Biology subjects

Paulsen, C. E.

Publications and source records attributed to Paulsen, C. E..

3 recordsLinked to original sources

bioRxiv2026

5'Untranslated regions provide a versatile toolkit for tunable exogenous protein expression

Transient transfection is widely used for protein expression in heterologous systems, yet uncontrolled overexpression frequently introduces artifacts that confound functional analyses. Although stable cell lines can mitigate these issues, generating lines for multiple constructs or variants is often impractical. Common alternatives, such as DNA titration, altered transfection conditions, or promoter swapping, provide only coarse and inconsistent control of protein abundance. Here, we establish a panel of ten human 5 untranslated regions (5UTRs) as a modular strategy to tune protein expression during transient transfection. Across three soluble proteins and three membrane proteins, these 5UTRs produce a reproducible dynamic range of expression, including fine-grained control of eYFP and the large sensory ion channel TRPA1. Notably, one 5UTR consistently suppresses expression across all proteins tested and alleviates overexpression-associated artifacts, improving functional analysis of a hyperactive channel variant, substantially reducing background in proximity biotinylation assays, and enhancing the specificity of a stress granule marker. In contrast, most 5UTRs enhance expression of the TRPV1 and TRPM8 sensory receptors, improving protein yield in heterologous systems. Together, this work identifies 5UTRs as a compact, versatile, and broadly applicable tool to fine-tune protein abundance, enabling more physiologically relevant and assay-optimized expression in transient transfection experiments. Significance StatementO_LIProtein overexpression remains a pervasive limitation in transient transfection experiments, as commonly used strategies provide coarse, labor-intensive, and often unpredictable control over protein abundance, frequently leading to artifacts or loss of physiological relevance. C_LIO_LIA compact, modular 5UTR toolkit enables fine-grained and scalable control of protein abundance in transient transfection, providing a reproducible dynamic range across soluble and membrane proteins outperforming promoter swapping for continuous tuning of expression levels. C_LIO_LITunable expression improves experimental fidelity across diverse applications, allowing investigators to match protein abundance to specific assay needs, reducing overexpression artifacts, preserving physiological readouts, and enhancing yield when high expression is required. C_LI

biochemistry↗

bioRxiv2024

Calmodulin binding is required for calcium mediated TRPA1 desensitization

Calcium (Ca2+) ions affect nearly all aspects of biology. Excessive Ca2+ entry is cytotoxic and Ca2+- mobilizing receptors have evolved diverse mechanisms for tight regulation that often include Calmodulin (CaM). TRPA1, an essential Ca2+-permeable ion channel involved in pain signaling and inflammation, exhibits complex Ca2+ regulation with initial channel potentiation followed by rapid desensitization. The molecular mechanisms of TRPA1 Ca2+ regulation and whether CaM plays a role remain elusive. We find that TRPA1 binds CaM best at basal Ca2+ concentration, that they co-localize in resting cells, and that CaM suppresses TRPA1 activity. Combining biochemical, biophysical, modeling, NMR spectroscopy, and functional approaches, we identify an evolutionarily conserved, high-affinity CaM binding element in the distal TRPA1 C-terminus (DCTCaMBE). Genetic or biochemical perturbation of Ca2+/CaM binding to the TRPA1 DCTCaMBE yields hyperactive channels that exhibit drastic slowing of desensitization with no effect on potentiation. Ca2+/CaM TRPA1 regulation does not require the N-lobe, raising the possibility that CaM is not the Ca2+ sensor, per se. Higher extracellular Ca2+ can partially rescue slowed desensitization suggesting Ca2+/CaM binding to the TRPA1 DCTCaMBE primes an intrinsic TRPA1 Ca2+ binding site that, upon binding Ca2+, triggers rapid desensitization. Collectively, our results identify a critical regulatory element in an unstructured TRPA1 region highlighting the importance of these domains, they reveal Ca2+/CaM is an essential TRPA1 auxiliary subunit required for rapid desensitization that establishes proper channel function with implications for all future TRPA1 work, and they uncover a mechanism for receptor regulation by Ca2+/CaM that expands the scope of CaM biology.

biochemistry↗

bioRxiv2022

Molecular Mechanism of Hyperactivation Conferred by a Truncated TRPA1 Disease Mutant Suggests New Gating Insights

The wasabi receptor, TRPA1, is a non-selective homotetrameric cation channel expressed in primary sensory neurons of the pain pathway, where it is activated by diverse chemical irritants. A direct role for TRPA1 in human health has been highlighted by the discovery of genetic variants associated with severe pain disorders. One such TRPA1 mutant was identified in a father-son pair with cramp fasciculation syndrome (CFS) and neuronal hyperexcitability-hypersensitivity symptoms that may be caused by aberrant channel activity, though the mechanism of action for this mutant is unknown. Here, we show the CFS-associated R919* TRPA1 mutant is functionally inactive when expressed alone in heterologous cells, which is not surprising since it lacks the 201 C-terminal amino acids that house critical channel gating machinery including the pore-lining transmembrane helix. Interestingly, the R919* mutant confers enhanced agonist sensitivity when co-expressed with wild type (WT) TRPA1. This channel hyperactivation mechanism is conserved in distant TRPA1 species orthologues and can be recapitulated in the capsaicin receptor, TRPV1. Using a combination of ratiometric calcium imaging, immunostaining, surface biotinylation, pulldown assays, fluorescence size exclusion chromatography, and proximity biotinylation assays, we show that the R919* mutant co-assembles with WT subunits into heteromeric channels. Within these heteromers, we postulate that R919* TRPA1 subunits contribute to hyperactivation by lowering energetic barriers to channel activation contributed by the missing regions. Additionally, we show heteromer activation can originate from the R919* TRPA1 subunits, which suggests an unexpected role for the ankyrin repeat and coiled coil domains in concerted channel gating. Our results demonstrate the R919* TRPA1 mutant confers gain-of-function thereby expanding the physiological impact of nonsense mutations, reveals a novel and genetically tractable mechanism for selective channel sensitization that may be broadly applicable to other receptors, and uncovers new gating insights that may explain the molecular mechanism of temperature sensing by some TRPA1 orthologues.

biochemistry↗