PDCD6IP Human

What is PDCD6IP and what are its primary cellular functions?

PDCD6IP (also known as ALIX) is a protein encoded by the PDCD6IP gene in humans. It serves multiple critical cellular functions, including:

• Participation in the ESCRT pathway, specifically in membrane scission processes and multivesicular body formation

• Essential role in the abscission stage of cytokinesis

• Regulation of programmed cell death, where overexpression can block apoptosis

• Interaction with endophilins, which regulate membrane shape during endocytosis

For comprehensive functional studies, researchers should employ complementary approaches including gene knockdown/knockout experiments, fluorescent tagging for localization studies, co-immunoprecipitation, and live-cell imaging to track its dynamics during various cellular processes.

What protein domains are present in PDCD6IP and what are their functions?

PDCD6IP contains several functional domains that mediate its diverse cellular activities:

The V domain is particularly significant as it recognizes the Short linear motif LYPxLxxL, which is mimicked by viral proteins such as the p6 late domain of HIV to facilitate viral hijacking of the ESCRT pathway . When designing experiments to study domain-specific functions, consider creating targeted deletion mutants or introducing point mutations in conserved residues.

What is the evidence linking PDCD6IP mutations to neurodevelopmental disorders?

Recent research has established connections between PDCD6IP mutations and neurodevelopmental conditions:

• A homozygous frameshift variant (c.154_158dup; p.Val54Profs*18) was identified in a consanguineous family with primary microcephaly, intellectual disability, and short stature

• Affected individuals presented with microcephaly, deep-set eyes, delayed speech and language, and moderate to severe intellectual disability

• The clinical features matched phenotypes observed in mouse and zebrafish models with PDCD6IP mutations

This evidence aligns with PDCD6IP's role in the ESCRT pathway, which is essential for neural progenitor cell division and neuronal development. For researchers studying these connections, comprehensive genetic screening of PDCD6IP in cohorts with unexplained microcephaly and development of functional assays to classify variants would be valuable approaches.

What are the phenotypic characteristics of patients with PDCD6IP mutations?

Based on documented cases, patients with pathogenic PDCD6IP mutations exhibit:

These findings were consistent among affected individuals in the reported family . Interestingly, heterozygous carriers in the same family showed no clinical abnormalities, suggesting a recessive inheritance pattern for these specific manifestations.

How does PDCD6IP contribute to the ESCRT pathway in cellular processes?

PDCD6IP serves as a bridge between early and late-acting components of the ESCRT machinery, playing crucial roles in:

• Multivesicular body (MVB) biogenesis

• Cytokinesis, particularly during the abscission stage

• Viral budding, when hijacked by viral proteins like HIV p6

• Membrane repair mechanisms

To effectively investigate PDCD6IP's role in the ESCRT pathway, researchers should employ electron microscopy to visualize MVB formation, live-cell imaging with fluorescently tagged ESCRT proteins to track recruitment kinetics, and reconstitution experiments using purified components. A systematic workflow might include generating PDCD6IP-depleted cell lines, rescuing with wild-type or domain mutants, and quantifying phenotypes in ESCRT-dependent processes.

What is the relationship between PDCD6IP's intrinsic disorder and its function?

PDCD6IP exhibits significant intrinsic disorder, with approximately 44.5% of its structure classified as intrinsically disordered . This high level of disorder likely contributes to:

• Flexibility in binding multiple protein partners

• Ability to undergo rapid conformational changes

• Susceptibility to regulation by post-translational modifications

• Adaptability to different cellular environments

For researchers investigating this aspect, various methodologies are recommended:

• Computational prediction using algorithms like PONDR or IUPred

• Experimental characterization via nuclear magnetic resonance (NMR) or circular dichroism (CD) spectroscopy

• Functional analysis through mutation of disordered regions

• Investigation of disorder-to-order transitions upon binding partner interaction

What approaches are most effective for studying PDCD6IP interactions with viral proteins?

PDCD6IP is targeted by several viruses, most notably HIV, which mimics the LYPxLxxL motif to hijack the ESCRT machinery for viral budding . To study these interactions:

• Structural biology approaches:

  • X-ray crystallography or cryo-EM of PDCD6IP in complex with viral peptides

  • NMR for dynamic interaction studies

• Binding assays:

  • Surface plasmon resonance (SPR) to determine binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

• Cellular assays:

  • Virus-like particle (VLP) budding assays with wild-type or mutant PDCD6IP

  • Fluorescence resonance energy transfer (FRET) to detect interactions in living cells

For comprehensive analysis, researchers should combine multiple approaches to confirm direct binding, validate interactions in cellular contexts, and assess functional consequences of disrupting these interactions.

How can researchers effectively analyze ESCRT pathway defects related to PDCD6IP dysfunction?

To systematically analyze ESCRT pathway defects resulting from PDCD6IP dysfunction:

• Cellular assays:

  • Multivesicular body formation quantification

  • EGF receptor degradation kinetics

  • Extracellular vesicle production measurement

  • Cytokinesis completion analysis

• Imaging approaches:

  • Electron microscopy for MVB morphology

  • Live-cell imaging with fluorescently tagged ESCRT components

  • Super-resolution microscopy for detailed structural analysis

• Biochemical methods:

  • Subcellular fractionation to assess membrane association

  • Immunoblotting for ESCRT recruitment

  • Ubiquitinated cargo sorting efficiency

A comprehensive workflow should include multiple ESCRT-dependent process assessments in cellular models with PDCD6IP dysfunction, standardized quantification of defects, and mechanistic studies to identify the molecular basis of observed abnormalities.

How can researchers differentiate between pathogenic and benign variants in PDCD6IP?

To distinguish pathogenic from benign PDCD6IP variants, implement a multi-faceted approach:

The specific frameshift variant c.154_158dup (p.Val54Profs*18) occurring in the first exon of PDCD6IP was predicted to be deleterious by all available bioinformatics tools and segregated with disease in the reported family .

What are the phenotypic consequences of PDCD6IP deficiency in animal models?

Animal models have provided valuable insights into PDCD6IP deficiency:

• Mouse models:

  • Microcephaly similar to human cases

  • Neurological dysfunction

  • Defects in neural progenitor cell division

• Zebrafish models:

  • Developmental abnormalities that parallel human conditions with PDCD6IP mutations

For comprehensive animal model studies, researchers should create multiple model types (knockout, knockdown, knock-in of human mutations), perform detailed phenotypic characterization across developmental stages, and conduct rescue experiments to confirm causality.

What cellular assays can be used to assess PDCD6IP function in cytokinesis?

Given PDCD6IP's critical role in the abscission stage of cytokinesis , these assays are recommended:

• Live-cell imaging approaches:

  • Time-lapse microscopy of dividing cells

  • Quantification of abscission timing and success rate

  • Tracking of midbody formation and resolution

  • Visualization of ESCRT component recruitment to the midbody

• Fixed-cell analysis:

  • Immunofluorescence for midbody markers

  • Quantification of multinucleated cells (indicating cytokinesis failure)

  • Analysis of persistent midbody remnants

• Functional assays:

  • Rescue experiments with domain mutants

  • Cell cycle synchronization followed by abscission analysis

A comprehensive protocol should include generation of PDCD6IP-depleted or mutant cell lines, synchronization at specific cell cycle stages, live imaging of division progression, and correlation with molecular markers of cytokinesis.

What approaches can be used to investigate PDCD6IP in neurodevelopment?

Based on the link between PDCD6IP mutations and microcephaly , researchers can investigate its role in neurodevelopment through:

• Animal models:

  • Conditional knockout in neural progenitors

  • Knock-in models of human disease mutations

  • Zebrafish for rapid assessment of neurodevelopmental phenotypes

• Cellular models:

  • Cerebral organoids from patient-derived or engineered iPSCs

  • Primary neuronal cultures

  • Neural stem cell proliferation and differentiation assays

• Imaging techniques:

  • Time-lapse imaging to track neural progenitor division

  • 3D imaging of brain development

  • Super-resolution microscopy for subcellular localization

A comprehensive research strategy should include characterization of phenotypes at cellular, tissue, and behavioral levels, identification of molecular mechanisms using omics approaches, and testing of potential rescue strategies.