Recombinant Chicken 55 kDa erythrocyte membrane protein (MPP1)

What is Chicken 55 kDa erythrocyte membrane protein (MPP1) and what are its key functions?

MPP1, also known as erythrocyte protein p55, is a membrane-associated protein that serves multiple critical functions in cellular processes. In its primary role, MPP1 functions as an essential regulator of neutrophil polarity by modulating AKT1 phosphorylation through a mechanism independent of PIK3CG activity .

MPP1 belongs to the MAGUK (Membrane-Associated Guanylate Kinase) family and serves as a crucial scaffolding protein. It interacts with components like flotillins and is significantly involved in the lateral organization of the erythroid plasma membrane . Additionally, MPP1 exhibits properties of a plus-end-directed kinesin-related protein with microtubule-binding and bundling capabilities, along with microtubule-stimulated ATPase activity .

During cell division, MPP1 plays a critical role in cytokinesis, where its suppression by RNA interference leads to failure of cell division during the late stages of this process .

How can researchers effectively detect and quantify MPP1 in biological samples?

For reliable detection and quantification of Chicken MPP1, sandwich ELISA represents the gold standard methodology. The protocol employs the following procedure:

• Antibody Coating: A microplate is pre-coated with an antibody specific for MPP1.

• Sample Application: Standards and samples are pipetted into wells where any MPP1 present binds to the immobilized antibody.

• Detection System: After washing, a biotin-conjugated antibody specific for MPP1 is added, followed by Streptavidin-conjugated Horseradish Peroxidase (HRP).

• Signal Generation: A substrate solution develops color proportional to the amount of bound MPP1.

• Measurement: The color reaction is stopped and intensity measured .

Current commercially available Chicken MPP1 ELISA kits demonstrate high specificity with no significant cross-reactivity between Chicken MPP1 and analogues. The assay reproducibility shows standard deviation less than 8% for standards repeated 20 times on the same plate, and less than 10% when measured across different operators .

What is the expression pattern of MPP1 in various tissues and developmental stages?

MPP1 exhibits a ubiquitous expression pattern across multiple tissues, unlike some other MPP family members such as MPP4, which is predominantly found in the retina .

During embryonic development, MPP1 expression has been detected from E14.5 onwards with varying tissue distribution:

Embryonic stages (E14.5-E18.5):

• Intense staining in liver and primitive gut

• Lower signal intensity in umbilical vein, ventricular layer of CNS, and jaw regions

• Expression in the neuroblastic layer of the eye, with intensity increasing from E16.5 to E18.5

• Strong expression in bone structures during ossification (e.g., femur at E16.5)

Postnatal stages (P7-P90):

• In the eye, expression localizes to the ganglion cell layer, inner nuclear layer, and photoreceptor cell layer

This expression pattern suggests MPP1 has multiple tissue-specific functions throughout development and in adult organisms.

What role does MPP1 play in cell division and how can it be studied experimentally?

MPP1, initially identified as M-Phase Phosphoprotein 1 through screening of proteins specifically phosphorylated at the G2/M transition, has been characterized as a plus-end-directed kinesin-related protein essential for cell division .

Experimental evidence for MPP1's role in cell division:

• Localization during cell cycle:

  • Interphase: Primarily nuclear localization

  • Metaphase: Diffuse throughout cytoplasm

  • Anaphase/Telophase: Localizes to the midzone

  • Cytokinesis: Concentrates on the midbody

• Functional experiments:

  • RNA interference targeting MPP1 results in failure of cell division during late cytokinesis

  • In vitro studies demonstrate MPP1 is a slow molecular motor (0.07 μm/s) that moves toward microtubule plus-ends

Recommended experimental approaches:

• Fluorescence microscopy with GFP-MPP1 fusion proteins to monitor dynamic localization

• In vitro motility assays using recombinant MPP1 and polarity-marked microtubules

• ATPase activity assays to measure microtubule-stimulated activity

• RNA interference or CRISPR-based approaches for functional studies

How can researchers produce and purify palmitoylated recombinant MPP1?

Palmitoylation of MPP1 plays a critical role in its function, particularly in the lateral organization of the erythroid plasma membrane . Obtaining properly palmitoylated MPP1 for in vitro studies represents a significant technical challenge. The following optimized protocol enables high-yield production of palmitoylated recombinant MPP1:

Expression System: Mammalian HEK-293F cells provide the optimal environment for proper post-translational modifications, particularly palmitoylation .

Purification Protocol:

• Clone the MPP1 coding sequence into an appropriate expression vector with a purification tag (e.g., FLAG, His)

• Transfect HEK-293F cells using optimized transfection conditions

• Harvest cells 48 hours post-transfection

• Lyse cells in appropriate buffer (e.g., 50mM Tris pH 8.0, 0.5M NaCl, 2mM MgCl₂)

• Purify using affinity chromatography based on the incorporated tag

• Verify palmitoylation status using Acyl-RAC methodology

This approach produces functional palmitoylated MPP1 suitable for studies of protein-protein and protein-membrane interactions, facilitating research on the molecular mechanisms of lateral membrane organization .

What is the significance of the MPP1-ABCC4 interaction in drug resistance, and how can it be targeted?

The interaction between MPP1 and ABCC4 has significant implications for drug resistance, particularly in Acute Myeloid Leukemia (AML). This protein complex plays a critical role in chemotherapeutic resistance through several mechanisms:

Experimental approaches for targeting this interaction:

• Genetic disruption of the PDZ-binding motif of ABCC4

• Small molecule screening to identify compounds that disrupt the protein complex

• Structure-based drug design targeting the interaction interface

Case study: High-throughput screening identified Antimycin A as a small molecule capable of disrupting the ABCC4-MPP1 protein complex, thereby reversing drug resistance in AML cell lines and primary patient AML cells .

How does MPP1 contribute to membrane organization and protein scaffolding?

MPP1 serves as a critical scaffolding protein in multiple cellular contexts, connecting various protein complexes to the membrane and cytoskeleton. Its scaffolding functions are primarily mediated through several key interactions:

In erythrocytes:

• Forms a complex with glycophorin C and protein 4.1, facilitating subcortical cytoskeleton-membrane linkage

• Interacts with flotillins, playing a crucial role in lateral organization of the erythroid plasma membrane

In neural tissues:

• Functions as a scaffold in post-synaptic regions of neurons

• Binds to MPP5 at the outer limiting membrane (OLM) of the retina

In epithelial polarity:

• Connects the Crumbs protein complex to the membrane

• May provide linkage between the Crumbs protein network and the actin cytoskeleton

Protein domains mediating interactions:

• SH3 domain

• HOOK domain

• GUK domain

• PDZ domain

The palmitoylation of MPP1 appears to be critical for these functions, particularly in membrane organization. Research suggests that this post-translational modification affects MPP1's ability to interact with membrane components and other proteins in the scaffolding network .

What methodologies are most effective for studying protein-protein interactions involving MPP1?

Investigating MPP1's interactions with other proteins requires a multi-faceted approach to capture both in vitro binding properties and in vivo functional relevance. The following methodologies have proven effective:

• Yeast two-hybrid screening:

  • Successfully identified MPP1-MPP5 interactions

  • Particularly effective for mapping specific interacting domains

  • Can be optimized using cell-to-cell mating protocols for higher efficiency

• Biochemical validation approaches:

  • GST-pull down assays to confirm interactions and determine binding affinities

  • Co-immunoprecipitation from tissue lysates (e.g., retinal lysates) to verify endogenous interactions

  • Western blotting to detect interacting partners

• Microscopy-based techniques:

  • Immunofluorescence to examine co-localization of MPP1 with partner proteins

  • Advanced techniques such as Förster Resonance Energy Transfer (FRET) or Proximity Ligation Assay (PLA) for detecting interactions in situ

• Functional validation:

  • Mutational analysis of interaction domains

  • RNA interference to examine consequences of protein depletion

  • Overexpression of wild-type versus mutant proteins

Case study: Research on MPP1-MPP5 interaction demonstrated that their binding is directional: the MPP1 prey containing the GUK domain interacts with the SH3+HOOK domain of MPP5, but the SH3+HOOK domain of MPP1 lacks binding affinity for the GUK domain in MPP5 .

What are the optimal conditions for recombinant MPP1 expression and purification?

Several expression systems have been utilized for producing recombinant MPP1, each with specific advantages depending on research needs:

Baculovirus Expression System:

• Suitable for producing full-length MPP1 (rMPP1) and truncated forms (e.g., rMC1)

• Protocol specifics:

  • Subclone MPP1 fragments into pFastBac HTb vector with appropriate tags (e.g., 6His, FLAG)

  • Generate recombinant viruses in Sf9 cells

  • Express proteins in High-Five cells

  • Harvest cells 48 hours post-infection (MOI=2)

  • Resuspend frozen cell pellets in lysis buffer (50 mM Tris, pH 8.0, 0.5 M NaCl, 2 mM MgCl₂)

  • Purify using affinity chromatography

Mammalian Expression System (HEK-293F cells):

• Optimal for producing palmitoylated MPP1

• Essential when post-translational modifications are required for functional studies

• Higher production costs but better biological activity

Choosing the right tags:

• FLAG epitope: Useful for immunodetection and purification

• 6His tag: Effective for metal affinity chromatography

• GFP fusion: Beneficial for localization studies

Careful consideration of the expression system based on experimental requirements is crucial for obtaining functional recombinant MPP1 with the necessary post-translational modifications.

How can researchers effectively study MPP1's role in cell polarity and cytoskeletal organization?

MPP1's involvement in cell polarity and cytoskeletal organization can be investigated through several complementary approaches:

Microtubule binding and motility assays:

• Prepare taxol-stabilized microtubules (MTs)

• Incubate recombinant MPP1 (0.1 μM) with taxol-MTs (1 μM) in BRB80 buffer with 10 μM taxol

• Fix with glutaraldehyde in MEM-50% sucrose

• Visualize using immunofluorescence with appropriate antibodies (e.g., anti-tubulin and anti-tag antibodies)

Motility assays with polarity-marked microtubules:

• Prepare flow cells coated with motor protein (0.15-0.3 μM)

• Wash with motility assay buffer (MAB: BRB80 buffer with 0.1 mg/ml casein, 1 mM ATP, 20 μM taxol)

• Add asymmetrically labeled MTs and observe movement

• Calculate velocity and directionality

Cell-based approaches:

• Generate GFP-MPP1 fusion constructs for live imaging

• Use RNA interference to deplete endogenous MPP1

• Employ fixed-cell immunofluorescence to examine effects on cytoskeletal organization

• Analyze changes in cell polarity markers following MPP1 manipulation

These methodologies provide complementary insights into MPP1's functions, from biochemical properties to cellular consequences of MPP1 modulation.

What are the key considerations for designing MPP1 knockout or knockdown experiments?

When designing genetic manipulation experiments targeting MPP1, researchers should consider several critical factors to ensure valid and interpretable results:

RNA Interference Approaches:

• siRNA design: Target conserved regions of MPP1 mRNA

• Validation: Confirm knockdown efficiency using qRT-PCR and western blotting

• Controls: Include non-targeting siRNA controls

• Phenotypic analysis: Focus on cytokinesis completion, as MPP1 suppression induces failure of cell division late in cytokinesis

CRISPR-Cas9 Gene Editing:

• Guide RNA selection: Choose targets with minimal off-target effects

• Verification: Sequence edit sites to confirm modifications

• Rescue experiments: Re-express wild-type or mutant MPP1 to confirm specificity

• Cell viability considerations: Given MPP1's role in cell division, complete knockout may affect cell viability

Tissue-Specific Approaches:

• Consider conditional knockouts for in vivo studies

• Use tissue-specific promoters for targeted expression

• Evaluate developmental timing for inducible systems

Readouts for Successful Manipulation:

• Cytokinesis defects: Binucleated cells, incomplete abscission

• Polarity disruption: Altered distribution of polarity markers

• Protein mislocalization: Changes in membrane protein distribution

• Functional assays: Drug sensitivity (particularly in cancer cells)

These considerations ensure that MPP1 manipulation experiments provide specific and interpretable insights into its biological functions.

How is MPP1 research contributing to our understanding of drug resistance in cancer?

MPP1 research has revealed significant insights into mechanisms of drug resistance, particularly in Acute Myeloid Leukemia (AML). Key findings include:

These findings suggest that targeting protein-protein interactions involving MPP1 may represent a novel strategy to overcome drug resistance in cancer therapy.

What are the emerging techniques for studying post-translational modifications of MPP1?

Post-translational modifications, particularly palmitoylation, are critical for MPP1 function. Several emerging techniques enable detailed investigation of these modifications:

• Acyl-RAC (Resin-Assisted Capture):

  • Allows specific isolation of palmitoylated proteins

  • Enables quantitative assessment of MPP1 palmitoylation levels

  • Compatible with downstream mass spectrometry analysis

• Click chemistry approaches:

  • Metabolic labeling with alkyne-modified palmitate analogs

  • Conjugation to azide-containing detection tags via click chemistry

  • Provides temporal resolution of palmitoylation dynamics

• Site-directed mutagenesis:

  • Targeted modification of putative palmitoylation sites

  • Functional assessment of mutants lacking specific modifications

  • Combined with cellular localization studies to determine impact

• Mass spectrometry techniques:

  • Direct identification of modified residues

  • Quantitative assessment of modification stoichiometry

  • Comparison across different cellular conditions

These methodologies provide complementary approaches to understand how post-translational modifications regulate MPP1's interactions, localization, and functions in different cellular contexts.

How can researchers leverage MPP1 studies for potential therapeutic applications?

Research on MPP1 has revealed several promising avenues for therapeutic development:

• Targeting drug resistance in cancer:

  • Disruption of the MPP1-ABCC4 complex sensitizes cancer cells to chemotherapy

  • Small molecules like Antimycin A provide proof-of-concept for this approach

  • Structure-guided design could yield more specific inhibitors

• Cell division modulators:

  • MPP1's essential role in cytokinesis suggests potential for anti-proliferative strategies

  • Targeting MPP1's motor or ATPase activity could inhibit cancer cell division

• Membrane organization therapeutics:

  • MPP1's role in membrane organization might be leveraged to alter cellular uptake of drugs

  • Modulating palmitoylation could provide a mechanism to control MPP1 function

• Biomarker applications:

  • High MPP1 expression correlates with poor prognosis in AML

  • Could serve as a prognostic biomarker or for patient stratification

• Polarity restoration approaches:

  • In diseases with disrupted cell polarity, modulating MPP1 function might help restore normal polarity

  • Potential applications in epithelial disorders or retinal diseases

Future research directions should focus on developing more specific modulators of MPP1 function and validating these approaches in relevant disease models.