Biology subjects

Lin, L. L.

Publications and source records attributed to Lin, L. L..

7 recordsLinked to original sources

bioRxiv2025

Functional integrity of the SEL1L-HRD1 complex is critical for ERAD and organismal viability

The SEL1L-HRD1 complex represents the most evolutionarily conserved branch of endoplasmic reticulum-associated degradation (ERAD), with SEL1L acting as a key cofactor for the E3 ubiquitin ligase HRD1. While the physiological relevance of this complex has been increasingly recognized, whether SEL1L is strictly required for HRD1 function in mammals has remained unclear. Here, using complementary in vivo and in vitro approaches, we define the architecture and physiological significance of the mammalian SEL1L-HRD1 ERAD complex. Our data demonstrate that direct binding between SEL1L and HRD1 is essential for ERAD function and neonatal survival in mice. In three knock-in mouse models harboring targeted mutations at the SEL1L-HRD1 interface, we show that the L709P variant - unlike the benign P699T mutation - results in complete neonatal lethality within 30 hours of birth, a phenotype more severe than that of the partially lethal S658P variant. Mechanistically, the L709P mutation abolishes SEL1L-HRD1 interaction, disrupting substrate engagement and impairing recruitment of the E2 enzyme UBE2J1, leading to the accumulation and aggregation of misfolded proteins in the ER. Notably, these defects can be partially rescued by HRD1 overexpression, echoing findings from yeast. Together, our results provide definitive evidence that the SEL1L-HRD1 interaction is essential for ERAD activity and neonatal viability in mammals, resolving a long-standing question in ERAD biology and identifying a new therapeutic strategy for modulating ERAD activity in humans. SIGNIFICANCE STATEMENTThe endoplasmic reticulum-associated degradation (ERAD) pathway is essential for maintaining cellular proteostasis and organismal health, yet its molecular regulation in mammals remains incompletely defined. The SEL1L-HRD1 complex constitutes the central axis of this conserved pathway, but whether SEL1L is required for HRD1 function in vivo has remained unresolved. Here, using genetic mouse models and biochemical analyses, we demonstrate that the physical interaction between SEL1L and HRD1 is essential for ERAD activity and neonatal viability. These findings resolve a long-standing question in ERAD biology, provide new insights into the structural and functional organization of the mammalian ERAD machinery, and highlight the SEL1L-HRD1 interface as a potential therapeutic target for modulating ERAD activity in diseases.

cell biology↗

bioRxiv2025

A molecular switch in NAC prevents mitochondrial protein mistargeting by SRP

The nascent polypeptide-associated complex (NAC) co-translationally screens all nascent proteins and regulates their access to the signal recognition particle (SRP) to ensure the fidelity of protein targeting to the endoplasmic reticulum (ER). However, the mechanism by which NAC prevents the mistargeting of nascent mitochondrial proteins remains unclear. Here, we identified a molecular switch in NAC that allows its central barrel domain to adopt a stabilized conformation on ribosomes exposing a mitochondrial targeting sequence (MTS). Mutations of the MTS on the nascent chain or in the NAC switch region increases NAC barrel dynamics and reduces its binding to the ribosome. This leads to an impaired ability of NAC to prevent mistargeting by SRP and causes ER stress in human cells. Our work reveals how NAC detects nascent mitochondrial proteins early in translation and prevents their promiscuous access to SRP, elucidating the structural basis that underlies this role and providing novel insights into protein targeting fidelity with broader implications for cellular proteostasis.

biophysics↗

bioRxiv2025

Structural basis and pathological implications of the dimeric OS9-SEL1L-HRD1 ERAD Core Complex

The SEL1L-HRD1 complex represents the most conserved branch of endoplasmic reticulum (ER)-associated degradation (ERAD), a critical pathway that clears misfolded proteins to maintain ER proteostasis. However, the molecular organization and pathogenic mechanisms of mammalian ERAD have remained elusive. Here, we report the first cryo-EM structure of the core mammalian ERAD complex, comprising the ER lectin OS9, SEL1L, and the E3 ubiquitin ligase HRD1. The structure, validated by mutagenesis and crosslinking assays, reveals a dimeric assembly of the core complex in which SEL1L and OS9 form a claw-like configuration in the ER lumen that mediates substrate engagement, while HRD1 dimerizes within the membrane to facilitate substrate translocation. Pathogenic SEL1L mutations at the SEL1L-OS9 (Gly585Asp) and SEL1L-HRD1 (Ser658Pro) interfaces disrupt complex formation and impair ERAD activity. A newly identified disease-associated HRD1 variant (Ala91Asp), located in transmembrane helix 3, impairs HRD1 dimerization and substrate processing, underscoring the functional necessity of this interface and HRD1 dimerization. Finally, the structure also reveals two methionine-rich crevices flanking the HRD1 dimer, suggestive of substrate-conducting channels analogous to those in the ER membrane protein complex (EMC). These findings establish a structural framework for mammalian ERAD and elucidate how mutations destabilizing this machinery contribute to human disease. SUMMARYThe dimeric structure of the human SEL1L-HRD1 ERAD core complex reveals key architectural and functional principles underlying the recognition and processing of misfolded proteins linked to human disease.

molecular biology↗

bioRxiv2025

SEL1L-HRD1 ER-Associated Degradation Facilitates Prohormone Convertase 2 Maturation and Glucagon Production in Islet α Cells

Proteolytic cleavage of proglucagon by prohormone convertase 2 (PC2) is required for islet cells to generate glucagon. However, the regulatory mechanisms underlying this process remain largely unclear. Here, we report that SEL1L-HRD1 endoplasmic reticulum (ER)-associated degradation (ERAD), a highly conserved protein quality control system responsible for clearing misfolded proteins from the ER, plays a key role in glucagon production by regulating turnover of the nascent proform of the PC2 enzyme (proPC2). Using a mouse model with SEL1L deletion in proglucagon-expressing cells, we observed a progressive decline in stimulated glucagon secretion and a reduction in pancreatic glucagon content. Mechanistically, we found that endogenous proPC2 is a substrate of SEL1L-HRD1 ERAD, and that degradation of misfolded proPC2 ensures the maturation of activation-competent proPC2 protein. These findings identify ERAD as a novel regulator of PC2 biology and an essential mechanism for maintaining cell function.

cell biology↗

bioRxiv2024

Purkinje Cell-specific Deficiency in SEL1L-HRD1 Endoplasmic Reticulum-Associated Degradation Causes Progressive Cerebellar Ataxia in Mice

Recent studies have identified multiple genetic variants of SEL1L-HRD1 ER-associated degradation (ERAD) in humans with neurodevelopmental disorders and locomotor dysfunctions, including ataxia. However, the relevance and importance of SEL1L-HRD1 ERAD in the pathogenesis of ataxia remain unexplored. Here we show that SEL1L deficiency in Purkinje cells leads to early-onset progressive cerebellar ataxia with progressive loss of Purkinje cells with age. Mice with Purkinje cell-specific deletion of SEL1L (Sel1LPcp2Cre) exhibit motor dysfunction beginning around 9 weeks of age. Transmission electron microscopy (TEM) analysis reveals dilated ER and fragmented nuclei in Purkinje cells of adult Sel1LPcp2Cre mice, indicative of altered ER homeostasis and cell death. Lastly, loss of Purkinje cells is associated with a secondary neurodegeneration of granular cells, as well as robust activation of astrocytes and proliferation of microglia, in the cerebellum of Sel1LPcp2Cre mice. These data demonstrate the pathophysiological importance of SEL1L-HRD1 ERAD in Purkinje cells in the pathogenesis of cerebellar ataxia. One-sentence summarySEL1L-HRD1 ERAD is indispensable for Purkinje cell function and cerebellar ataxia pathogenesis in mice.

neuroscience↗

bioRxiv2023

SEL1L-HRD1 ER-associated degradation is a new ataxia gene

The SEL1L-HRD1 protein complex represents the most conserved branch of endoplasmic reticulum (ER)-associated degradation (ERAD); however, definitive evidence for the importance of SEL1L in HRD1 ERAD is lacking. Here we report that attenuation of the interaction between SEL1L and HRD1 impairs HRD1 ERAD function and has pathological consequences in mice. Our data show that SEL1L variant p.Ser658Pro (SEL1LS658P) previously identified in Finnish Hound suffering cerebellar ataxia is a recessive hypomorphic mutation, causing partial embryonic lethality, developmental delay, and early-onset cerebellar ataxia in homozygous mice carrying the bi-allelic variant. Mechanistically, SEL1LS658P variant attenuates the SEL1L-HRD1 interaction and causes HRD1 dysfunction by generating electrostatic repulsion between SEL1L F668 and HRD1 Y30 residues. Proteomic screens of SEL1L and HRD1 interactomes revealed that the SEL1L-HRD1 interaction is prerequisite for the formation of a functional HRD1 ERAD complex, as SEL1L recruits not only the lectins OS9 and ERLEC1, but the E2 UBE2J1 and retrotranslocon DERLIN, to HRD1. These data underscore the pathophysiological importance and disease relevance of the SEL1L-HRD1 complex, and identify a key step in organizing the HRD1 ERAD complex.

cell biology↗

bioRxiv2019

Dynamic evolution of cervical cancer mutations during chemoradiation using novel sampling approach

ObjectiveThe aim of this study was to validate a whole exome sequencing approach to longitudinally characterize the tumor mutational profile of cervical cancer patients undergoing chemoradiation (CRT). Experimental DesignCervical cancer tumor specimens from twenty-seven patients undergoing chemoradiation were collected before and throughout CRT and whole exome sequencing (WES) was performed to characterize individual mutations and alterations in unique genes. WES data were analyzed from cervical cancer patients in The Cancer Genome Atlas (TCGA) as a comparison group. ResultsOver 93% of mutated genes detected at baseline were present in TCGA. Tumor purity from collected swabs correlated with MRI tumor volumes during the course of treatment (R2=0.969). CDK4/CDK6/cyclin D1-related gene mutations involved in the ERK1/2, p16INK4, and p53 pathway and G1/S checkpoint most commonly persisted at the end of CRT. ConclusionThis non-invasive swab technique to serially sample tumor during CRT will allow new discoveries of dynamic tumor mutational profile changes during chemoradiation for mucosal tumors. Mutations that survived or increased during the initial weeks of radiation treatment are potential drivers of radiation resistance including the CDL4/CDK6/cyclin D1-related pathway. Statement of Translational RelevanceThere are no established biomarkers to predict chemoradiation (CRT) response for cervical cancer patients. Serial biopsies cannot be performed due to risks of bleeding and fistula. We used a novel non-invasive swab-based biopsy technique to obtain serial samples from a cohort of twenty-seven patients through the course of treatment, and validated this approach to obtain whole exome sequencing data. We analyzed dynamic tumor mutation profiles during CRT. Results from this study show that mutations in CDK4/CDK6/cyclin D1-related genes increased at the end of CRT, suggesting this pathway as a potential driver of radiation resistance.

cancer biology↗