Biology subjects

Lemberg, M. K.

Publications and source records attributed to Lemberg, M. K..

11 recordsLinked to original sources

bioRxiv2026

Proteolytic Remodeling of Cargo Receptor Networks by RHBDL4 Tunes Secretory Pathway Flux

Cargo receptors are central organizers of the secretory pathway, yet the mechanisms controlling their abundance remain poorly understood. The endoplasmic reticulum (ER)-resident intramembrane protease RHBDL4 promotes substrate turnover via a non-canonical branch of ER-associated degradation and has recently been implicated in regulating secretory pathway components. We previously identified the p24 cargo receptor TMED7 as an RHBDL4 substrate, suggesting that cargo receptor turnover contributes to secretory pathway regulation. Here, quantitative proteomics identify members of the ER-Golgi intermediate compartment (ERGIC) cargo receptor family as endogenous RHBDL4 substrates, demonstrating that RHBDL4 targets multiple cargo receptor families within the early secretory pathway. Accordingly, RHBDL4 modulates multiple ERGIC-dependent transport pathways. In addition, unbiased secretome analysis reveals increased secretion of lysosomal precursor proteins upon RHBDL4 ablation. Mechanistically, we show that this phenotype is mediated, at least in part, by RHBDL4-dependent cleavage of the lysosomal cargo receptor sortilin/SORT1. Together, these findings identify cargo receptors as a major class of RHBDL4 substrates and establish proteolytic remodeling of cargo receptor networks as a mechanism for regulating secretory pathway flux.

cell biology↗

bioRxiv2026

A Golgi-bypass secretion mechanism for proTGFα involving TMED9 and GRASP65

We recently demonstrated that the endoplasmic reticulum export of proTGF is tightly gated, and that proteostasis factors, including the intramembrane protease RHBDL4, can promote its release by an unknown mechanism. While investigating a potential link between RHBDL4-regulated p24/TMED family proteins and proTGF trafficking, we uncovered an RHBDL4-independent pathway in which the cargo receptor TMED9 induces unconventional protein secretion (UcPS) of ectopically expressed proTGF. This pathway selectively requires GRASP65, but not the closely related GRASP55, and depends on components of the autophagic machinery and the ESCRT-associated protein ALIX, consistent with a Golgi-bypass secretion route. Although largely based on overexpression systems, our findings identify a previously unrecognized UcPS mechanism and provide insight into alternative protein trafficking pathways that may be exploited under conditions of proteostasis stress and in pathological contexts such as cancer and inflammation.

cell biology↗

bioRxiv2024

An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

Rhomboid proteases are ubiquitous intramembrane serine proteases that can cleave transmembrane substrates within lipid bilayers. They exhibit many and diverse functions, such as but not limited to, growth factor signaling, immune and inflammatory response, protein quality control, and parasitic invasion. Human rhomboid protease RHBDL4 has been demonstrated to play a critical role in removing misfolded proteins from the Endoplasmic Reticulum and is implicated in severe diseases such as various cancers and Alzheimers disease. Therefore, RHBDL4 is expected to constitute an important therapeutic target for such devastating diseases. Despite its critical role in many biological processes, the enzymatic properties of RHBDL4 remain largely unknown. To enable a comprehensive characterization of RHBDL4s kinetics, catalytic parameters, substrate specificity, and binding modality we expressed and purified recombinant RHBDL4, and employed it in a Forster Resonance Energy Transfer-based cleavage assay. Until now, kinetic studies have been limited mostly to bacterial rhomboid proteases. Our in vitro platform offers a new method for studying RHBDL4s enzymatic function and substrate preferences. Furthermore, we developed and tested potential inhibitors using our assay and successfully identified peptidyl -ketoamide inhibitors of RHBDL4 that are highly effective against recombinant RHBDL4. We utilize ensemble docking and molecular dynamics (MD) simulations to explore the binding modality of substrate-derived peptides bound to RHBDL4. Our analysis focused on key interactions and dynamic movements within RHBDL4s active site that contributed to binding stability, offering valuable insights for optimizing the non-prime side of RHBDL4 ketoamide inhibitors. In summary, our study offers fundamental insights into RHBDL4s catalytic activities and substrate preferences, laying the foundation for downstream applications such as drug inhibitor screenings and structure-function studies, which will enable the identification of lead drug compounds for RHBDL4.

biochemistry↗

bioRxiv2021

Cleavage of PGAM5 by the intramembrane protease PARL is governed by transmembrane helix dynamics and oligomeric state

The intramembrane protease PARL is a crucial mitochondrial safeguard by cleaving the mitophagy regulators PINK1 and PGAM5. PGAM5 substrate determinates have not been rigorously investigated and it is unclear how uncoupling the mitochondrial membrane potential regulates its processing inversely to PINK1. Here we show that in PGAM5 several hydrophilic residues distant from the cleavage site serve as key determinant for PARL- catalyzed cleavage. NMR analysis indicates that a short N-terminal amphipathic helix, followed by a kink and a C-terminal helix harboring the scissile peptide bond, is key for a productive interaction with PARL. In difference to PINK1, PGAM5 is stably inserted into the inner mitochondrial membrane until uncoupling the membrane potential triggers its disassembly into monomers that are vulnerable to PARL-catalyzed processing. We suggest a model in which PGAM5 is a slowly processed substrate with PARL-catalyzed cleavage that is influenced by multiple hierarchical substrate features including a membrane-potential- dependent oligomeric switch.

cell biology↗

bioRxiv2021

Intramembrane protease SPP defines a cholesterol-regulated switch of the mevalonate pathway

Intramembrane proteolysis regulates important processes such as signaling and transcriptional and posttranslational abundance control of proteins with key functions in metabolic pathways. This includes transcriptional control of mevalonate pathway genes, thereby ensuring balanced biosynthesis of cholesterol and other isoprenoids. Our work shows that, at high cholesterol levels, signal peptide peptidase (SPP) cleaves squalene synthase (SQS), an enzyme that defines the branching point for allocation of isoprenoids to the sterol and non-sterol arms of the mevalonate pathway. This intramembrane cleavage releases SQS from the membrane and targets it for proteasomal degradation. Regulation of this mechanism is achieved by the E3 ubiquitin ligase TRC8 that, in addition to ubiquitinating SQS in response to cholesterol levels, acts as an allosteric activator of SPP-catalyzed intramembrane cleavage of SQS. Cellular cholesterol levels increase in the absence of SPP activity. Hence, SPP-TRC8 mediated abundance control of SQS acts as a regulation step within the mevalonate pathway.

cell biology↗

bioRxiv2020

Insights into the catalytic properties of the mitochondrial rhomboid protease PARL

The rhomboid protease PARL is a critical regulator of mitochondrial homeostasis through its cleavage of substrates such as PINK1, PGAM5, and Smac, which have crucial roles in mitochondrial quality control and apoptosis. To gain insight into the catalytic properties of the PARL protease, we expressed human PARL in yeast and used FRET-based kinetic assays to measure proteolytic activity in vitro. We show PARL activity in detergent is enhanced by cardiolipin. Significantly higher turnover rates are observed for PARL reconstituted in proteoliposomes, with Smac being cleaved most rapidly at a rate of 1 min-1. PGAM5 is cleaved with the highest efficiency compared to PINK1 and Smac. In proteoliposomes, a truncated {beta}-cleavage form of PARL is more active than the full-length enzyme for hydrolysis of PINK1, PGAM5 and Smac. Multiplex substrate profiling reveals a substrate preference for PARL with a bulky side chain Phe in P1, which is distinct from small side chain residues typically found with bacterial rhomboid proteases. This study using recombinant PARL provides fundamental insights into its catalytic activity and substrate preferences.

biochemistry↗

bioRxiv2019

Intramembrane protease RHBDL4 induces ER-associated degradation of the oligosaccharyltransferase complex

The Endoplasmic Reticulum (ER)-resident intramembrane rhomboid protease RHBDL4 generates metastable protein fragments and together with the ER-associated degradation (ERAD) machinery provides a clearance mechanism for aberrant and surplus proteins. However, the endogenous substrate spectrum and with that the role of RHBDL4 in physiological ERAD is mainly unknown. Here, we use a substrate trapping approach in combination with quantitative proteomics to identify physiological RHBDL4 substrates. This revealed oligosacharyltransferase (OST) complex subunits such as the catalytic active subunit STT3A as substrates for the RHBDL4-dependent ERAD pathway. RHBDL4-catalyzed cleavage inactivates OST subunits by triggering dislocation into the cytoplasm and subsequent proteasomal degradation. Thereby, RHBDL4 controls the abundance and activity of OST, suggesting a novel link between the ERAD machinery and glycosylation tuning.

cell biology↗

bioRxiv2019

Intramembrane protease RHBDL4 interacts with erlin complex to target unstable soluble proteins for degradation

Protein degradation is fundamentally important to ensure cell homeostasis. In the endoplasmic reticulum (ER), the ER-associated degradation (ERAD) pathway targets incorrectly folded and unassembled proteins into the cytoplasm for turnover by the proteasome. In contrast, lysosomal degradation serves as a failsafe mechanism for removing proteins that resist ERAD by forming aggregates. Previously, we showed that the ER- resident rhomboid protease RHBDL4, together with p97, mediates membrane protein degradation. However, whether RHBDL4 acts in concert with additional ERAD components is unclear, and its full substrate spectrum remains to be defined. Here, we show that besides membrane proteins, RHBDL4 cleaves aggregation-prone luminal ERAD substrates. Because RHBDL4 with mutations in the rhomboid domain leads to stabilization of substrates at the cytoplasmic side, we hypothesize that analogue to the homologue ERAD factor derlin, RHBDL4 is directly involved in substrate retrotranslocation. RHBDL4s interaction with the erlin ERAD complex and reciprocal interaction of rhomboid substrates with erlins suggest that RHBDL4 and erlins form a complex that clips substrates and thereby rescues aggregation-prone peptides in the ER lumen from terminal aggregation.

cell biology↗

bioRxiv2018

CRISPR/Cas12a-assisted PCR tagging of mammalian genes

Here we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g. GFP), a Cas12a CRISPR RNA for cleavage of the target locus and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artefacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein tagged genes

cell biology↗

bioRxiv2018

Metastable XBP1u transmembrane domain mediates insertion into the ER membrane and intramembrane proteolysis by the signal peptide peptidase

Unspliced XBP1 mRNA encodes XBP1u, the transcriptionally inert variant of the unfolded protein response (UPR) transcription factor XBP1s. XBP1u targets its mRNA-ribosome-nascent-chain-complex to the endoplasmic reticulum (ER) to facilitate UPR activation and prevents overactivation. Yet, its membrane association is controversial. Here, we use cell-free translocation and cellular assays to define a moderately hydrophobic stretch in XBP1u that is sufficient to mediate insertion into the ER membrane. Mutagenesis of this transmembrane (TM) region reveals residues that facilitate XBP1u turnover by an ER-associated degradation route that is dependent on signal peptide peptidase (SPP). Furthermore, the impact of these mutations on TM helix dynamics was assessed by residue-specific amide exchange kinetics, evaluated by a semi-automated algorithm. Based on our results, we suggest that SPP-catalyzed intramembrane proteolysis of TM helices is not only determined by their conformational flexibility, but also by side chain interactions near the scissile peptide bond with the enzymes active site.

cell biology↗

bioRxiv2017

A genome-wide resource for high-throughput genomic tagging of yeast ORFs

Here we describe a C-SWAT library for high-throughput tagging of Saccharomyces cerevisiae ORFs. It consists of 5661 strains with an acceptor module inserted after each ORF, which can be efficiently replaced with tags or regulatory elements. We validate the library with targeted sequencing and demonstrate its use by tagging the yeast proteome with bright fluorescent proteins, determining how sequences downstream of ORFs influence protein expression and localizing previously undetected proteins.

genomics↗