HARS Human, His

What is HARS and what is its primary function in human cells?

Histidyl-tRNA synthetase (HARS) is a critical enzyme responsible for catalyzing the aminoacylation of tRNAs with their corresponding amino acid, specifically histidine. This enzyme is essential for the incorporation of histidine into proteins during translation, making it a fundamental component of protein synthesis machinery . HARS belongs to the class II family of aminoacyl-tRNA synthetases, characterized by specific structural motifs and catalytic mechanisms . The enzyme functions as a homodimer with subunits consisting of 420-550 amino acid residues, which is considered relatively short compared to other aminoacyl-tRNA synthetases that can range from 300 to 1100 amino acid residues .

What are the key structural domains of human HARS protein?

Human HARS protein exhibits a three-domain architecture that is crucial for its functionality:

• N-terminal catalytic domain: Contains the six-stranded antiparallel β-sheet and three motifs characteristic of class II aminoacyl-tRNA synthetases. This domain is primarily responsible for the aminoacylation reaction and substrate binding .

• HisRS-specific helical domain: This domain is inserted between motifs 2 and 3 of the catalytic domain and is believed to interact with the acceptor stem of tRNA during the aminoacylation process .

• C-terminal α/β domain: This domain likely plays a role in the recognition of the anticodon stem and loop of tRNA^His, facilitating proper positioning of the tRNA for aminoacylation .

The integrated function of these domains enables HARS to properly recognize histidine and tRNA^His and catalyze the formation of histidyl-tRNA, which is essential for protein synthesis.

What is the significance of the His-tag in recombinant HARS protein?

The His-tag in recombinant HARS protein is a sequence of 6-10 histidine residues added to either the N- or C-terminus of the protein during recombinant production. In commercial preparations of HARS Human Recombinant protein, a 23 amino acid His tag is typically fused at the N-terminus when produced in E. coli expression systems . This tag serves multiple critical research purposes:

• Purification efficiency: The His-tag enables single-step purification using metal affinity chromatography (typically nickel or cobalt-based resins), resulting in high purity (>95%) protein preparations .

• Detection versatility: The His-tag allows for easy detection using anti-His antibodies in various experimental techniques such as Western blotting, immunoprecipitation, and immunofluorescence.

• Minimal impact on structure: The relatively small size of the His-tag generally minimizes interference with protein folding and function, making it an ideal tag for functional studies of HARS.

• Option for tag removal: If necessary for specific experimental applications, the His-tag can be removed using specific proteases when cleavage sites are incorporated into the construct design.

What are the optimal conditions for storing and handling recombinant HARS protein?

For optimal preservation of recombinant HARS protein activity and stability, the following storage and handling guidelines should be implemented:

Short-term storage (2-4 weeks):

• Store at 4°C in the buffer provided (typically 20mM Tris-HCl buffer, pH 8.0, containing 10% glycerol, 1mM DTT, and 0.1M NaCl) .

Long-term storage:

• Store frozen at -20°C for extended periods .

• For maximum stability during prolonged storage, addition of a carrier protein (0.1% HSA or BSA) is recommended .

• Avoid repeated freeze-thaw cycles as they can significantly compromise protein stability and activity .

Handling considerations:

• Always work with the protein on ice when thawed.

• Centrifuge the vial briefly before opening to ensure all liquid is at the bottom.

• Use sterile techniques to prevent contamination.

• Consider dividing the stock into single-use aliquots to minimize freeze-thaw cycles.

How can the aminoacylation activity of HARS be measured in experimental settings?

Measuring the aminoacylation activity of HARS requires monitoring the formation of histidyl-tRNA. Several methodologies can be employed:

Radioactive assay:

• Incubate HARS with tRNA^His, ATP, and [³H]- or [¹⁴C]-labeled histidine.

• Allow the aminoacylation reaction to proceed at physiological pH (usually 7.5) and temperature (37°C).

• Precipitate the aminoacyl-tRNA with trichloroacetic acid.

• Collect precipitates on filter papers and wash to remove unreacted labeled amino acids.

• Measure radioactivity to quantify the amount of histidyl-tRNA formed.

Non-radioactive pyrophosphate release assay:

• Couple the aminoacylation reaction to pyrophosphate (PPi) release.

• Use a pyrophosphatase to convert PPi to inorganic phosphate.

• Detect inorganic phosphate using colorimetric methods (malachite green assay).

• This method allows for continuous monitoring of the reaction.

HPLC-based assay:

• Conduct the aminoacylation reaction with unlabeled histidine.

• Extract and analyze the aminoacyl-tRNA by HPLC.

• Detect the aminoacylated tRNA by its characteristic elution profile compared to uncharged tRNA.

Each method offers different advantages in terms of sensitivity, ease of use, and compatibility with high-throughput screening.

What expression systems are most effective for producing recombinant human HARS?

E. coli expression systems are predominantly used for producing recombinant human HARS due to several advantages :

Bacterial expression advantages:

• High yield and cost-effectiveness

• Rapid growth and protein production

• Well-established protocols and expression vectors

• Compatibility with His-tag purification strategies

Optimized expression protocol:

• Clone the HARS coding sequence (amino acids 1-509) into a bacterial expression vector with an N-terminal His-tag.

• Transform into an appropriate E. coli strain (BL21(DE3) or similar).

• Culture in LB or TB media until mid-log phase (OD₆₀₀ = 0.6-0.8).

• Induce with IPTG (typically 0.5-1mM) at lowered temperature (16-25°C) to enhance solubility.

• Harvest cells after 16-20 hours of induction.

• Lyse cells in buffer containing 20mM Tris-HCl (pH 8.0), 10% glycerol, 1mM DTT, and 0.1M NaCl.

• Purify using nickel affinity chromatography.

Alternative expression systems:
For applications requiring post-translational modifications or when solubility is problematic in E. coli, consider:

• Mammalian cell lines (HEK293, CHO)

• Insect cell expression systems (Sf9, High Five)

• Yeast expression systems (Pichia pastoris)

Each alternative system requires specific optimization but may provide protein with characteristics more closely resembling the native human enzyme.

How does the structure of human HARS compare to HARS from other organisms?

Human HARS shares significant structural similarities with HARS from other organisms, particularly in conserved catalytic domains, while exhibiting species-specific variations:

Comparative structural analysis:

What is the mechanism of the aminoacylation reaction catalyzed by HARS?

The aminoacylation reaction catalyzed by HARS follows a two-step mechanism that is characteristic of class II aminoacyl-tRNA synthetases:

Step 1: Amino acid activation

• Histidine and ATP bind to the active site of HARS.

• HARS catalyzes the formation of histidyl-adenylate (His-AMP), an activated intermediate, with the release of pyrophosphate (PPi).

• This reaction requires magnesium ions as cofactors.

• The histidyl-adenylate remains bound to the enzyme.

Step 2: Transfer to tRNA

• The 3'-OH of the terminal adenosine (A76) of tRNA^His attacks the carbonyl carbon of the histidyl-adenylate.

• This results in the transfer of the histidine to the tRNA, forming histidyl-tRNA^His.

• AMP is released as a byproduct.

The reaction can be represented as:

• Histidine + ATP + HARS → HARS·His-AMP + PPi

• HARS·His-AMP + tRNA^His → Histidyl-tRNA^His + AMP + HARS

Research has shown that HARS can also catalyze the synthesis of diadenosine tetraphosphate (Ap₄A), a signaling molecule involved in various cellular regulatory mechanisms . This side reaction occurs when ATP attacks the enzyme-bound histidyl-adenylate instead of tRNA^His.

What are the key residues involved in substrate recognition and catalysis in human HARS?

The human HARS protein contains several critical residues and motifs that are essential for substrate recognition and catalysis:

Histidine recognition:

• A binding pocket with specific residues that form hydrogen bonds with the imidazole ring of histidine.

• Conserved residues that interact with the α-amino and α-carboxyl groups of histidine.

ATP binding and activation:

• Class II signature motifs 1, 2, and 3 form the ATP binding site.

• Motif 2 contains a conserved arginine that stabilizes the transition state during amino acid activation.

• Motif 3 contributes to positioning the 3' end of the tRNA.

tRNA recognition:

• The C-terminal domain contains residues that interact with the anticodon loop of tRNA^His.

• Specific residues recognize the unique G-1 residue at position -1 in tRNA^His (an identifying feature of histidine tRNAs).

• The helical domain interacts with the acceptor stem of tRNA^His.

Catalytic residues:

• Conserved residues position the 3'-OH of the terminal adenosine of tRNA for nucleophilic attack on the histidyl-adenylate.

• Metal-coordinating residues position magnesium ions essential for catalysis.

Mutagenesis studies have demonstrated that alterations to these key residues can significantly impact enzyme activity, substrate binding affinity, and specificity, providing insights into the molecular basis of HARS function and potential implications for disease-causing mutations.

How is HARS implicated in autoimmune diseases?

HARS has been identified as a significant autoantigen in several autoimmune diseases, with particularly strong associations to inflammatory myopathies:

HARS in myositis:

• HARS is the primary antigen recognized by anti-Jo-1 antibodies, which are the most common myositis-specific autoantibodies .

• Anti-Jo-1 antibodies are found in 20-30% of patients with polymyositis and in a smaller percentage of dermatomyositis patients.

• The presence of anti-Jo-1 antibodies defines a distinct clinical entity known as "antisynthetase syndrome," characterized by myositis, interstitial lung disease, arthritis, Raynaud's phenomenon, and mechanic's hands.

Epitope mapping:

• Research has successfully identified specific epitopes on HARS that are recognized by autoantibodies from patients with myositis .

• These epitopes are often located in surface-exposed regions of the protein that become accessible to the immune system.

• Conformational epitopes appear to be particularly important in the autoimmune response against HARS.

Pathogenic mechanisms:

• Several theories exist regarding how HARS becomes an autoantigen:

  • Enzymatic activity modification during inflammation

  • Abnormal subcellular localization and exposure to the immune system

  • Post-translational modifications creating neo-epitopes

  • Molecular mimicry with viral or bacterial proteins

Understanding the interaction between anti-Jo-1 antibodies and HARS has significant implications for diagnostic testing, disease monitoring, and potential therapeutic interventions in myositis and related autoimmune conditions.

What role do mutations in HARS play in human diseases?

Mutations in the HARS gene have been associated with several neurological disorders, particularly peripheral neuropathies:

Charcot-Marie-Tooth disease (CMT):

• Missense mutations in HARS have been identified in patients with CMT, a hereditary peripheral neuropathy.

• These mutations typically affect conserved residues important for enzyme function or stability.

• The pathogenic mechanism appears to involve both loss-of-function and potential gain-of-toxic-function effects.

Involvement in other neurological disorders:

• Certain HARS variants have been associated with peripheral neuropathy with or without additional neurological features.

• The phenotypic spectrum ranges from pure motor neuropathies to more complex syndromes with additional CNS involvement.

Biochemical consequences of disease-associated mutations:

• Reduced aminoacylation activity

• Impaired protein stability

• Altered tRNA binding affinity

• Abnormal subcellular localization

The link between HARS mutations and neurological disorders highlights the critical importance of accurate tRNA charging for neuronal function and maintenance. These findings have expanded our understanding of how defects in the translation machinery can lead to tissue-specific pathologies despite the ubiquitous expression of aminoacyl-tRNA synthetases.

How can HARS be utilized in studies of Human Accelerated Regions (HARs)?

While HARS (Histidyl-tRNA synthetase) and HARs (Human Accelerated Regions) are distinct entities, they intersect in exciting ways that open novel research directions:

Conceptual connections:

• Human Accelerated Regions (HARs) are genomic segments that have evolved rapidly in humans compared to other species, potentially contributing to human-specific traits .

• Approximately half of all HARs are active in brain development, particularly as enhancers that boost gene expression .

• Mutations in HARs have been associated with neurodevelopmental and cognitive disorders .

Research applications combining HARS and HARs:

• Evolutionary proteomics: Investigate whether HARs influence the expression or structure of translation machinery components, including HARS, during human brain development.

• Comparative systems biology: Examine how the aminoacylation activity of HARS may be regulated differently in human neural tissues compared to other primates, potentially due to HAR-mediated gene expression changes.

• Neurodevelopmental models: Utilize recombinant HARS protein in cell culture systems where HAR function has been modified (through CRISPR-based approaches) to assess the downstream impact on protein synthesis rates and patterns.

• Histidine metabolism in brain evolution: Explore how changes in histidine incorporation into proteins (mediated by HARS) might contribute to human-specific neural properties potentially influenced by HARs.

This interdisciplinary approach combining molecular enzymology with evolutionary genomics offers promising avenues for understanding human-specific aspects of brain development and function.

What are the latest methodological advances in studying HARS interactions with potential inhibitors?

Recent methodological advances have significantly enhanced our ability to study HARS interactions with potential inhibitors, offering new avenues for both basic research and drug discovery:

Biophysical techniques:

• Surface Plasmon Resonance (SPR): Enables real-time, label-free detection of HARS-inhibitor interactions with precise kinetic and thermodynamic characterization.

• Isothermal Titration Calorimetry (ITC): Provides direct measurement of binding energetics between HARS and potential inhibitors, revealing enthalpy and entropy contributions.

• Microscale Thermophoresis (MST): Allows for inhibitor binding studies using minimal protein amounts and working in complex biological matrices.

Structural approaches:

• Cryo-electron microscopy (Cryo-EM): Enables visualization of HARS-inhibitor complexes without crystallization, particularly valuable for studying conformational changes upon inhibitor binding.

• Fragment-based screening: Identifies low molecular weight compounds that bind to specific HARS pockets, which can be further developed into high-affinity inhibitors.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Maps inhibitor-induced changes in HARS protein dynamics and solvent accessibility.

Computational approaches:

• Molecular dynamics simulations: Provide insights into the dynamic behavior of HARS-inhibitor complexes over time.

• Machine learning-based virtual screening: Utilizes AI algorithms to predict potential HARS inhibitors from large compound libraries.

• Quantitative structure-activity relationship (QSAR) models: Establish mathematical relationships between inhibitor structural properties and their activity against HARS.

These advanced methodologies are essential for understanding the structure-activity relationships of HARS inhibitors and developing compounds with improved specificity and potency.

How can HARS be engineered to incorporate non-canonical amino acids into proteins?

Engineering HARS to incorporate non-canonical amino acids (ncAAs) represents an advanced research direction with significant implications for protein engineering and synthetic biology:

Current engineering strategies:

• Active site engineering:

  • Rational modification of the histidine binding pocket to accommodate histidine analogs with altered side chains.

  • Directed evolution approaches selecting for HARS variants that efficiently charge tRNA with specific ncAAs.

  • Computer-aided design combining structural insights with predictive algorithms to guide mutagenesis.

• tRNA engineering in conjunction with HARS modifications:

  • Development of orthogonal HARS-tRNA pairs that function independently from the host's translation machinery.

  • Modification of the tRNA anticodon to reassign specific codons for ncAA incorporation.

  • Engineering the tRNA body to enhance interactions with modified HARS variants.

• Cell-free translation systems:

  • Reconstitution of translation machinery with engineered HARS and tRNAs for in vitro synthesis of proteins containing ncAAs.

  • Optimization of reaction conditions to favor ncAA incorporation over competing natural amino acids.

Applications of engineered HARS systems:

This research direction combines principles from enzyme engineering, synthetic biology, and chemical biology to expand the chemical diversity of proteins beyond the constraints of the canonical 20 amino acids.

What are the most promising future directions in HARS research?

The field of HARS research is evolving rapidly, with several promising directions emerging at the intersection of fundamental biochemistry, clinical medicine, and technological innovation:

• Systems biology integration: Exploring how HARS functions within the broader context of the cellular aminoacylation network and protein synthesis machinery. This includes investigating potential regulatory interactions with other synthetases and translation factors.

• Non-canonical functions: Further characterization of HARS functions beyond aminoacylation, including potential roles in gene regulation, immune signaling, and cell-cell communication that may contribute to human-specific traits.

• Therapeutic targeting: Development of small molecule modulators of HARS activity, either as potential antimicrobials (targeting pathogen-specific features) or as treatments for HARS-related autoimmune conditions and neuropathies.

• Structural dynamics: Application of advanced biophysical techniques to understand the conformational changes and molecular motions critical for HARS function, particularly during tRNA recognition and aminoacylation.

• Evolutionary implications: Further investigation into how changes in HARS structure and function may have contributed to human-specific traits, potentially in connection with Human Accelerated Regions and brain development.

These research directions promise to deepen our understanding of this essential enzyme while opening new avenues for therapeutic intervention in HARS-related disorders.