ssm4 Antibody
Description
Introduction to SSM4 Antibody
SSM4 antibodies are immunological reagents designed to recognize and bind to SSM4 proteins found in various yeast species. Currently, commercial antibodies are available for detecting SSM4 in both Saccharomyces cerevisiae (baker's yeast) and Schizosaccharomyces pombe (fission yeast) . These antibodies have enabled researchers to investigate the diverse functions of SSM4 proteins across different yeast models, enhancing our understanding of evolutionary conservation and functional diversity in protein quality control and splicing mechanisms.
The antibodies are primarily available as polyclonal preparations purified through antigen affinity techniques, offering high specificity for their target proteins . While sharing the same name, it's important to note that SSM4 proteins in different yeast species may have distinct functions and characteristics, necessitating species-specific antibody preparations.
SSM4 in Saccharomyces cerevisiae
In Saccharomyces cerevisiae, SSM4 (also known as Doa10) functions as an E3 ubiquitin ligase involved in the endoplasmic reticulum-associated degradation (ERAD) pathway . This protein plays a critical role in protein quality control, specifically targeting misfolded or aberrant proteins for degradation. Recent research has shown that SSM4/Doa10 interacts with Ypf1, a homolog of signal peptide peptidase (SPP), in topology surveillance mechanisms . This interaction is particularly important for identifying and eliminating proteins with incorrect membrane topology, such as the lanosterol demethylase CYP51A1 when its amphipathic helix is aberrantly inserted into the membrane .
SSM4 in Schizosaccharomyces pombe
In contrast, SSM4 in Schizosaccharomyces pombe appears to be involved in meiotic regulation. It is classified among meiotic genes that are typically silenced during vegetative growth . Research indicates that SSM4 expression is regulated through mechanisms involving 3'-end formation and is subjected to surveillance by the Dhp1/Rat1/Xrn2 factor, which triggers premature transcription termination as a means of gene silencing .
SSm4 in Trypanosomes
Remarkably, a distinct SSm4 protein has been identified in trypanosomes, where it functions as a U4-specific Sm core protein essential for RNA splicing . This SSm4 plays a critical role in both trans- and cis-splicing mechanisms. Silencing of SSm4 in trypanosomes results in dramatic growth inhibition and general splicing defects, highlighting its essential nature . Furthermore, SSm4 is required for the integrity of the U4 snRNA and U4/U6 di-snRNP complexes, which are fundamental components of the splicing machinery .
Investigation of Splicing Mechanisms
SSm4 antibodies have proven invaluable in splicing research, particularly in trypanosome studies. Researchers have employed these antibodies to demonstrate that SSm4 is sufficient for specifying U4 Sm core assembly in vitro . Through reconstitution experiments, scientists showed that replacing SmD3 with SSm4 in the Sm core is sufficient to switch RNA binding specificity between canonical and U4-specific Sm sites . This finding has significant implications for understanding the specificity determinants in RNA-protein interactions during splicing.
Protein Quality Control Studies
In S. cerevisiae, SSM4/Doa10 antibodies have contributed to our understanding of protein quality control mechanisms. Research utilizing these antibodies has revealed how the E3 ubiquitin ligase collaborates with other proteins in the ERAD pathway to identify and eliminate aberrant proteins. Recent studies highlight that SSM4/Doa10 is particularly involved in topology surveillance, targeting proteins that have incorrect membrane orientation .
Meiotic Regulation Research
In fission yeast studies, ssm4 antibodies have facilitated investigations into meiotic gene regulation mechanisms. Researchers have used these antibodies to track ssm4 expression patterns and protein localization during different growth phases and meiosis . These studies have contributed to understanding how meiotic genes are silenced during vegetative growth and activated during sexual development.
SSm4 in RNA Splicing
One of the most significant findings regarding SSm4 comes from trypanosome studies, which revealed its essential role in RNA splicing . Through in vitro reconstitution experiments, researchers demonstrated that SSm4 is both necessary and sufficient for U4 snRNP assembly. The exchange of a single Sm polypeptide, SmD3 with SSm4, was found to be sufficient to switch the RNA binding specificity between canonical and U4-specific Sm sites . This discovery highlights the remarkable specificity determinants within the spliceosomal machinery.
Silencing of SSm4 in trypanosomes leads to characteristic phenotypes including:
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Dramatic growth inhibition
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General trans- and cis-splicing defects
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Accumulation of unspliced pre-mRNAs
These findings establish SSm4 as an essential component of the splicing machinery in trypanosomes, with potential implications for understanding analogous mechanisms in other organisms.
SSM4/Doa10 in Protein Quality Control
Recent research has expanded our understanding of SSM4/Doa10's role in protein quality control beyond simple misfolded protein detection. Studies have revealed that this E3 ubiquitin ligase, in cooperation with the SPP homolog Ypf1, participates in monitoring the topological orientation of transmembrane proteins . This surveillance mechanism specifically targets proteins with incorrectly oriented membrane domains, such as CYP51A1 molecules with amphipathic helices inserted in a type II orientation .
This discovery highlights a sophisticated quality control mechanism that extends beyond simple protein folding assessment to include topological evaluation of membrane proteins.
Regulation of Meiotic Genes
In fission yeast, ssm4 has been identified among meiotic genes regulated by the Dhp1/Rat1/Xrn2 factor, which triggers premature transcription termination . Researchers have mapped the 3' ends of ssm4 meiotic gene transcripts in vegetative cells to investigate mechanisms of silencing. This work contributes to our understanding of how cells regulate the expression of stage-specific genes and prevent their inappropriate activation.
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Ssm4p, a p150-Glued protein, is specifically upregulated in response to pheromone and plays a crucial role in this nuclear movement. PMID: 15509865
KEGG: spo:SPAC27D7.13c
STRING: 4896.SPAC27D7.13c.1
Q&A
What is SSM4 and how does it differ from SSTR4?
SSM4 (P40318) is a protein found in Saccharomyces cerevisiae (Baker's yeast) that functions in yeast cellular processes . This is distinct from SSTR4 (Somatostatin Receptor Type 4), which is a 388 amino acid multi-pass transmembrane glycoprotein receptor for somatostatin-14 found in mammals. SSTR4 is part of the G-protein coupled receptor superfamily that mediates inhibitory effects on hormone secretion and cell proliferation . These proteins share similar nomenclature but represent entirely different biological systems with distinct evolutionary origins and functions.
What are the major structural characteristics of SSTR4?
SSTR4 is a multi-pass transmembrane glycoprotein receptor that belongs to the G-protein coupled receptor superfamily. The receptor contains 388 amino acids and functions primarily as a receptor for somatostatin-14. It demonstrates similar affinities for natural SST-14 and SST-28 but exhibits marked differences in affinities toward synthetic analogues. Structurally important regions include the second extracellular loop, containing amino acid residues 182-194, which serves as an immunogenic epitope for antibody production .
In which tissue types is SSTR4 typically expressed?
Somatostatin receptors, including SSTR4, are expressed by various tissues, notably:
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Neuronal cells (particularly in the hippocampal pyramidal layer)
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Endocrine tissues
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Gastrointestinal tract
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Immune cells
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Certain tumor types
Immunohistochemical staining has demonstrated SSTR4 expression in the rat hippocampus, particularly in the pyramidal layer, with distinct localization patterns separate from parvalbumin-expressing interneurons . Flow cytometry has also confirmed SSTR4 expression on the cell surface of human THP-1 monocytic leukemia cells .
Antibody Selection and Characterization
Methodological validation of SSTR4 antibodies should include:
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Blocking peptide experiments: Compare antibody reactivity with and without pre-incubation with SSTR4 blocking peptide (e.g., Somatostatin Receptor Type 4 extracellular Blocking Peptide #BLP-SR004)
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Western blot analysis: Verify band size corresponds to the predicted molecular weight of SSTR4 (approximately 40-45 kDa)
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Cross-reactivity testing: Confirm the antibody does not react with other SSTR subtypes (particularly important as Mouse anti-Human Somatostatin Receptor 4 antibody specifically recognizes SSTR type 4, but none of the other SSTR subtypes)
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Multiple detection methods: Validate findings using orthogonal techniques such as WB, IHC, and flow cytometry
What are optimal protocols for using SSTR4 antibodies in immunohistochemistry?
For successful SSTR4 immunohistochemistry, researchers should consider:
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Fixation: 4% paraformaldehyde is recommended for tissue preservation
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Antigen retrieval: For formalin-fixed, paraffin-embedded tissues, perform antigen retrieval with either TE buffer (pH 9.0) or citrate buffer (pH 6.0)
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Blocking: 2% BSA for 1 hour at room temperature to reduce non-specific binding
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Primary antibody dilution:
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Detection systems: Fluorescent secondary antibodies for co-localization studies or HRP-conjugated systems for chromogenic detection
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Controls: Include negative controls (secondary antibody only) and positive controls (tissues known to express SSTR4)
What methodologies enable functional analysis of SSTR4 in experimental systems?
When studying SSTR4 function, researchers should consider:
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G-protein signaling assays: SSTR4 is functionally coupled to inhibition of adenylate cyclase and activation of arachidonate release and MAP kinase cascade
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Cell proliferation assays: SSTR4 mediates antiproliferative actions of somatostatin in tumor cells
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Receptor binding studies: Competitive binding assays can be utilized similar to those developed for other somatostatin receptors (like the PSGL-1 competitive binding assay designed for SSL5)
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Functional inhibition studies: Similar to approaches used with other receptor systems, antibodies can be tested for their ability to block receptor function
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Gene expression analysis: Evaluate downstream effects of receptor activation/inhibition on target gene expression
How can scFv antibody technology be applied to receptor studies similar to SSTR4?
Phage display technology for developing single-chain variable fragment (scFv) antibodies represents an advanced approach that could be applied to SSTR4 research:
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Selection process: Similar to the methodology used for SSL proteins, researchers can perform multiple rounds of panning against SSTR4, alternating between different immobilization strategies to reduce non-specific binding
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Functional screening: Selected scFvs can be tested for their ability to bind the target and inhibit receptor function in biological assays
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Structural analysis: Promising scFv candidates can be analyzed through protein-protein docking and molecular dynamics simulations to predict binding modes
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Binding energy calculations: Computational analysis can predict favorable binding interactions and help optimize antibody design
This approach has successfully yielded inhibitory antibodies against other target proteins and could be adapted for SSTR4 research applications.
What are the challenges in developing therapeutic antibodies targeting SSTR4?
Developing therapeutic antibodies against SSTR4 presents several research challenges:
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Receptor accessibility: As a multi-pass transmembrane protein, SSTR4 has limited extracellular domains accessible for antibody binding
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Cross-reactivity: The antibody must distinguish between SSTR4 and other SSTR family members that share structural similarities
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Functional outcomes: Determining whether an antibody should act as an antagonist or agonist depends on the desired therapeutic effect
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Internalization dynamics: Understanding how antibody binding affects receptor internalization and recycling is critical for therapeutic efficacy
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Epitope selection: Targeting the second extracellular loop (as with the peptide DTRPARGGEAVAC, corresponding to amino acid residues 182-194 of rat SSTR4) has proven effective for detection but may have different requirements for therapeutic applications
How do SSTR4 antibodies compare with other tools for studying somatostatin signaling?
When designing experiments to study somatostatin signaling pathways, researchers should consider multiple approaches:
| Approach | Advantages | Limitations | Applications |
|---|---|---|---|
| SSTR4 antibodies | Direct protein detection, compatible with fixed samples | May have cross-reactivity, epitope masking | Protein localization, co-expression studies |
| Synthetic SST analogues | Longer half-lives than natural SSTs, receptor subtype selectivity | Different binding affinities compared to natural ligands | Functional assays, receptor activation studies |
| Genetic models | Complete elimination of receptor function | Compensatory mechanisms, developmental effects | In vivo functional studies, pathway analysis |
| Receptor binding assays | Quantitative affinity measurements | Limited to binding without downstream functional data | Drug screening, comparative pharmacology |
This multi-faceted approach allows for comprehensive characterization of somatostatin receptor biology and pharmacology .
What methodological approaches address contradictory findings in SSTR4 research?
When facing contradictory results in SSTR4 studies, consider implementing these methodological strategies:
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Antibody validation: Confirm antibody specificity using multiple techniques including blocking peptides, Western blotting with positive and negative controls, and comparison with genetic knockdown models
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Cell type considerations: SSTR4 expression and function may vary significantly between cell types; document and compare the exact cellular contexts used
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Species differences: Account for species-specific variations in SSTR4 sequence and function when comparing studies across different model organisms
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Experimental conditions: Standardize experimental conditions including ligand concentrations, incubation times, and detection methods
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Quantitative analysis: Employ rigorous statistical approaches and report quantitative data with appropriate controls to facilitate cross-study comparisons
This systematic approach helps resolve apparent contradictions and builds a more coherent understanding of SSTR4 biology.
