GFP localization as a readout for compound activity

• Membrane integrity assays in PEF1-manipulated cells to identify protective compounds

• CRISPR-based screens to identify synthetic lethal interactions with PEF1 inhibition

• Reporter gene assays for downstream calcium signaling pathways affected by PEF1

In silico approaches:

• Structure-based virtual screening targeting the calcium-binding pockets or protein-protein interaction interfaces

• Molecular dynamics simulations to identify allosteric modulators of PEF1 function

• Systems biology approaches to predict compound effects on PEF1-associated networks

Validation methods:

• Analog-sensitive kinase approaches (similar to those used with Pef1-as) to validate compound specificity

• Cellular thermal shift assays (CETSA) to confirm target engagement in cells

• Functional rescue experiments to verify mechanism of action

These screening strategies should be designed with consideration for PEF1's calcium-binding properties and protein interaction network, focusing on compounds that specifically modulate these functions with minimal off-target effects.

What are the emerging areas of PEF1 research in human biology?

Several emerging research areas are expanding our understanding of PEF1 in human biology:

• Membrane repair mechanisms: Building on findings from fungal models , researchers are investigating PEF1's role in human cell membrane repair pathways, particularly in the context of mechanical stress and pathogen interaction.

• Calcium signaling networks: Research is exploring how PEF1 integrates into broader calcium signaling networks, particularly through its interaction with PDCD6/ALG-2 and potential connections to other calcium-sensitive pathways .

• Tissue-specific functions: Given the differential expression patterns observed in brain tissues , researchers are examining tissue-specific roles of PEF1, particularly in neuronal function and development.

• Cell cycle regulation: Based on the role of Pef1 in chromosome cohesion , investigations are exploring potential functions of human PEF1 in cell cycle progression and chromosome maintenance.

• Host-pathogen interactions: Building on findings that PEF1 orthologs contribute to pathogen virulence , researchers are studying how human PEF1 responds to pathogen-induced membrane damage and contributes to cellular defense mechanisms.

• Protein interaction network: Advanced proteomics approaches are mapping the complete interaction network of PEF1 to better understand its functional context within cellular signaling cascades.

These emerging areas represent frontiers in PEF1 research with significant potential for uncovering novel biological principles and therapeutic opportunities.

How do current research findings about PEF1 compare across different model organisms?

Current research reveals both conserved and divergent aspects of PEF1 function across different model organisms:

This comparative analysis reveals evolutionary conservation of PEF1's fundamental role in membrane integrity and calcium signaling, while suggesting species-specific adaptations in cellular localization and detailed molecular functions. The membrane-protective function appears to be a universal feature, while specific roles in polarized growth and pathogenicity may represent adaptive specializations in certain organisms. These cross-species insights provide valuable direction for human PEF1 research, highlighting both basic mechanisms likely to be conserved and potential specialized functions that may have evolved in humans .

What are the key takeaways for researchers beginning to study PEF1?

Researchers beginning to study PEF1 should consider the following key takeaways:

• PEF1 is a calcium-binding protein belonging to the penta-EF-hand family with five conserved helix-loop-helix motifs essential for calcium binding .

• It functions in calcium signaling pathways through interaction with PDCD6/ALG-2 and plays critical roles in membrane protection during cellular stress .

• The protein demonstrates evolutionary conservation across species, with orthologs in fungi showing similar membrane-protective functions .

• PEF1 localizes to sites of membrane perturbation, functioning as a "first responder" to stabilize membranes requiring repair .

• Expression patterns vary across tissues, particularly in brain regions, suggesting tissue-specific functions .

• Research approaches should combine protein localization, interaction studies, and functional assays focused on membrane integrity.

• Evidence from fungal models suggests potential roles in pathogenesis and stress response that may have parallels in human disease contexts .

• Future research directions should explore PEF1's role in membrane repair mechanisms, calcium signaling networks, and potential therapeutic applications.