MTUS1 Antibody

What is MTUS1 and what are its key cellular functions?

MTUS1 (Microtubule Associated Tumor Suppressor 1) is a novel tumor suppressor protein involved in regulating cell proliferation, migration, and tumor growth. It's also known by several other names, including ATIP (Angiotensin-II Type 2 Receptor-Interacting Protein), MTSG1, and ATBP . MTUS1 functions primarily by:

• Cooperating with AGTR2 (Angiotensin-II Type 2 Receptor) to inhibit ERK2 activation and cell proliferation

• Potentially mediating AGTR2 cell surface expression

• Inducing UBE2V2 expression upon angiotensin-II stimulation (working with PTPN6)

• Inhibiting cancer cell proliferation (particularly isoform 1), delaying mitosis progression by prolonging metaphase, and reducing tumor growth

Recent research has revealed that MTUS1/ATIP1 localizes to the outer mitochondrial membrane and exerts anticancer effects through ROS-induced pyroptosis, particularly in head and neck squamous cell carcinoma (HNSCC) .

What cellular localizations are reported for MTUS1?

MTUS1 exhibits complex subcellular distribution patterns, with different isoforms showing distinct localizations:

• Cell membrane

• Cytoplasm

• Golgi apparatus

• Mitochondrion (specifically the outer mitochondrial membrane)

• Nucleus

• Centrosome

This diverse localization pattern enables MTUS1 to interact with various cellular signaling pathways and likely contributes to its multifaceted tumor suppressor functions .

What applications are MTUS1 antibodies validated for?

MTUS1 antibodies have been validated for multiple laboratory applications, though specific validations vary by antibody:

When selecting an antibody, researchers should verify the specific applications validated for their antibody of choice and confirm reactivity with their species of interest .

What are the expected molecular weights for MTUS1 detection?

When working with MTUS1 antibodies, researchers should be aware of multiple potential band sizes due to various isoforms:

The discrepancy between calculated and observed molecular weights likely reflects post-translational modifications and/or the complex tertiary structure of the protein .

How does MTUS1 expression correlate with cancer prognosis?

Research has demonstrated significant correlations between MTUS1 expression and cancer prognosis:

In colorectal adenocarcinoma:

These findings suggest MTUS1 functions as a tumor suppressor, and its expression levels could serve as a potential prognostic biomarker in colorectal and other cancers .

What is the significance of mitochondrial localization of MTUS1/ATIP1?

Recent studies have revealed MTUS1/ATIP1's localization to the outer mitochondrial membrane has significant functional implications:

• MTUS1/ATIP1 interacts directly with MFN2 (Mitofusin 2) in the outer mitochondrial membrane

• This interaction impacts:

  • Mitochondrial morphology

  • Mitochondrial movement

  • Mitochondrial metabolism

  • Cellular oxidative stress levels

Functional consequences observed include:

• Stimulation of reactive oxygen species (ROS)

• Recruitment of Bax to mitochondria

• Facilitation of cytochrome c release to the cytosol

• Activation of caspase-3

• Induction of GSDME-dependent pyroptotic death, particularly in HNSCC cells

These findings suggest MTUS1/ATIP1's mitochondrial localization plays a critical role in its tumor suppressor functions through regulation of cellular energy metabolism and programmed cell death pathways .

What methodological considerations should be made for MTUS1 immunohistochemistry?

For optimal MTUS1 immunohistochemical staining, researchers should consider the following methodological approaches based on published protocols:

Tissue Preparation:

• Formalin-fixed paraffin-embedded (FFPE) sections at 4-μm thickness

• Use of tissue microarrays (TMAs) with 3-mm-diameter tissue cylinders from representative cancer areas can enable high-throughput analysis

Staining Protocol:

• Automated systems (e.g., Benchmark XT System, Ventana Medical Systems) can provide consistent results

• Primary antibody recommendations:

  • Polyclonal rabbit anti-MTUS1 antibody at 1:100 dilution (Aviva)

  • Alternative: ab198176 at 1:40 dilution for human tissues

Scoring System:

• Signal intensity scoring on 0-3 scale:

  • 0: negative

  • 1: weak

  • 2: moderate

  • 3: strong

• Record percentage of cells at each intensity (in units of 10% using eyeballing method)

• Calculate H-score using: H-score = (1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+))

• Optimal cutoff determination using ROC curve analysis with survival data (e.g., H-score of 60)

Quality Control:

• Blinding of pathologists to clinical parameters and outcomes

• Use of multiple evaluators to ensure reproducibility

• Include proper positive controls based on known MTUS1 expression patterns

How can MTUS1 antibodies be used to investigate its role in cellular signaling?

To investigate MTUS1's role in cellular signaling pathways, researchers can employ the following methodological approaches:

Co-immunoprecipitation (Co-IP):

• Use MTUS1 antibodies with agarose conjugation (e.g., sc-393121 AC) to pull down protein complexes

• Investigate interactions with known partners like:

  • AGTR2 (AT2 receptor)

  • MFN2 (in mitochondrial studies)

  • PTPN6 and other signaling proteins

Dual Immunofluorescence/Co-localization:

• Combine MTUS1 antibodies with markers for specific cellular compartments (e.g., MitoTracker for mitochondria)

• Various conjugated forms available (FITC, PE, Alexa Fluor conjugates)

• Can help determine precise subcellular localization in different contexts

Functional Assays:

• Combine MTUS1 antibody detection with:

  • Mitochondrial function assays (membrane potential, ATP production)

  • ROS detection methods

  • Cell death assays (e.g., LDH release for pyroptosis)

  • ERK2 activation assays to assess tumor suppressor functions

Protease Protection Assays:

• Combine with Protease K assays to determine topology of MTUS1 in membranes

• Particularly useful for mitochondrial localization studies

These approaches can help elucidate MTUS1's roles in multiple signaling pathways, including the AT2 receptor pathway, mitochondrial function, and cell death mechanisms.

How do I troubleshoot inconsistent MTUS1 detection in Western blotting?

When facing inconsistent MTUS1 detection in Western blotting, consider these methodological troubleshooting approaches:

Sample Preparation:

• Ensure complete protein extraction using appropriate buffers for specific cellular compartments (particularly important for membrane-bound and mitochondrial pools of MTUS1)

• Use protease inhibitors to prevent degradation

• Consider phosphatase inhibitors if studying post-translationally modified forms

Antibody Selection:

• Match antibody to the specific MTUS1 isoform of interest (amino acid regions differ)

• Consider the following validated antibodies and their properties:

Transfer and Detection:

• For high molecular weight isoforms (120-180 kDa), ensure sufficient transfer time

• Consider reducing SDS-PAGE gel percentage for better resolution of high MW proteins

• Test different blocking agents if background is an issue

Positive Controls:

• Use validated positive controls including:

  • Human skeletal muscle tissue

  • Mouse uterus tissue

  • SH-SY5Y cells

Validation Approaches:

• Compare detection with multiple antibodies targeting different epitopes

• Include MTUS1 knockdown/overexpression controls to confirm specificity

• Consider species reactivity (human, mouse, rat) when selecting controls

What approaches can be used to study MTUS1's role in mitochondrial function and ROS-induced pyroptosis?

Recent research has revealed MTUS1/ATIP1's involvement in mitochondrial function and ROS-induced pyroptosis. To study these processes, researchers can employ these methodological approaches:

Mitochondrial Function Analysis:

• MitoTracker staining to assess mitochondrial morphology and distribution

• Seahorse assays to measure:

  • Oxygen Consumption Rate (OCR)

  • Extracellular Acidification Rate (ECAR)

  • ATP production capacity

Oxidative Stress Assessment:

• Intracellular ROS level measurement using fluorescent probes

• Mitochondrial membrane potential assays (measure red/green fluorescence intensity ratio)

• ATP level quantification in MTUS1-overexpressed vs. knockdown cells

Pyroptosis Detection:

• High-content microscopic imaging for morphological changes

• Immunofluorescence staining for pyroptosis markers including:

  • Bax recruitment to mitochondria

  • Cytochrome c release to cytosol

  • Caspase-3 activation

  • GSDME expression/cleavage

• Lactate dehydrogenase (LDH) release assay to quantify pyroptotic cell death

In Vivo Models:

• HNSCC cell-derived xenograft (CDX) models

• Patient-derived xenograft (PDX) models

• Combined with MTUS1/ATIP1 overexpression or knockdown to assess functional effects

These approaches can provide comprehensive insights into MTUS1's role in mitochondrial function and cell death pathways, potentially revealing new therapeutic targets for cancers with altered MTUS1 expression.

What are the current knowledge gaps in MTUS1 antibody applications for cancer research?

Despite significant advances in understanding MTUS1's role in cancer biology, several knowledge gaps remain that could benefit from further antibody-based research:

• Comprehensive mapping of all MTUS1 isoform-specific expression patterns across cancer types

• Development of isoform-specific antibodies for all five known transcript variants

• Standardized scoring systems for MTUS1 immunohistochemistry across different cancer types

• Better understanding of post-translational modifications affecting antibody detection

• Development of therapeutic antibodies targeting MTUS1 pathway components

Future research using MTUS1 antibodies should focus on these areas to advance our understanding of this important tumor suppressor and potentially develop new diagnostic and therapeutic approaches for cancers with altered MTUS1 expression .

How can MTUS1 antibodies contribute to developing new therapeutic strategies?

MTUS1 antibodies can play critical roles in developing new cancer therapeutic strategies through:

• Facilitating patient stratification based on MTUS1 expression patterns

• Identifying biomarkers predictive of response to therapies targeting MTUS1-associated pathways

• Enabling high-throughput screening of compounds that modulate MTUS1 expression

• Supporting development of therapeutic approaches targeting the ROS-induced pyroptosis pathway in cancers with low MTUS1 expression

• Validating in vivo responses in patient-derived xenograft models

Recent evidence showing MTUS1/ATIP1's role in mitochondrial function and ROS-induced pyroptosis provides a particularly promising avenue for therapeutic development in HNSCC and potentially other cancers .