ARFIP2 Human

What is ARFIP2 and what are its fundamental structural characteristics?

ARFIP2 is a protein encoded by the ARFIP2 gene located on human chromosome 11 . The human recombinant ARFIP2 is a single polypeptide chain containing 341 amino acids with a molecular mass of approximately 40.2 kDa . Structurally, ARFIP2 is characterized by:

• An N-terminal domain (1-108) critical for protein-protein interactions

• A conserved AH-BAR (Amphipathic Helix-Bin/Amphiphysin/Rvs) domain (109-341) that mediates membrane binding and shaping

• Membrane-binding capabilities, particularly to PI4P-enriched membrane domains

The BAR domain is particularly significant as it enables ARFIP2 to sense and induce membrane curvature, a function essential for its role in vesicular trafficking and membrane dynamics.

What are the primary cellular functions of ARFIP2?

ARFIP2 serves multiple critical cellular functions across different pathways:

• Membrane trafficking: Regulates cargo exit from the Golgi apparatus and is involved in endocytosis through the trans-Golgi network in a PI4P-dependent manner

• Autophagy regulation: Acts as a cofactor for ATG9A-mediated autophagosome formation

• Lysosomal homeostasis: Cooperates with ATG9A to regulate PI4P levels for lysosomal membrane integrity after damage and during bacterial infection

• Mitochondrial quality control: Facilitates mitophagy through the PINK1/Parkin pathway, particularly in specialized cells like podocytes

• Cytoskeletal organization: As a Rac1 binding protein, it is essential for Rac-mediated actin polymerization leading to membrane ruffling and lamellipodia formation

How can researchers effectively isolate and purify ARFIP2 for in vitro studies?

For effective isolation and purification of ARFIP2:

• Expression system selection: E. coli-based expression systems have been successfully used to produce recombinant human ARFIP2

• Protein tagging strategy: N-terminal His-tagging (typically 23 amino acids) facilitates purification while maintaining protein functionality

• Purification protocol:

  • Use proprietary chromatographic techniques for optimal purity

  • A purification protocol yielding >90% purity (as determined by SDS-PAGE) has been documented

• Buffer composition for stability:

  • Recommended buffer: 20mM Tris-HCl buffer (pH 8.0), 0.2M NaCl, 1mM DTT, and 40% glycerol

  • This formulation maintains protein stability during storage and handling

Which protein-protein interactions are most significant for ARFIP2 function?

ARFIP2 engages in multiple protein-protein interactions that define its cellular functions:

• Small GTPases:

  • ARF family: Interacts with Arf6, ARF3, and ARF5 in a GTP-dependent manner

  • RAC1: Functions as a binding partner essential for actin cytoskeleton dynamics

• Autophagy-related proteins:

  • ATG9A: ARFIP2 facilitates ATG9A trafficking during autophagosome formation

  • AP-3S1: The N-terminal domain of ARFIP2 mediates this interaction, which is crucial for vesicle coat assembly

• Lipid metabolism enzymes:

  • PI4K2A: ARFIP2 interacts with PI4K2A through its N-terminal domain, influencing PI4P production at membrane sites

  • ORP9: ARFIP2 can inhibit ORP9/11-mediated lipid transport, particularly when donor liposomes are enriched in PI4P

These interactions collectively enable ARFIP2 to coordinate membrane trafficking, autophagy, and lipid homeostasis across multiple cellular compartments.

What methodological approaches can researchers use to study ARFIP2's role in autophagy?

Investigating ARFIP2's role in autophagy requires multi-faceted methodological approaches:

• Genetic manipulation strategies:

  • CRISPR/Cas9-mediated ARFIP2 knockout in cell lines (e.g., immortalized podocytes) to assess autophagy flux

  • Domain-specific mutants to dissect the contribution of N-terminal versus AH-BAR domains

  • Chimeric constructs (e.g., ARFIP2-N-terminal fused with ARFIP1-BAR domain) to investigate domain-specific functions

• Autophagy flux assessment:

  • LC3 puncta formation and conversion (LC3-I to LC3-II) by immunoblotting

  • p62/SQSTM1 accumulation as an indicator of impaired autophagy

  • Tandem fluorescent-tagged LC3 (mRFP-GFP-LC3) to distinguish between autophagosome formation and lysosomal fusion

• Protein trafficking visualization:

  • Live-cell imaging of fluorescently tagged ATG9A to track vesicular movement between compartments

  • Immunofluorescence colocalization studies of ARFIP2 with ATG9A and lysosomal/Golgi markers

  • Electron microscopy to visualize ultrastructural changes in autophagosome formation

• Biochemical interaction assays:

  • GFP-trap experiments to identify ARFIP2 interaction partners in the autophagy pathway

  • Immunoisolation of LAMP-1-positive membranes to assess ARFIP2 recruitment to lysosomes upon damage

How does ARFIP2 regulate PI4P levels at lysosomal membranes, and what techniques can assess this function?

ARFIP2 plays a critical role in regulating PI4P levels at lysosomal membranes through several mechanisms:

• Regulatory mechanism:

  • ARFIP2 is recruited to PI4P-enriched lysosomal domains through its AH-BAR domain after lysosomal membrane permeabilization

  • It inhibits PI4P lipid transfer mediated by ORP9-10-11 heterodimers, preventing excessive PI4P depletion

  • ARFIP2 interacts with PI4K2A and AP-3 to facilitate coat assembly on PI4P-enriched regions, promoting vesicle formation and retrieval to the Golgi

• Experimental techniques for assessment:

  • In vitro lipid transport assays: FRET-based assays measuring ORP9/11-mediated PI4P transport between donor and acceptor liposomes, with and without ARFIP2 addition

  • PI4P visualization: Specific PI4P probes (e.g., P4C domain from SidC) fused to fluorescent proteins

  • Liposome binding assays: Using purified ARFIP2 and PI4P-containing liposomes to assess direct binding

  • Lysosomal damage models: LLOMe treatment to induce lysosomal membrane permeabilization followed by immunoisolation of LAMP-1 positive membranes

What role does ARFIP2 play in bacterial infection defense, and how can this be experimentally investigated?

ARFIP2 contributes to cellular defense against bacterial pathogens through lysosomal membrane repair mechanisms:

• Defense mechanism:

  • Upon bacterial infection (e.g., M. tuberculosis and Salmonella), ARFIP2 helps restrict pathogen proliferation

  • ARFIP2 acts as a modulator of the PITT (PI4P-regulated interorganelle transport) pathway, maintaining lysosomal membrane integrity during bacterial challenge

  • Following lysosomal membrane permeabilization, ARFIP2 works with ATG9A to deliver PI4K2A to lysosomes, facilitating PI4P production necessary for ER-lysosome contact site formation and lipid exchange for repair

• Experimental approaches:

  • Bacterial infection models: M. tuberculosis and Salmonella infection in control versus ARFIP2-deficient cells to assess pathogen restriction

  • Bacterial survival assays: Colony-forming unit (CFU) measurements in infected cells with normal or depleted ARFIP2

  • Lysosomal damage assessment: Galectin-3 puncta formation as a marker of ruptured vesicles

  • Time-course studies: Analysis of ARFIP2 recruitment to damaged lysosomes during infection progression

  • Contact site visualization: Techniques to visualize ER-lysosome contact sites during infection and repair processes

• Advanced analysis methods:

  • Super-resolution microscopy to precisely track ARFIP2 localization during infection

  • Proteomics analysis of ARFIP2-associated protein complexes in infected versus uninfected cells

  • Phospholipid profiling of lysosomes during bacterial infection in the presence or absence of ARFIP2

How does ARFIP2 contribute to mitochondrial quality control, and what are the recommended methods to study this function?

ARFIP2 plays a significant role in mitochondrial quality control, particularly through the regulation of mitophagy:

• Functional involvement:

  • ARFIP2 deficiency interferes with ATG9A trafficking and the PINK1-Parkin pathway

  • This leads to compromised mitochondrial fission and affects mitochondrial respiration

  • ARFIP2 facilitates ATG9A trafficking during PINK1/Parkin-regulated mitophagy

• Recommended methodological approaches:

  • Mitochondrial morphology analysis: Fluorescence microscopy using MitoTracker or mitochondrially-targeted fluorescent proteins to assess fission/fusion dynamics

  • Mitochondrial function assessment:

    • Oxygen consumption rate (OCR) measurements

    • Mitochondrial membrane potential analysis using JC-1 or TMRM dyes

    • ATP production assays

  • Mitophagy flux quantification:

    • mt-Keima or mito-QC reporter systems for mitophagy visualization

    • Immunoblotting for mitochondrial proteins (e.g., TOMM20, COX4) to assess mitochondrial mass

    • Colocalization of mitochondrial markers with autophagy/lysosomal markers

• PINK1/Parkin pathway analysis:

  • PINK1 stabilization assessment on depolarized mitochondria

  • Parkin recruitment to damaged mitochondria

  • Ubiquitination of outer mitochondrial membrane proteins

• In vivo validation:

  • Streptozotozin-induced diabetic mouse models with Arfip2 deficiency to assess mitochondrial changes in podocytes

  • Ultrastructural analysis of mitochondria in tissue samples using electron microscopy

What are the methodological considerations when studying ARFIP2 in hepatocellular carcinoma (HCC)?

Research on ARFIP2 in hepatocellular carcinoma requires specialized approaches:

• Expression analysis methods:

  • Quantitative real-time PCR (qPCR) using gene-specific primers for ARFIP2 with β-actin as internal control

  • Western blot analysis to quantify protein levels in tumor versus normal tissue

  • Immunohistochemistry (IHC) of paraffin-embedded HCC specimens to assess protein expression patterns

• Clinical correlation approaches:

  • Stratification of patient samples based on recurrence/metastasis (R/M) timing:

    • NR/M group (no recurrence/metastasis)

    • R/M ≤12 months group

    • R/M 12-24 months group

  • Correlation analysis between ARFIP2 expression and clinicopathological features:

    • Tumor number (single vs. multiple)

    • Microvascular invasion

    • Tumor differentiation

    • TNM staging

• Functional studies in HCC models:

  • Gain-of-function and loss-of-function experiments in HCC cell lines

  • Analysis of EMT (epithelial-mesenchymal transition) markers

  • Migration and invasion assays

  • Assessment of PI3K/Akt signaling pathway components

How can researchers effectively differentiate between the functions of ARFIP1 and ARFIP2?

Distinguishing between ARFIP1 and ARFIP2 functions requires specialized experimental approaches:

• Domain-specific analysis:

  • The AH-BAR domains of ARFIP1 and ARFIP2 are highly conserved, while their N-terminal domains differ

  • Creation of chimeric proteins (e.g., N-terminal ARFIP2 (1-92) fused with AH-BAR domain of ARFIP1 (94-341)) can help isolate domain-specific functions

• Interaction partner profiling:

  • ARFIP2 interacts with AP-3S1, ORP9, and PI4K2A through its N-terminal domain, while ARFIP1 does not share these interactions

  • ARFIP1 is not involved in ATG9A vesicle trafficking, unlike ARFIP2

  • Comparative interactome analysis using techniques like BioID or proximity labeling

• Subcellular localization:

  • While both proteins localize to the Golgi, ARFIP2 is specifically recruited to lysosomes upon damage

  • The N-terminal ARFIP2-ΔBAR (1-108) domain does not retain Golgi localization when overexpressed, unlike the full-length protein

• Functional rescue experiments:

  • Knockout of one family member followed by expression of the other to test functional complementation

  • Domain swapping experiments to identify regions responsible for specific functions

What are the critical factors for successful ARFIP2 protein storage and handling?

For optimal ARFIP2 protein stability and functionality:

• Storage conditions:

  • Store at 4°C if the entire vial will be used within 2-4 weeks

  • For longer periods, store frozen at -20°C

  • Add a carrier protein (0.1% HSA or BSA) for long-term storage

  • Avoid multiple freeze-thaw cycles

• Buffer formulation:

  • Optimal buffer: 20mM Tris-HCl buffer (pH 8.0), 0.2M NaCl, 1mM DTT, and 40% glycerol

  • DTT is critical for maintaining the protein in a reduced state

  • High glycerol percentage helps prevent protein aggregation during freeze-thaw cycles

• Working concentration considerations:

  • Typical working concentration of 0.25mg/ml has been documented as functional

  • Dilution should be performed in the same buffer to maintain stability

What experimental controls are essential when studying ARFIP2 in cellular systems?

When designing experiments to study ARFIP2 function:

• Essential controls for genetic manipulation studies:

  • Empty vector controls for overexpression studies

  • Non-targeting siRNA/shRNA for knockdown experiments

  • Wildtype cell lines alongside CRISPR/Cas9-generated knockout lines

  • Rescue experiments with wildtype ARFIP2 to confirm phenotype specificity

• Domain-specific controls:

  • AH-BAR domain only constructs

  • N-terminal domain only constructs

  • Chimeric constructs (ARFIP1/ARFIP2) to isolate domain-specific functions

• Interaction controls:

  • GTP-locked and GDP-locked mutants of ARF proteins to test GTP-dependency of interactions

  • Use of ARFIP1 as a control for ARFIP2-specific interactions

  • Co-immunoprecipitation with irrelevant proteins to confirm binding specificity

• Functional assays:

  • Positive and negative controls for autophagy induction (e.g., rapamycin, bafilomycin A1)

  • Controls for lysosomal damage (e.g., LLOMe treatment) with time course analyses

  • Mitochondrial function controls (e.g., CCCP for depolarization)