PSMD10 Human

What is the basic structure of human PSMD10 and how does it relate to its function?

Human PSMD10 (also known as Gankyrin) is a 226 amino acid protein that functions as a non-ATPase regulatory subunit of the 26S proteasome . The protein contains multiple ankyrin repeats that mediate protein-protein interactions, particularly with components of autophagy machinery and the proteasome . Crystallographic studies have revealed that PSMD10 has a structure with channels accessible to small molecules, as demonstrated in the high-resolution crystal structure (PDB ID: 7VO6) .

The functional significance of this structure lies in how the ankyrin repeat domains enable selective binding with partners like ATG7, ATG10, and ATG12 . The first ankyrin repeat is particularly critical for interactions with ATG7 and ATG10, while the last three ankyrin repeats directly interact with ATG7 . Interestingly, PSMD10's interactions with ATG12 involve the third to fifth ankyrin repeats, illustrating how different structural regions mediate specific protein interactions .

How does PSMD10 homodimerization occur and what techniques can detect this phenomenon?

PSMD10 homodimerization is primarily stabilized through disulfide bonds at the N-terminus, with Cysteine 4 (Cys4) playing a crucial role in this stabilization . Researchers have demonstrated this using multiple complementary techniques:

• Size-exclusion chromatography (SEC): Purified PSMD10 separates into two distinct peaks corresponding to dimers and monomers, which can be confirmed using multiangle laser light scattering analysis .

• Native gel electrophoresis: This technique can visualize the dimer-monomer distribution under non-denaturing conditions .

• Genetically incorporated crosslinkers: Using proximity-enabled unnatural amino acids (Uaas) like Azi allows detection of intermolecular proximities in living cells .

• Chemical crosslinking with disuccinimidyl suberate (DSS): This approach can capture transient interactions even when traditional disulfide bonding is disrupted .

When conducting dimerization studies, researchers should note that reducing agents such as DTT and β-mercaptoethanol disrupt the PSMD10 homodimer, confirming the disulfide-mediated nature of the interaction . For mutational analysis, the PSMD10 C4S mutant fails to form stable homodimers in native gels but can still be captured with crosslinking techniques, suggesting Cys4 stabilizes rather than initiates dimerization .

What are the most reliable approaches to distinguish between PSMD10's proteasomal and non-proteasomal functions?

To differentiate between PSMD10's proteasomal and non-proteasomal functions, researchers should consider these methodological approaches:

• Selective mutational analysis: The PSMD10 C4S mutant maintains interaction with the proteasome component Rpt3 but loses interactions with autophagy proteins ATG7 and ATG10 . This selective disruption provides a powerful tool to separate these functional pathways.

• Domain-specific deletions: Removing the first ankyrin repeat prevents PSMD10 interaction with ATG7 and ATG10 without affecting ATG12 binding, allowing further functional discrimination .

• Co-immunoprecipitation assays: These can assess PSMD10 binding to proteasomal components (like Rpt3) versus autophagy machinery under various conditions, revealing pathway-specific interactions .

• Proximity-enabled crosslinking: This technique can capture dynamic interactions under physiological conditions, providing insights into which complexes form in specific cellular contexts .

When designing experiments to distinguish these functions, researchers should monitor both ubiquitination levels and autophagy markers (such as LC3-II formation) to comprehensively assess pathway involvement. The evidence suggests PSMD10 has distinct structural elements mediating its diverse functions, making it possible to experimentally separate its roles in proteasomal degradation versus autophagy regulation .

What are the optimal methods for detecting PSMD10 protein-protein interactions in living cells?

For studying PSMD10 protein-protein interactions in living cells, several complementary approaches have proven effective:

• Genetically incorporated proximity-enabled crosslinking: This technique provides exceptional specificity for detecting intermolecular proximities in physiological conditions. Incorporating unnatural amino acids (Uaas) like Azi at specific positions in PSMD10 enables covalent crosslinking with interacting proteins when the groups are in close proximity . This method has successfully captured interactions between PSMD10 and partners like ATG7, revealing that the last three ankyrin repeats directly interact with ATG7 .

• Immunoprecipitation assays: Traditional co-IP approaches remain valuable for detecting PSMD10 complexes, particularly when combined with specific antibodies against endogenous proteins. These assays have demonstrated interactions between PSMD10 and multiple partners including ATG7, ATG10, ATG12, and HSF1 .

• Native gel electrophoresis: This technique preserves protein complexes and has been successfully used to visualize PSMD10 homodimers and their disruption by interacting proteins like NleE .

• Fluorescence colocalization: Microscopy-based approaches can reveal the spatial relationship between PSMD10 and its partners. This approach has shown that NleE suppresses colocalization of ATG7 with LC3 puncta, providing functional context to biochemical interaction data .

When designing interaction studies, researchers should include appropriate controls for specificity, such as testing mutant versions of PSMD10 (e.g., PSMD10 C4S) or interaction partners. This multi-method approach provides robust validation of protein-protein interactions in the complex cellular environment.

How can researchers distinguish between direct and indirect PSMD10 interactions in complex cellular pathways?

Distinguishing between direct and indirect PSMD10 interactions requires a hierarchical experimental approach:

• In vitro reconstitution with purified components: This represents the gold standard for demonstrating direct interactions. Size-exclusion chromatography with purified PSMD10 and potential partners can confirm binary interactions without cellular cofactors .

• Domain mapping through deletion mutants: By systematically removing specific domains of PSMD10 (such as individual ankyrin repeats), researchers can identify regions necessary for particular interactions. This approach revealed that PSMD10's first ankyrin repeat is crucial for ATG7/ATG10 binding, while ATG12 requires the third to fifth repeats .

• Site-specific crosslinking: Incorporating photoactivatable crosslinkers at defined positions in PSMD10 allows precise mapping of interaction interfaces. This technique has successfully demonstrated direct contact between PSMD10's last three ankyrin repeats and ATG7 .

• Competition assays: If two proteins compete for binding to PSMD10, this suggests they interact with the same surface, indicating direct rather than scaffolded interactions.

• Structural studies: High-resolution crystal structures, like that available for PSMD10 (PDB ID: 7VO6), can be used to inform docking studies and predict interaction interfaces .

When interpreting interaction data, researchers should consider that PSMD10 functions in multiple cellular complexes. The C4S mutation specifically disrupts interactions with ATG7 and ATG10 while preserving binding to proteasomal component Rpt3, demonstrating how carefully designed mutations can dissect complex interaction networks .

What are the most effective methods for studying PSMD10 in the context of autophagy regulation?

To investigate PSMD10's role in autophagy regulation, researchers should employ these methodological approaches:

• LC3 puncta formation assays: Monitoring LC3 puncta by fluorescence microscopy provides a reliable readout of autophagosome formation. This approach has demonstrated that NleE suppresses the colocalization of ATG7 with LC3 puncta in a PSMD10-dependent manner .

• Biochemical assessment of LC3 lipidation: Western blotting for LC3-II formation offers quantitative measurement of autophagy activation. This can be combined with treatments that modulate autophagy (starvation, rapamycin) to assess PSMD10's role under different conditions .

• Genetic manipulation approaches:

  • PSMD10 knockout cells provide a clean background for reconstitution experiments

  • Structure-guided mutations (e.g., C4S or ankyrin repeat deletions) allow dissection of specific interaction requirements

  • Inducible expression systems help control timing and expression levels

• Interactome analysis of PSMD10 during autophagy induction: Proximity labeling combined with mass spectrometry can reveal dynamic changes in PSMD10's interaction partners during autophagy activation.

• Functional reconstitution: Reintroducing wild-type or mutant PSMD10 into knockout cells allows assessment of which structural features are necessary for autophagy regulation .

When designing these experiments, researchers should consider that PSMD10 affects autophagy through multiple mechanisms, including direct interaction with ATG7/ATG10 and potential transcriptional effects via HSF1 . Therefore, comprehensive assessment requires monitoring both protein-protein interactions and transcriptional outputs to fully characterize PSMD10's role in autophagy regulation.

How does PSMD10 function at the intersection of proteasomal degradation and autophagy?

PSMD10 represents a unique regulatory node connecting the proteasomal and autophagic degradation systems. Based on current research, PSMD10 employs distinct structural domains and molecular mechanisms to participate in these pathways:

In proteasomal degradation:

• PSMD10 functions as a non-ATPase subunit of the 26S proteasome

• It interacts with the AAA-ATPase subunit Rpt3, a core component of the proteasome

• Unlike many proteasome-associated proteins, PSMD10 does not appear to promote degradation of all binding partners (e.g., NleE protein levels and ubiquitination were not affected by PSMD10 knockout)

In autophagy:

• PSMD10 directly interacts with ATG7 (an E1-like enzyme) and ATG10, promoting LC3 lipidation and autophagosome formation

• PSMD10 homodimerization is crucial for these interactions, as the C4S mutant fails to bind ATG7/ATG10 while maintaining Rpt3 interaction

• The first ankyrin repeat is essential for ATG7/ATG10 binding, while the last three ankyrin repeats directly contact ATG7

Methodologically, researchers can distinguish between these functions by:

• Using the C4S mutation to selectively disrupt autophagy interactions while preserving proteasomal binding

• Monitoring pathway-specific markers (ubiquitination vs. LC3-II) in response to PSMD10 manipulation

• Employing domain-specific mutations to selectively impair particular protein interactions

These findings suggest PSMD10 may serve as a switch between degradation pathways, potentially coordinating cellular responses to different types of stress or substrates.

What mechanisms explain PSMD10's involvement in pathogen defense and how can these be experimentally validated?

PSMD10 plays a critical role in host defense against bacterial pathogens through its autophagy-promoting functions. Research has revealed a mechanism where pathogens directly target PSMD10 to suppress host defense:

• Pathogen effector protein NleE directly interacts with PSMD10's N-terminus, as demonstrated through pairwise chemical crosslinking with genetically incorporated unnatural amino acids .

• This interaction suppresses PSMD10 homodimerization, which is essential for PSMD10's autophagy-promoting functions. Native gel electrophoresis and crosslinking approaches have confirmed this suppression mechanism .

• By preventing PSMD10 dimerization, NleE specifically blocks PSMD10's interaction with ATG7 and ATG10, but not with ATG12 or HSF1, demonstrating selective pathway inhibition .

• Functionally, this leads to reduced ATG7-LC3 colocalization and attenuated autophagosome formation, benefiting pathogen survival .

To experimentally validate these mechanisms, researchers should:

• Employ proximity-enabled crosslinking to map the precise interaction interface between NleE and PSMD10

• Use structural biology approaches to determine how NleE binding prevents PSMD10 dimerization

• Develop PSMD10 mutants resistant to NleE binding and test their ability to maintain autophagy during infection

• Assess whether forced dimerization of PSMD10 (e.g., through engineered disulfide bonds) can overcome NleE inhibition

• Evaluate the broader relevance by testing whether other bacterial pathogens employ similar mechanisms

These approaches would provide mechanistic insight into pathogen-host interactions and potentially reveal therapeutic targets to enhance host defense responses .

How does PSMD10 homodimerization regulate its diverse cellular functions?

PSMD10 homodimerization serves as a molecular switch that selectively regulates its diverse cellular functions. The following mechanisms and experimental evidence highlight this regulatory role:

• Structural basis of dimerization:

  • Disulfide bonds at the N-terminus, particularly involving Cysteine 4, stabilize PSMD10 homodimers

  • Size-exclusion chromatography and native gel electrophoresis confirm the existence of stable dimers in physiological conditions

  • The C4S mutation prevents stable dimer formation while maintaining monomeric PSMD10

• Functional consequences of dimerization:

  • Dimerization is essential for interactions with autophagy machinery components ATG7 and ATG10, but not for ATG12 binding

  • The PSMD10 C4S mutant fails to interact with ATG7 and ATG10 while maintaining binding to proteasomal component Rpt3

  • This selective requirement suggests dimerization creates unique interaction surfaces or conformations needed for specific protein-protein interactions

• Regulation of dimerization:

  • Pathogen effector NleE suppresses PSMD10 homodimerization by interacting with its N-terminus

  • This provides a mechanism for pathogens to selectively inhibit PSMD10's autophagy-promoting functions while potentially preserving its proteasomal roles

  • Reducing agents disrupt PSMD10 dimers, suggesting potential redox regulation in cellular contexts

To further investigate the regulatory impact of dimerization, researchers could:

• Develop constitutively dimeric PSMD10 variants through engineered disulfide bonds

• Use hydrogen-deuterium exchange mass spectrometry to identify conformational changes induced by dimerization

• Perform comparative interactome analyses of monomeric versus dimeric PSMD10 to comprehensively map dimerization-dependent interactions

These approaches would provide deeper insights into how PSMD10 dimerization serves as a regulatory mechanism controlling its participation in distinct cellular pathways .

What are the technical challenges in resolving contradictory findings about PSMD10's role in proteasomal versus non-proteasomal pathways?

Resolving contradictory findings regarding PSMD10's diverse functions requires addressing several technical challenges:

• Context-dependent activity:

  • PSMD10 function may vary across cell types, stress conditions, and disease states

  • Methodological approach: Perform comparative studies across multiple cell lines and primary cells, using consistent experimental conditions and quantitative readouts

  • Include time-course analyses to capture dynamic changes in PSMD10 complex formation

• Stoichiometric considerations:

  • The balance between monomeric and dimeric PSMD10 likely influences pathway engagement

  • Methodological approach: Develop techniques to quantify the dimer/monomer ratio in living cells

  • Use single-molecule approaches to determine if individual PSMD10 molecules shuttle between pathways

• Post-translational modifications:

  • PTMs could explain seemingly contradictory functions by directing PSMD10 to specific pathways

  • Methodological approach: Apply mass spectrometry to map PTMs under various conditions

  • Generate modification-specific antibodies or biosensors to track modified PSMD10 pools

• Technical artifacts in protein interaction studies:

  • Overexpression can drive non-physiological interactions; cell lysis can disrupt or create artificial complexes

  • Methodological approach: Use CRISPR-Cas9 to tag endogenous PSMD10

  • Apply proximity labeling techniques (BioID, TurboID) to capture interactions in intact cells

  • Validate key interactions with multiple complementary techniques (co-IP, crosslinking, microscopy)

• Reconciliation strategies for contradictory data:

  • Systematically test whether differences arise from experimental conditions or biological variables

  • Create a comprehensive interaction map incorporating temporal and spatial dynamics

  • Apply network analysis to identify potential coordinating mechanisms between pathways

By addressing these challenges through rigorous methodology and integrative analysis, researchers can develop a unified model of PSMD10 function that accommodates its diverse cellular roles .

How can researchers effectively study the dual role of PSMD10 in both normal physiology and pathological conditions?

Studying PSMD10's dual roles in normal physiology and pathology requires sophisticated experimental approaches that can distinguish context-dependent functions:

• Physiologically relevant model systems:

  • Develop conditional knockout models allowing temporal control of PSMD10 deletion

  • Use patient-derived cells and tissues to study disease-specific alterations

  • Employ human intestinal organoids (HIOs) for investigating PSMD10 in gut physiology and infection responses

  • Create knock-in models expressing structure-based mutants (e.g., C4S) to selectively disrupt specific functions

• Quantitative proteomics approaches:

  • Apply SILAC or TMT labeling to compare PSMD10 interactomes in normal versus pathological states

  • Use thermal proteome profiling to identify proteins whose stability depends on PSMD10 interaction

  • Perform systematic ubiquitinome analysis to identify PSMD10-dependent degradation substrates

• Systems biology integration:

  • Combine transcriptomics, proteomics, and functional assays to build comprehensive network models

  • Apply mathematical modeling to predict how PSMD10 perturbation affects different cellular pathways

  • Validate model predictions using targeted interventions

• Pathway-specific reporters:

  • Develop biosensors that specifically monitor PSMD10 activity in proteasomal versus autophagic contexts

  • Create dual-reporter systems to simultaneously track pathway engagement in living cells

  • Apply spatial proteomics to determine compartment-specific functions

• Therapeutic relevance:

  • Screen for compounds that selectively modulate specific PSMD10 functions

  • Use the high-resolution crystal structure (PDB ID: 7VO6) to identify potential small molecule binding sites

  • Test whether targeting PSMD10 can restore normal pathway function in disease models

When interpreting data from these approaches, researchers should carefully consider that PSMD10 may simultaneously affect multiple pathways through its diverse interactions. Rigorous controls and complementary methodologies are essential for distinguishing direct from indirect effects and for establishing causal relationships in complex cellular systems .

What novel approaches can reveal the structural determinants of PSMD10's selective protein interactions?

Understanding the structural basis of PSMD10's selective protein interactions requires integrating cutting-edge structural biology with functional approaches:

• High-resolution structural analysis:

  • Leverage the existing crystal structure (PDB ID: 7VO6) as a foundation for interaction studies

  • Apply cryo-electron microscopy to resolve PSMD10 in complex with partner proteins

  • Use NMR to characterize the dynamic aspects of PSMD10 interactions, particularly the conformational changes associated with dimerization

  • Perform hydrogen-deuterium exchange mass spectrometry to map binding interfaces and conformational changes

• Structure-guided mutational analysis:

  • Create a comprehensive library of surface mutations across PSMD10's structure

  • Systematically test how these mutations affect individual protein interactions

  • Develop deep mutational scanning approaches to simultaneously assess thousands of variants

  • Focus particularly on the ankyrin repeat regions that mediate specific interactions

• Advanced crosslinking strategies:

  • Apply genetically incorporated photoactivatable crosslinkers at specific positions throughout PSMD10

  • Use mass spectrometry to identify crosslinked peptides, precisely mapping interaction interfaces

  • Combine with molecular dynamics simulations to model transient interaction states

  • Develop time-resolved crosslinking to capture the dynamics of complex assembly/disassembly

• Engineered protein design:

  • Create chimeric proteins swapping domains between PSMD10 and related ankyrin repeat proteins

  • Design PSMD10 variants with altered specificity to test interaction models

  • Engineer artificial dimerization systems to control PSMD10 oligomeric state

  • Develop biosensors that report on specific PSMD10 conformational states

• Small molecule probes:

  • Identify compounds that bind to specific pockets in PSMD10 using the crystal structure

  • Test how these molecules affect individual protein interactions

  • Develop chemical genetics approaches using engineered binding pockets

These approaches would provide unprecedented insight into how PSMD10's structure determines its selective interactions with diverse partners, potentially revealing targetable features for therapeutic intervention .

What are common pitfalls in PSMD10 protein purification and how can they be addressed?

PSMD10 protein purification presents several challenges that researchers should anticipate and address:

• Maintaining native oligomeric state:

  • Challenge: PSMD10 exists as both monomers and dimers stabilized by disulfide bonds that can be disrupted during purification

  • Solution: Carefully control redox conditions during extraction and purification

  • Methodological approach: Use non-reducing buffers when preserving dimers is desired

  • Validation: Confirm oligomeric state by size-exclusion chromatography coupled with multi-angle light scattering

• Protein solubility issues:

  • Challenge: PSMD10 may aggregate or show poor solubility, particularly at high concentrations

  • Solution: Optimize buffer conditions (pH, salt concentration, additives)

  • Methodological approach: Test fusion tags that enhance solubility (MBP, SUMO)

  • Consider purifying individual domains if full-length protein proves problematic

• Preserving functional activity:

  • Challenge: Purified PSMD10 may lose interaction capability with partners

  • Solution: Validate functionality through in vitro binding assays with known partners (ATG7, ATG10)

  • Methodological approach: Include activity assays at each purification step to monitor functional integrity

• Preventing non-specific disulfide formation:

  • Challenge: The critical Cys4 residue can form non-native disulfide bonds during purification

  • Solution: Include appropriate concentrations of reducing agents when isolating monomeric PSMD10

  • Alternative approach: Use the C4S mutant when studying functions that don't require dimerization

• Crystallization challenges:

  • Challenge: Obtaining diffraction-quality crystals for structural studies

  • Solution: Screen multiple constructs with varying boundaries

  • Methodological approach: Consider surface entropy reduction mutations to promote crystal contacts

  • Success example: The high-resolution crystal structure (PDB ID: 7VO6) demonstrates that well-diffracting crystals are achievable

When developing purification protocols, researchers should carefully consider their experimental goals – whether preserving native dimers or working with defined monomeric forms – and adjust conditions accordingly. Rigorous quality control using multiple analytical techniques (SEC, native gels, activity assays) is essential to ensure the purified protein accurately represents the physiological state .

How can researchers overcome technical challenges in studying PSMD10 protein complexes?

Studying PSMD10 protein complexes presents several technical challenges that require specialized approaches:

• Preserving transient or weak interactions:

  • Challenge: PSMD10 forms dynamic complexes that may dissociate during standard isolation procedures

  • Solution: Apply crosslinking strategies prior to complex isolation

  • Methodological approach: Use genetically incorporated photo-crosslinkers like Azi positioned at key interfaces

  • Alternative: Chemical crosslinkers like disuccinimidyl suberate (DSS) can capture interactions without genetic modification

• Distinguishing direct from indirect interactions:

  • Challenge: Co-immunoprecipitation can pull down entire complexes, obscuring direct binding partners

  • Solution: Employ pairwise in vitro binding assays with purified components

  • Methodological approach: Map interaction domains through systematic deletion analysis

  • Validation: Confirm direct interactions through proximity-enabled crosslinking in cells

• Capturing condition-specific complexes:

  • Challenge: PSMD10 forms different complexes under various cellular conditions

  • Solution: Apply proximity labeling techniques (BioID, TurboID) under defined conditions

  • Methodological approach: Compare PSMD10 interactomes in normal, stress, and pathological states

  • Example: Compare PSMD10 complexes during bacterial infection versus normal conditions

• Analyzing membrane-associated complexes:

  • Challenge: Some PSMD10 complexes may form at membranes during autophagosome formation

  • Solution: Use membrane fractionation approaches combined with crosslinking

  • Methodological approach: Apply proximity labeling specifically at membrane interfaces

  • Validation: Fluorescence microscopy to confirm colocalization at relevant membranes

• Quantifying complex stoichiometry:

  • Challenge: Determining the composition and stoichiometry of native PSMD10 complexes

  • Solution: Apply quantitative proteomics with absolute quantification standards

  • Methodological approach: Native mass spectrometry of intact complexes

  • Alternative: Single-molecule approaches to analyze complex composition

For any complex analysis, researchers should employ multiple orthogonal techniques and include appropriate controls, such as interaction-deficient mutants (e.g., C4S for dimerization-dependent complexes or ankyrin repeat deletions for specific partner interactions). The combination of biochemical, structural, and cellular approaches provides the most comprehensive understanding of PSMD10's diverse protein complexes .

What are best practices for ensuring reproducibility in PSMD10 functional studies?

Ensuring reproducibility in PSMD10 functional studies requires rigorous attention to methodological details:

• Experimental model standardization:

  • Challenge: Cell line heterogeneity and passage effects can influence PSMD10 function

  • Solution: Use early-passage, authenticated cell lines with documented mycoplasma testing

  • Methodological approach: Create and validate PSMD10 knockout cell lines as clean backgrounds for functional studies

  • Consider using human intestinal organoids (HIOs) for more physiologically relevant systems

• Expression level considerations:

  • Challenge: Overexpression can drive non-physiological interactions and pathway perturbations

  • Solution: Use inducible expression systems to control PSMD10 levels

  • Methodological approach: Generate cell lines with CRISPR-edited endogenous PSMD10 (tagged or mutated)

  • Validation: Quantify expression levels relative to endogenous PSMD10 in all experiments

• Functional assay standardization:

  • Challenge: Autophagy and proteasome assays are sensitive to culture conditions

  • Solution: Develop standardized protocols with appropriate positive and negative controls

  • Methodological approach: Include time-course analyses to capture dynamic responses

  • Validation: Use multiple independent readouts for each pathway (e.g., for autophagy: LC3 puncta, LC3-II formation, and substrate degradation)

• Controlling for off-target effects:

  • Challenge: siRNA or CRISPR approaches may have unintended consequences

  • Solution: Use multiple independent targeting sequences

  • Methodological approach: Confirm phenotypes with rescue experiments using siRNA-resistant constructs

  • Validation: Compare phenotypes obtained with genetic versus chemical inhibition approaches

• Data analysis and reporting:

  • Challenge: Selective reporting and analytical variations can limit reproducibility

  • Solution: Pre-register experimental plans when possible

  • Methodological approach: Report all experimental attempts, including negative results

  • Provide detailed methods including cell culture conditions, antibody sources/dilutions, and statistical approaches

  • Share raw data when possible

• Interaction studies reproducibility:

  • Challenge: Protein-protein interactions can be influenced by experimental conditions

  • Solution: Validate key interactions with multiple techniques (co-IP, crosslinking, proximity labeling)

  • Methodological approach: Include interaction-deficient mutants as negative controls

  • Test interactions under different cellular conditions to assess robustness

By adhering to these best practices, researchers can enhance the reproducibility of PSMD10 functional studies and build a more reliable foundation for understanding its complex roles in cellular physiology and pathology .