PRMT1 Human

What is the structural organization of PRMT1 and how does it relate to function?

PRMT1 exhibits a two-domain structure consisting of an AdoMet (S-adenosyl-L-methionine) binding domain and a barrel-like domain, with the active site pocket located between these two domains . Crystallographic studies have revealed three peptide binding channels: two positioned between the domains and one on the surface perpendicular to the strands forming the β barrel .

The enzyme functions as a dimer, and this dimerization is essential for AdoMet binding and subsequent enzymatic activity . Mutagenesis studies have confirmed that two active site glutamates are crucial for catalytic activity, with the E153Q mutation specifically disrupting enzymatic function . The active site architecture precisely positions substrate arginine residues for methylation, ensuring specific and efficient catalysis.

What are the known PRMT1 splicing variants and their functional implications?

PRMT1 exhibits multiple splicing variants, particularly at the N-terminus which is the least conserved region among PRMT1 proteins across species . In humans, at least six splicing variants (v1-v6) have been documented, producing proteins between 353-371 amino acids with different N-terminal regions .

Mouse studies have demonstrated that variants v1 and v2 display different substrate specificities despite high sequence similarity . The vast majority of human and mouse ESTs represent splicing version 1, suggesting its predominance in vivo . In Xenopus, at least two versions corresponding to mammalian v1 and v2 can also be found .

How is PRMT1 expression regulated in different human tissues?

PRMT1 is expressed at detectable levels in all examined tissues, with particularly high expression in developing neural structures during embryogenesis . The gene is highly conserved across eukaryotes, with sequence identity exceeding 90% among mammals, zebrafish, and Xenopus, and approximately 50% between human and S. cerevisiae .

In human colon tissues, specific expression patterns have been documented:

• PRMT1 is detected in all colonic mucosa crypt cells

• Strong PRMT1 staining appears in both submucosal and myenteric plexuses

• Specific and intense expression occurs in neuron cell bodies, confirmed by co-localization with the neuron-specific marker HuC/D

• PRMT1 is present in the same neuronal cells expressing neuronal nitric oxide synthase (NOS)

This expression pattern suggests tissue-specific roles for PRMT1 in neural development and function, particularly in the enteric nervous system.

What experimental methodologies are commonly used to study PRMT1 activity?

Several established methodologies are employed to investigate PRMT1 activity:

• In vitro methylation assays: Utilizing purified PRMT1 enzyme with substrate proteins (such as hnRNP A1) or peptides containing arginine residues . The activity is measured using radioactively labeled [³H]-AdoMet or [¹⁴C]-AdoMet as methyl donors.

• Western blot analysis: Detecting PRMT1 protein levels or asymmetrically dimethylated arginine using specific antibodies . This method was crucial in demonstrating reduced PRMT1 protein levels in aganglionosis segments of Hirschsprung disease patients.

• Immunohistochemistry and immunofluorescence: Localizing PRMT1 expression in tissues or cells, often with double-labeling to identify co-expression with cell-type specific markers .

• Molecular dynamic simulation: Used to predict and improve inhibitory activities of potential PRMT1 inhibitors .

• Mutagenesis studies: Identifying critical residues for enzymatic activity, such as active site glutamates and residues involved in dimerization .

These techniques collectively provide comprehensive insights into PRMT1's structure, function, and biological roles.

What is the evolutionary conservation pattern of PRMT1 across species?

PRMT1 exhibits remarkable evolutionary conservation, indicating its fundamental biological importance:

The high conservation extends to genomic structure as well. For instance, human and A. thaliana PRMT1 genes share identical positions for seven of eight introns, despite their evolutionary distance . This conservation underscores PRMT1's essential cellular functions maintained throughout eukaryotic evolution.

How does PRMT1 contribute to neurodegenerative diseases?

PRMT1 has been implicated in several neurodegenerative conditions, particularly amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) . The mechanism involves several pathways:

• Neural development: PRMT1 is essential for the development of neurons, astrocytes, and oligodendrocytes in the central nervous system . Disruption of these developmental processes may contribute to neurodegenerative vulnerability.

• Protein methylation: PRMT1 methylates various neuronal proteins, modifying their function, localization, and interactions . Altered methylation patterns may contribute to protein aggregation and neural dysfunction.

• Transcriptional regulation: As a regulator of gene expression, PRMT1 influences neuronal homeostasis and response to cellular stress .

• Nitric oxide regulation: PRMT1 co-localizes with neuronal nitric oxide synthase (NOS) in enteric neurons, suggesting a potential role in regulating nitric oxide signaling, which is crucial for neuronal function .

Further investigation using cell type-specific PRMT1-deficient animal models is required to precisely understand its roles in neurodegenerative pathogenesis . The continued relevance of PRMT1 in neurodegenerative diseases remains an active and promising research area.

What is the role of PRMT1 in cancer biology, particularly in glioblastoma?

PRMT1 plays significant roles in cancer biology through multiple mechanisms:

• Myc interaction: PRMT1 forms a protein complex with Myc and PRMT5 in glioblastoma stem cells (GSCs) . Within this complex, PRMT1 asymmetrically dimethylates Myc, while PRMT5 symmetrically dimethylates it. These modifications differentially regulate Myc stability and binding to target promoters .

• TGF-β/SMAD signaling: PRMT1 functions as an essential mediator of TGF-β/SMAD signaling, promoting TGF-β-induced epithelial-mesenchymal transition (EMT) through SMAD7 methylation . This mechanism contributes to cancer cell invasion and metastasis.

• Altered expression: Enhanced PRMT1 expression has been documented in various cancers, including lung cancer, correlating with poor prognosis through promotion of tumor cell growth, proliferation, invasion, and metastasis .

The ratio between symmetrically and asymmetrically dimethylated Myc changes in GSCs grown in stem versus differentiating conditions, suggesting a regulatory mechanism in cancer stemness . These findings identify PRMT1 as a potential therapeutic target and dimethylated Myc species as possible diagnostic and prognostic markers for glioblastoma multiforme (GBM) .

How can PRMT1 activity be targeted for therapeutic intervention?

Several approaches have been developed to target PRMT1 activity for therapeutic purposes:

• Small molecule inhibitors: Compounds such as WCJ-394 have been identified as potent PRMT1 inhibitors . WCJ-394 significantly affects expression of PRMT1-related proteins and inhibits TGF-β1-induced EMT in A549 lung cancer cells, leading to significant inhibition of cancer cell invasion and metastasis .

• Rational drug design strategies: Novel PRMT1 inhibitors have been designed by introducing hydrogen donor groups (such as amide, hydrazide, amino, and N-hydroxyamidino) on benzene rings of lead compounds . Molecular dynamic simulation has been used to predict and improve inhibitory activities .

• Structure-based targeting: The crystal structure of PRMT1 provides insights for designing inhibitors targeting the active site pocket between its two domains . The two essential glutamate residues in the active site present potential targets for inhibitor design .

• Dimerization interference: Since dimerization is essential for PRMT1 activity, compounds disrupting this process could serve as effective inhibitors .

WCJ-394 has emerged as an important leading compound for future PRMT1-guided drug discovery, with demonstrated efficacy in preclinical models .

What is the significance of PRMT1 in the enteric nervous system and Hirschsprung disease?

PRMT1 plays critical roles in the enteric nervous system (ENS) with particular relevance to Hirschsprung disease (HSCR):

The significance of these patterns includes:

• Diagnostic potential: Western blot analyses reveal reduced PRMT1 protein levels in aganglionosis segments of HSCR patients . The absence of PRMT1 staining in plexuses of aganglionosis segments makes it a potential marker for HSCR .

• Disease specificity: The absence of PRMT1 staining is specific to the megacolon of HSCR, as strong PRMT1 staining is observed in plexuses of rectal ectasia segments from anorectal malformation patients .

• Co-expression with NOS: PRMT1 is present in the same neuronal cells expressing neuronal NOS in plexuses, suggesting a potential regulatory relationship between arginine methylation and nitric oxide synthesis, which is crucial for intestinal motility .

These findings establish PRMT1 as a useful marker for HSCR and provide a foundation for investigating PRMT1's function in ENS development and intestinal motility .

How do PRMT1 (Type I) and PRMT5 (Type II) methylation patterns differ in their biological impact?

PRMT1 and PRMT5 catalyze distinct types of arginine methylation with different biological consequences:

The biological significance of these differences is exemplified in the case of Myc regulation in glioblastoma:

• Differential stability regulation: Asymmetrically dimethylated Myc (by PRMT1) and symmetrically dimethylated Myc (by PRMT5) exhibit different stability properties .

• Dynamic equilibrium: The ratio between these two methylation states changes during cellular differentiation in glioblastoma stem cells, suggesting a regulatory switch mechanism .

• Transcriptional impact: The different methylation patterns differentially affect Myc binding to its target promoters, influencing downstream gene expression .

This dual methylation system represents a previously unrecognized layer of post-translational regulation for Myc and potentially other proteins, opening new avenues for understanding cellular differentiation and disease processes .

What are the critical amino acid residues and structural features for PRMT1 enzymatic activity?

Several key residues and structural features are essential for PRMT1 enzymatic activity:

• Active site glutamates: Two glutamate residues in the active site are critical for catalytic activity . The E153Q mutation specifically disrupts enzymatic function, highlighting its importance .

• Dimerization interface: Specific residues at the dimerization interface are essential, as dimerization is required for AdoMet binding and subsequent enzymatic activity .

• AdoMet binding pocket: Located in the AdoMet binding domain, this pocket contains residues that interact with the methyl donor and position it for methyl transfer .

• Substrate binding channels: Three peptide binding channels have been identified that accommodate substrate proteins and position arginine residues for methylation :

  • Two channels located between the two domains

  • One channel on the surface perpendicular to the strands forming the β barrel

• N-terminal region: While the N-terminus is the least conserved region among PRMT1 proteins, different N-terminal variants can affect substrate specificity .

Understanding these critical structural features provides a basis for rational drug design and for predicting how mutations might affect PRMT1 function in disease states.

How does PRMT1-mediated arginine methylation interact with other post-translational modifications?

PRMT1-mediated methylation exists within a complex network of post-translational modifications (PTMs) that collectively regulate protein function:

• Crosstalk with phosphorylation: Methylation by PRMT1 can influence phosphorylation of nearby residues and vice versa. This interplay is particularly important in signal transduction pathways .

• Interaction with ubiquitination: PRMT1-mediated methylation can affect protein stability by modulating ubiquitination, as observed with Myc in glioblastoma stem cells .

• Histone modification crosstalk: As a histone modifier, PRMT1 participates in the "histone code," where different modifications interact to regulate chromatin structure and gene expression .

• Competition with other PRMTs: PRMT1 may compete with other arginine methyltransferases (like PRMT5) for the same substrate, creating a dynamic balance between different methylation patterns .

• Enzymatic regulation: PRMT1 activity itself can be regulated by various PTMs, including phosphorylation, which adds another layer of complexity to this regulatory network .

This intricate interplay between different PTMs creates a sophisticated regulatory system that fine-tunes protein function in response to cellular needs and environmental signals.

What methods can be used to specifically monitor asymmetric arginine dimethylation by PRMT1 in complex biological samples?

Several advanced methodologies can specifically detect PRMT1-mediated asymmetric arginine dimethylation:

• Antibody-based approaches:

  • Western blotting with antibodies specific for asymmetrically dimethylated arginine (ADMA)

  • Immunoprecipitation followed by mass spectrometry (IP-MS) to enrich and identify methylated proteins

  • Immunohistochemistry and immunofluorescence for tissue and cellular localization

• Mass spectrometry-based methods:

  • Targeted multiple reaction monitoring (MRM) MS for quantitative analysis of specific methylated peptides

  • Global proteomics approaches with ADMA-specific enrichment strategies

  • Heavy isotope labeling to track methylation dynamics

• In vitro enzymatic assays:

  • Radioactive methylation assays using purified PRMT1 and [³H]-AdoMet or [¹⁴C]-AdoMet

  • Fluorescence-based assay systems that specifically detect asymmetric dimethylation

• Genetic approaches:

  • PRMT1 knockdown/knockout followed by comparative methylome analysis

  • Expression of substrate mutants lacking specific arginine residues to confirm methylation sites

These methodologies, often used in combination, provide comprehensive insights into the PRMT1 methylome and its dynamic changes in development and disease states .