Recombinant Human Patatin-like phospholipase domain-containing protein 5 (PNPLA5)

What is the domain structure of PNPLA5 and how does it relate to its function?

PNPLA5 is a 429 amino acid protein containing two main domains: an N-terminal patatin α/β hydrolase domain and a C-terminal domain with distinct functions. The patatin domain (residues 12-181) harbors the catalytic dyad Ser49-Asp168 with the lipase motifs GSSSG and DGA, respectively . This structural arrangement is critical for its enzymatic function as a triacylglycerol lipase.

The domain organization of PNPLA5 directly influences its functional capabilities:

• The N-terminal catalytic domain confers enzymatic activity

• The C-terminal domain (residues 286-429) is essential for lipid droplet targeting

• Specific residues in the C-terminal domain (positions 358-361) contain a conserved arginine-rich motif (RSRRLV) critical for lipid droplet binding

Experimental truncation studies have demonstrated that constructs lacking the C-terminal domain (e.g., PNPLA5(1-286)) lose lipid droplet localization and exhibit a dominant-negative effect on lipid metabolism, despite retaining an intact catalytic domain .

What specific sequence motifs in PNPLA5 are responsible for lipid droplet targeting?

The lipid droplet targeting mechanism of PNPLA5 has been characterized through systematic truncation experiments. These studies reveal that:

• The C-terminal third of PNPLA5 (residues 286-429) is both necessary and sufficient for lipid droplet targeting

• Truncation analysis demonstrated that:

  • Removal of residues up to 364 from the C-terminus (constructs PNPLA5(286-364) and PNPLA5(286-376)) had no appreciable effect on lipid droplet binding

  • Further truncation to residue 353 (PNPLA5(286-352)) completely prevented lipid droplet association

• The key determinant is a basic patch region containing conserved arginine or positively charged amino acids at positions 358-361 in the amino acid sequence (RSRRLV motif)

This lipid targeting motif (LTM) appears to be a common feature among PNPLA family members, though the exact sequence varies. Researchers investigating other PNPLA proteins should consider these C-terminal regions as critical for proper subcellular localization and function.

How does PNPLA5 contribute to autophagosome formation at the molecular level?

PNPLA5 plays a critical role in autophagy initiation through its ability to mobilize lipids from lipid droplets for autophagosomal membrane formation. The molecular mechanisms include:

• TG mobilization: PNPLA5 acts as a triglyceride lipase that hydrolyzes stored triglycerides in lipid droplets

• Lipid intermediate generation: The diglycerides produced by PNPLA5's lipase activity can be converted to phospholipids necessary for autophagosomal membrane expansion

• Coordination with complementary enzymes: PNPLA5 functions in concert with:

  • CPT1 (cholinephosphotransferase), which transfers choline to DAG to form phosphatidylcholine

  • LPCAT2 (lysophosphatidylcholine acyltransferase 2), which remodels phospholipids

Experimental evidence supporting this role includes:

• PNPLA5 knockdown inhibits LC3-II conversion (a key marker of autophagosome formation)

• PNPLA5 overexpression increases autophagosome formation as measured by LC3 puncta

• PNPLA5 colocalizes with ATG16L1 (an early autophagy protein) on lipid droplets

This mechanism explains how PNPLA5 affects diverse autophagic substrates, including protein aggregates, mitochondria, and intracellular microbes, beyond just lipid droplet degradation (lipophagy) .

How does PNPLA5 function and structure compare to other members of the PNPLA family?

The PNPLA family consists of nine members (PNPLA1-9) with varying functions in lipid metabolism. Key comparisons include:

What are the optimal experimental approaches for studying PNPLA5 localization and activity?

Several complementary techniques have proven effective for investigating PNPLA5:

For Localization Studies:

• Fluorescence microscopy with tagged constructs:

  • N-terminal GFP-tagged PNPLA5 constructs combined with lipid droplet staining (e.g., LipidTox Red)

  • Advantage: Allows visualization of subcellular localization

  • Method: Transfect cells with GFP-PNPLA5 constructs, treat with oleic acid to induce lipid droplet formation, fix and stain

• Truncation and mutation analysis:

  • Generate systematic truncation constructs (e.g., PNPLA5(1-286), PNPLA5(286-429))

  • Site-directed mutagenesis of key residues (e.g., the basic patch region 358-361)

  • Advantage: Identifies critical regions for localization

• Co-localization with organelle markers:

  • Double labeling with lipid droplet markers and PNPLA5

  • Co-localization with autophagy proteins (e.g., ATG16L1)

  • Advantage: Provides context for functional studies

For Activity Studies:

• Triglyceride hydrolase assays:

  • Measure release of fatty acids from triglyceride substrates

  • Compare wild-type vs. catalytic mutants (e.g., S49A)

  • Advantage: Directly measures enzymatic activity

• Autophagy flux assays:

  • LC3-II conversion by western blot in the presence/absence of bafilomycin A1

  • High-content imaging analysis of LC3 puncta formation

  • Long-lived protein degradation assays

  • Advantage: Connects PNPLA5 activity to autophagy function

• Lipid droplet quantification:

  • Measure changes in lipid droplet size/number following PNPLA5 manipulation

  • Techniques include flow cytometry with neutral lipid dyes or microscopy-based analysis

  • Advantage: Assesses physiological impact on lipid stores

These approaches can be applied to both recombinant protein studies and cellular models expressing endogenous or overexpressed PNPLA5.

How is PNPLA5 expression regulated in different physiological conditions?

PNPLA5 expression exhibits tissue-specific and condition-dependent regulation:

Tissue Distribution:

• Expressed in almost every tissue in humans, with peak expression in the brain and pituitary gland

• This pattern differs from other PNPLA family members, suggesting specialized functions

Nutritional and Metabolic Regulation:

• Expression is low during fasting states

• Increases during adipocyte differentiation, similar to PNPLA3

• This pattern suggests a role in energy storage rather than mobilization during fasting

Chemical and Environmental Regulation:

Several compounds affect PNPLA5 expression as demonstrated in rodent models:

• Increased by:

  • 1-Naphthylisothiocyanate

  • Bisphenol A

• Decreased by:

  • 17β-estradiol

  • 4,4'-sulfonyldiphenol (bisphenol S)

  • Acetamide

Epigenetic Regulation:

• Benzo[a]pyrene affects methylation of the PNPLA5 promoter and increases methylation of PNPLA5 exons

These regulatory patterns provide insights into the physiological contexts where PNPLA5 function may be particularly important. The differential regulation compared to other PNPLA family members suggests non-redundant functions.

What evidence suggests connections between PNPLA5 and disease processes?

While direct evidence linking PNPLA5 to specific diseases is limited compared to other PNPLA family members, several lines of evidence suggest potential pathological connections:

Metabolic Disorders:

• PNPLA5's role in triglyceride metabolism suggests potential involvement in obesity and related metabolic disorders

• The established role of PNPLA5 in autophagy links it to conditions where autophagy dysfunction contributes to pathology

Comparative Evidence from Related PNPLAs:

Other PNPLA family members have established disease associations:

• PNPLA2 mutations: triglyceride accumulation in multiple tissues

• PNPLA3 (I148M variant): non-alcoholic fatty liver disease (NAFLD)

• PNPLA6/PNPLA9 mutations: neurological disorders and neurodegeneration

Experimental Evidence:

• PNPLA5 knockdown inhibits autophagic clearance of various substrates, including:

  • Protein aggregates (p62 accumulation)

  • Mitochondria (increased mitochondrial mass)

  • Intracellular microbes (reduced clearance)

Chemical Exposures:

• Multiple environmental compounds affect PNPLA5 expression , suggesting its potential involvement in toxicant-induced pathology

The connections between PNPLA5 and disease remain an active area of investigation, with particular relevance to metabolic disorders, neurodegenerative diseases (given high brain expression), and conditions associated with autophagy dysfunction.

How can researchers optimize the production and purification of recombinant PNPLA5?

Based on available protocols for PNPLA5 and related proteins, researchers can consider several approaches for optimal recombinant production:

Expression Systems:

Different expression systems have been successfully used:

• HEK-293 cells: Produce well-folded, mammalian post-translationally modified PNPLA5

• E. coli: Suitable for producing specific domains or fragments

• Cell-free protein synthesis (CFPS): Alternative for producing constructs that might be toxic in living cells

Tagging Strategies:

Several tags have been successfully employed:

• His-tag: Common for one-step affinity purification

• Strep-tag: Alternative for mild elution conditions

• GFP fusion proteins: Useful for localization studies

• His-Sumo tag: May improve solubility (as used for related PNPLA2)

Purification Considerations:

• Purification yields of >90% purity can be achieved using appropriate affinity chromatography

• For full-length PNPLA5, consider detergent screening to maintain solubility of the lipid droplet-binding domain

• Analytical SEC (HPLC) is recommended for quality control

Storage Recommendations:

• Store at -80°C to maintain activity

• For liquid formulations, Tris/PBS-based buffers with 5-50% glycerol help maintain stability

• For lyophilized preparations, consider Tris/PBS-based buffer with 6% Trehalose, pH 8.0 before lyophilization

• Avoid repeated freeze-thaw cycles

Functional Validation:

• Verify enzymatic activity through triacylglycerol hydrolase assays

• Confirm proper folding through circular dichroism or limited proteolysis

• For localization studies, verify lipid droplet binding capability in cellular assays

What methods can be used to study PNPLA5 interactions with other proteins in the autophagy pathway?

Several complementary approaches have been employed to study PNPLA5 protein interactions:

Microscopy-Based Methods:

• Co-localization studies:

  • Fluorescently tagged PNPLA5 combined with immunostaining of potential interacting partners

  • Example: PNPLA5 co-localization with ATG16L1 on lipid droplets

  • Advantage: Provides spatial context for interactions

• Förster Resonance Energy Transfer (FRET):

  • Can detect direct protein-protein interactions at nanometer scale

  • Similar approaches have been used to study PNPLA3 interactions with ABHD5

  • Methodology includes acceptor photobleaching or sensitized emission FRET

  • Advantage: Detects interactions in living cells

• Fluorescence Cross-Correlation Spectroscopy (FCCS):

  • Analyzes co-diffusion of fluorescently labeled proteins

  • Has been used successfully for analyzing related PNPLA family interactions

  • Advantage: Quantitative measurement of binding dynamics

Biochemical Methods:

• Co-immunoprecipitation:

  • Pull-down of PNPLA5 followed by detection of interacting partners

  • Can be performed with tagged recombinant proteins or endogenous proteins

  • Advantage: Identifies stable interactions

• Proximity-based labeling:

  • BioID or APEX2 fused to PNPLA5 to identify proximal proteins

  • Particularly useful for transient interactions on membrane surfaces

  • Advantage: Identifies interaction networks in native cellular contexts

• Surface Plasmon Resonance (SPR) or Biolayer Interferometry (BLI):

  • In vitro measurements of binding kinetics using purified proteins

  • Advantage: Provides quantitative binding parameters

Functional Validation Methods:

• Mutational analysis:

  • Generate interaction-deficient mutants based on structural predictions

  • Test effects on both binding and downstream functional outcomes

  • Example: Testing lipid droplet binding domain mutations on autophagy function

• Pharmacological disruption:

  • Use specific inhibitors of PNPLA5 or potential interacting partners

  • Measure effects on complex formation and downstream signaling

• Domain-swapping experiments:

  • Exchange functional domains between PNPLA family members

  • Determine which domains are necessary and sufficient for specific interactions

When investigating PNPLA5 interactions, researchers should consider the membrane/lipid droplet context, as these interactions may depend on specific lipid environments.

How do mutations in PNPLA5 affect autophagy, and what methodologies can detect these effects?

Although naturally occurring disease-associated mutations in PNPLA5 have not been extensively characterized, experimental mutations have revealed important structure-function relationships:

Key PNPLA5 Mutations and Their Effects:

• Catalytic site mutations:

  • Mutations in the Ser49-Asp168 catalytic dyad abolish enzymatic activity

  • These mutations maintain lipid droplet localization but prevent triglyceride hydrolysis

• Lipid droplet targeting motif mutations:

  • Alterations to the RSRRLV motif in the C-terminal domain disrupt lipid droplet localization

  • This prevents PNPLA5 from accessing its triglyceride substrates

  • Overexpression of PNPLA5 with altered LD-targeting motif decreases autophagic proteolysis of long-lived proteins during starvation

• Domain deletion mutations:

  • Deletion of the C-terminal domain (residues 286-429) prevents lipid droplet targeting

  • N-terminal domain constructs (e.g., PNPLA5(1-286)) localize to cytoplasm and nucleus instead of lipid droplets

  • These constructs fail to reduce lipid droplet size and can have dominant-negative effects

Methodologies to Assess Autophagy Defects:

• Autophagic flux measurements:

  • LC3-II immunoblotting with/without lysosomal inhibitors (e.g., bafilomycin A1)

  • Flow cytometry-based measurement of GFP-LC3 degradation

  • Tandem fluorescent-tagged LC3 (mRFP-GFP-LC3) to assess autophagosome maturation

• Substrate-specific autophagy assays:

  • p62/SQSTM1 degradation assays for selective autophagy

  • Mitochondrial mass measurement using MitoTracker Green for mitophagy

  • Long-lived protein degradation assays for bulk autophagy

• Microscopy-based analysis:

  • Quantification of LC3 puncta number and area using high-content imaging

  • Co-localization analysis between autophagosomes and specific cargo

• Lipid metabolism assessments:

  • Lipid droplet consumption during autophagy induction

  • Lipidomic analysis to detect changes in phospholipid profiles

• In vivo models:

  • PNPLA5 knockout or knockdown in animal models

  • Assessment of autophagy-dependent phenotypes in tissue-specific contexts