OLA1 Human

What is OLA1 and what makes it unique among GTPases?

OLA1 (Obg-like ATPase 1) is a highly conserved protein encoded by the OLA1 gene located on chromosome 2 (locus 2q31.1) in humans. What makes OLA1 exceptional is that while it belongs to the TRAFAC class, Obg family, and YchF subfamily of P-loop GTPases, it has evolved altered nucleotide specificity. Unlike most GTPases, OLA1 binds adenosine triphosphate (ATP) with higher affinity than guanosine triphosphate (GTP) and possesses both GTPase and ATPase activities . This dual nucleotide-hydrolyzing capability suggests specialized functions in cellular metabolism and stress responses that distinguish it from other Obg-family proteins.

Methodologically, researchers should note that when studying OLA1's enzymatic activity, assays must be designed to measure both GTP and ATP hydrolysis to fully characterize its function in experimental settings.

How is OLA1 structured and what are its key domains?

OLA1 protein consists of three main structural domains:

• A central guanidine domain

• A flanking coiled-coil ATPase domain

• A C-terminal TGS domain

This structural arrangement is critical for its diverse cellular functions . The central domain is responsible for nucleotide binding, while the other domains mediate protein-protein interactions and regulatory functions.

For structural studies, crystallography and cryo-EM approaches have proven effective for analyzing OLA1's conformation changes during nucleotide binding and hydrolysis.

What is the role of OLA1 in mitochondrial function and bioenergetics?

OLA1 plays a crucial role in maintaining mitochondrial content, structure, and function. Research has demonstrated that:

• OLA1 depletion leads to decreased expression of nuclear-encoded mitochondrial proteins related to mitochondrial structure, protein complexes, and anion channels

• OLA1 knockout results in fewer mitochondria with structural abnormalities, including cystic dilatations and absence of cristae and matrix proteins

• OLA1-depleted cells show decreased maximum respiratory capacity and ATP-linked oxygen consumption, with increased extracellular acid production

Methodologically, researchers investigating OLA1's role in mitochondrial function should employ multiple complementary approaches:

• Oxygen consumption rate (OCR) measurements

• Extracellular acidification rate (ECAR) analysis

• Electron microscopy for structural assessment

• qPCR analysis of mitochondrial genes

How does OLA1 phosphorylation affect its cellular localization and function?

OLA1 phosphorylation at specific residues governs its subcellular localization and enzymatic function:

• Phosphorylation at Ser232/Tyr236 triggers OLA1 translocation from the cytoplasm and mitochondria into the nucleus

• Subsequent phosphorylation at Thr325 effectively changes its biochemical function from ATPase to GTPase activity

• This phosphorylation cascade is regulated by ERK1/2 (extracellular-regulated kinases 1 and 2) and restrained by PP1A (protein phosphatase 1A) when stress abates

For researchers studying OLA1 phosphorylation, site-directed mutagenesis of these phosphorylation sites, combined with subcellular fractionation and immunofluorescence microscopy, provides valuable insights into the regulatory mechanisms of OLA1 function.

What is the relationship between OLA1 and translational regulation, particularly regarding p21?

OLA1 functions as a translational regulator of p21, a cyclin-dependent kinase inhibitor. Research has shown that:

• OLA1-null mouse embryonic fibroblasts (MEFs) show impaired proliferation due to defective cell cycle progression

• These defects are associated with reduced cyclins D1 and E1, attenuated Rb phosphorylation, and increased p21 Cip1/Waf1

• The accumulation of p21 in OLA1-null cells is due to enhanced mRNA translation through an eIF2-dependent mechanism

• Reconstitution of OLA1 expression or treatment with an eIF2α dephosphorylation inhibitor can prevent p21 accumulation

For experimental approaches, polysome profiling to assess p21 mRNA translation efficiency, combined with Western blotting to measure p21 protein levels, provides comprehensive insight into OLA1's translational regulatory function.

What mutations in OLA1 have been identified in cardiovascular diseases?

Researchers have identified several mutations in the OLA1 gene in patients with cardiovascular diseases, particularly heart failure:

The non-synonymous 5144A>G mutation resulting in 254Tyr>Cys in exon 8 is particularly significant as it affects protein function . For this mutation, researchers have developed a cost-effective Tetra-ARMS PCR-based screening test that can differentiate between homozygous (AA and GG) and heterozygous (A/G) genotypes using easily accessible cells such as blood cells .

How does OLA1 expression differ between normal and pathological states?

Expression of OLA1 varies significantly between normal and disease states:

• OLA1 is significantly downregulated in failing human heart tissue compared to non-failing hearts

• In cancer, OLA1 often shows aberrant expression, with levels positively correlating with tumor progression in several malignancies

• Decreased pulmonary OLA1 expression is reported in patients with persistent pulmonary hypertension of the newborn (PPHN)

These differential expression patterns suggest context-dependent roles of OLA1 in different tissues and disease states. For researching OLA1 expression, quantitative RT-PCR, Western blotting, and immunohistochemistry provide complementary information about mRNA and protein levels in tissues of interest.

What are the optimal experimental approaches for studying OLA1 function in human cells?

For comprehensive analysis of OLA1 function in human cells, researchers should consider multiple complementary approaches:

• Genetic manipulation:

  • CRISPR/Cas9-mediated knockout or knockin of wild-type or mutant OLA1

  • siRNA or shRNA-mediated knockdown for transient reduction

  • mRNA-based reconstitution for rescue experiments

• Functional assays:

  • Cell proliferation and cell cycle analysis

  • Mitochondrial function assays (OCR, ECAR, membrane potential)

  • Stress response assays (oxidative, heat, ER stress)

  • Translation efficiency measurements (polysome profiling, SUnSET method)

• Biochemical analyses:

  • Co-immunoprecipitation to identify interaction partners

  • In vitro ATPase/GTPase activity assays

  • Subcellular fractionation to track localization

  • Phosphorylation site mapping using mass spectrometry

Researchers should be aware that different cell types may exhibit varying responses to OLA1 manipulation, as observed with the contrasting effects of OLA1 knockdown in tumor cells versus vascular cells .

How can researchers effectively address the dual enzymatic activity of OLA1?

To effectively study OLA1's dual ATPase and GTPase activities:

• Purification strategy: Use bacterial or mammalian expression systems with appropriate tags (His, GST) that don't interfere with nucleotide binding

• Activity measurement:

  • Thin-layer chromatography to separate ATP/GTP from ADP/GDP

  • Malachite green assay for phosphate release quantification

  • Real-time kinetic measurements using fluorescent nucleotide analogs

• Substrate specificity determination:

  • Competition assays with varying ATP:GTP ratios

  • Structural studies of nucleotide-bound states

  • Mutational analysis of nucleotide-binding sites

• Physiological relevance:

  • Correlation of enzymatic activity with cellular functions

  • Identification of cellular conditions that favor one activity over the other

  • Analysis of the phosphorylation state at Thr325, which switches activity from ATPase to GTPase

What phenotypes are observed in OLA1 knockout mouse models?

OLA1 knockout mouse models display several distinctive phenotypes:

• Growth and development:

  • Stunted growth (35% decrease in body weight by E18.5)

  • Delayed development with immature organs

  • Uniform developmental delay of 0.75-1.0 day starting at E8.5

• Viability:

  • High perinatal lethality (approximately 80% dead within 24h of birth)

  • Death associated with respiratory distress and cyanosis

  • Survivors remain significantly smaller than wild-type mice

• Organ-specific effects:

  • Proportionate restriction in growth across most organs

  • Particularly immature lungs with reduced saccular formations

  • Heart weight showing a trend toward higher weight (not statistically significant)

For researchers planning to use OLA1 knockout models, these severe phenotypes necessitate careful experimental design and may require tissue-specific or inducible knockout strategies for studying adult functions.

How do cell-based OLA1 knockdown models compare with organism-level knockout models?

Cell-based knockdown and organism-level knockout of OLA1 show important differences:

Interestingly, spontaneously immortalized OLA1-null MEFs show normalized growth rates and loss of p53/p21 accumulation, suggesting adaptation to OLA1 deficiency . This highlights the importance of using primary cells or early-passage cells when studying OLA1's physiological functions.

How can researchers address the seemingly contradictory roles of OLA1 in different cell types?

OLA1 exhibits seemingly contradictory functions in different cell types:

• In tumor cells:

  • OLA1 promotes mitochondrial energy metabolism

  • Increases glycolysis by downregulating oxidative phosphorylation enzymes

  • OLA1 knockdown reduces glycolysis and tumor progression

• In vascular cells:

  • OLA1 is protective

  • OLA1 knockdown increases glycolysis and cell death

To address these contradictions, researchers should:

• Use multiple cell types in parallel experiments

• Conduct comprehensive metabolic profiling (glycolysis, OXPHOS, fatty acid oxidation)

• Examine cell type-specific interaction partners

• Investigate post-translational modifications that might differ between cell types

• Consider the role of OLA1 in the context of the tissue microenvironment

A unified experimental approach might reveal that OLA1's biochemical function varies based on cellular context, potentially due to different phosphorylation states or binding partners.

What are the key challenges in translating OLA1 research from animal models to human applications?

Translating OLA1 research from animal models to human applications faces several challenges:

• Phenotypic severity:

  • The severe developmental phenotype of OLA1 knockout mice complicates adult-focused studies

  • Researchers must develop tissue-specific or inducible models for studying post-developmental functions

• Species differences:

  • While OLA1 is highly conserved, regulatory mechanisms may differ between species

  • Human-specific interaction partners or post-translational modifications should be considered

• Disease complexity:

  • OLA1's involvement in multiple cellular processes makes it challenging to target therapeutically

  • Tissue-specific effects require careful consideration when developing interventions

• Technical considerations:

  • High-quality antibodies specific to human OLA1 are essential

  • Methods for modulating OLA1 activity (rather than expression) are needed for potential therapeutic applications

For human studies, researchers should focus on patient-derived cells and samples, correlating OLA1 variants with clinical outcomes across diverse populations.

What ethical considerations should researchers address when conducting OLA1 studies with human subjects?

When conducting OLA1 research involving human subjects, researchers must address several ethical considerations:

• All research involving human subjects must receive prior approval from an Institutional Review Board (IRB)

• IRB review ensures research is conducted ethically, risks to participants are minimized, selection of participants is equitable, and participants are fully informed

• Even pilot studies involving only one human subject require the same scrutiny as full-scale research projects

• When collecting genetic information related to OLA1 variants, researchers must consider potential implications for participants and related family members

For international OLA1 research, researchers should note that human subjects in foreign countries merit the same level of protection as subjects in the United States, though acceptable practices for informed consent and recruitment may vary across locations .

How should researchers approach the collection and storage of human samples for OLA1 genetic analysis?

For proper collection and storage of human samples for OLA1 genetic analysis:

• Obtain appropriate informed consent, clearly explaining:

  • The potential discovery of disease-associated variants

  • How data will be stored and shared

  • Whether results will be returned to participants

• Follow standardized protocols for sample collection:

  • For blood samples (most common for OLA1 analysis), minimize risks of brief pain, bruising, dizziness, and infection

  • Process samples promptly to maintain DNA integrity

• Implement secure data management:

  • Use coding systems to protect participant identity

  • Apply appropriate security measures for genetic data storage

  • Consider whether collection of identifiers like Social Security numbers is necessary

• Plan for long-term storage and potential future use:

  • Include provisions in consent forms for future research

  • Establish clear timelines for sample retention

Researchers should also be aware that though basic facts such as race, ethnic group, and sex are important for understanding group differences in OLA1 variants, these data must be handled responsibly to prevent misuse that could support harmful ideas about groups .