Biology subjects

Hermanson, J. N.

Publications and source records attributed to Hermanson, J. N..

3 recordsLinked to original sources

bioRxiv2025

Public Cohort Analysis Identifies Thyroglobulin Variants as Hypothyroidism Risk Factors

Hypothyroidism is a prevalent endocrine disorder characterized by insufficient thyroid hormone (T3T4) production. Thyroglobulin (Tg) serves as the prohormone for T3 and T4 production, with many variants of uncertain clinical significance due to genetic diversity in the Tg gene. We leveraged the large-scale All of Us biobank to investigate the disease association of prevalent yet undercharacterized Tg variants. We related variant presence to thyroid-stimulating hormone levels and levothyroxine (LT4) usage as proxies for thyroid function. This identified R152H, Q870H, A993T, P1012L, and P1494L variants linked to increased LT4 usage and decreased thyroid function, while the R320C variant was associated with decreased thyroid function. Molecular characterization in Fisher rat thyroid cells revealed decreased secretion efficiency of R152H, Q870H, and R320C variants. Affinity purification-mass spectrometry demonstrated that secretion-deficient variants showed higher engagement with the protein homeostasis network, indicating protein quality control defects as the pathophysiology mechanism. In contrast, secretion-competent A993T and P1494L variants showed elevated interactions with degradation and antigen-presentation pathways, suggesting an alternative pathophysiology possibly linked to Hashimotos disease, an autoimmune condition with overproduction of autoantibodies that target thyroid proteins. In support, participants carrying the A993T or P1494L variants had elevated anti-TPO antibody levels. We estimate [~]150,000 US individuals currently taking levothyroxine could benefit from precision medicine targeting these variants, with [~]100,000 carrying Q870H. Our findings highlight the power of combining large public biobank data with molecular characterization to understand Tg genotype-to-phenotype relationships. Q870H represents a candidate for molecular therapies to restore secretion, offering precision medicine beyond LT4 replacement therapy. Significance StatementHypothyroidism affects millions of Americans who typically receive levothyroxine hormone replacement therapy, yet some patients continue experiencing symptoms despite treatment. Correlating genomic and health data from the large-cohort All of Us biobank identified specific variants in the thyroglobulin gene, which produces thyroid hormones, that contribute to thyroid dysfunction through distinct biological pathways. Molecular characterization and interactomics revealed that some variants impair hormone secretion due to protein misfolding, while others associate with immune presentation that may link to autoimmune thyroid disease. The Q870H variant emerged as a promising target for precision medicine, potentially benefiting [~]100,000 Americans currently taking levothyroxine. This research demonstrates how combining large-scale genomic data with molecular characterization can identify new therapeutic targets beyond standard hormone replacement therapy.

biochemistry↗

bioRxiv2025

Dissecting Branch-Specific Unfolded Protein Response Activation in Drug-Tolerant BRAF-Mutant Melanoma using Data-Independent Acquisition Mass Spectrometry

Cells rely on the Unfolded Protein Response (UPR) to maintain ER protein homeostasis (proteostasis) when faced with elevated levels of misfolded and aggregated proteins. The UPR is comprised of three main branches--ATF6, IRE1, and PERK--that coordinate the synthesis of proteins involved in folding, trafficking, and degradation of nascent proteins to restore ER function. Dysregulation of the UPR is linked to numerous diseases, including neurodegenerative disorders, cancer, and diabetes. Despite its importance, identifying UPR targets has been challenging due to their heterogeneous induction, which varies by cell type and tissue. Additionally, defining the magnitude and range of UPR-regulated genes is difficult because of intricate temporal regulation, feedback between UPR branches, and extensive cross-talk with other stress-signaling pathways. To comprehensively identify UPR-regulated proteins and determine their branch specificity, we developed a data-independent acquisition (DIA) liquid-chromatography mass spectrometry (LC-MS) pipeline. Our optimized workflow improved identifications of low-abundant UPR proteins and leveraged an automated SP3-based protocol on the Biomek i5 liquid handler for label-free peptide preparation. Using engineered stable cell lines that enable selective pharmacological activation of each UPR branch without triggering global UPR activation, we identified branch-specific UPR proteomic targets. These targets were subsequently applied to investigate proteomic changes in multiple patient-derived BRAF-mutant melanoma cell lines treated with a BRAF inhibitor (PLX4720, i.e., vemurafenib). Our findings revealed differential regulation of the XBP1s branch of the UPR in the BRAF-mutant melanoma cell lines after PLX4720 treatment, likely due to calcium activation, suggesting that the UPR plays a role as a non-genetic mechanism of drug tolerance in melanoma. In conclusion, the validated branch-specific UPR proteomic targets identified in this study provide a robust framework for investigating this pathway across different cell types, drug treatments, and disease conditions in a high-throughput manner.

systems biology↗

bioRxiv2025

Development of an Adaptive, Economical, and Easy-to-Use SP3-TMT Automated Sample Preparation Workflow for Quantitative Proteomics

Liquid handling robots have been developed to automate various steps of the bottom-up proteomics workflow, however, protocols for the generation of isobarically labeled peptides remain limited. Existing methods often require costly specialty devices and are constrained by fixed workflows. To address this, we developed a cost-effective, flexible, automated sample preparation protocol for TMT-labeled peptides using the Biomek i5 liquid handler. Our approach leverages Single-Pot Solid-Phase-Enhanced Sample Preparation (SP3) with paramagnetic beads to streamline protein cleanup and digestion. The protocol also allows for adjustment of trypsin concentration and peptide-to-TMT ratio to increase throughput and reduce costs, respectively. We compared our automated and manual 18-plex TMT-Pro labeling workflows by monitoring select protein markers of the Unfolded Protein Response (UPR) in pharmacologically activatable, engineered cell lines. Overall, the automated protocol demonstrated equivalent performance in peptide and protein identifications, digestion and labeling efficiency, and an enhancement in the dynamic range of TMT quantifications. Compared to the manual method, the Biomek protocol significantly reduces hands-on time and minimizes sample handling errors. The 96-well format additionally allows for the number of TMT reactions to be scaled up quickly without a significant increase in user interaction. Our optimized automated workflow enhances throughput, reproducibility, and cost-effectiveness, making it a valuable tool for high-throughput proteomics studies.

systems biology↗