Biology subjects

Adoff, H.

Publications and source records attributed to Adoff, H..

4 recordsLinked to original sources

bioRxiv2024

DNAJC13 localization to endosomes is opposed by its J domain and its disordered C-terminal tail

Endosomes are a central sorting hub for membrane cargos. DNAJC13/RME-8 plays a critical role in endosomal trafficking by regulating the endosomal recycling or degradative pathways. DNAJC13 localizes to endosomes through its N-terminal Plekstrin Homology (PH)-like domain, which directly binds endosomal phosphoinositol-3-phosphate (PI(3)P). However, little is known about how DNAJC13 localization is regulated. Here, we show that two regions within DNAJC13, its J domain and disordered C-terminal tail, act as negative regulators of its PH-like domain. Using a structure-function approach combined with quantitative proteomics, we mapped these control points to a conserved YLT motif in the C-terminal tail as well as the catalytic HPD triad in its J domain. Mutation of either motif enhanced DNAJC13 endosomal localization in cells and increased binding to PI(3)P in vitro. Further, these effects required the N-terminal PH-like domain. We show that, similar to other PI(3)P binding domains, the N-terminal PH-like domain binds PI(3)P weakly in isolation and requires oligomerization for efficient PI(3)P binding and endosomal localization. Together, these results demonstrate that interaction between DNAJC13 and PI(3)P serves as a molecular control point for regulating DNAJC13 localization to endosomes. Significance StatementO_LIDNAJC13 controls endosomal sorting by regulating proteins which mediate the endosomal recycling and degradative subdomains. C_LIO_LIHere we show that subcellular localization of DNAJC13 is regulated through the coordinated action of three of its domains: the PH-like domain which has low affinity for PI(3)P, the J domain, and a YLT motif in its disordered C-terminus. C_LIO_LIThis study defines a novel mechanism by which DNAJC13 function is regulated. C_LI

cell biology↗

bioRxiv2024

Retromer Opposes Opioid-Induced Downregulation of the Mu Opioid Receptor

The mu opioid receptor (MOR) is protected from opioid-induced trafficking to lysosomes and proteolytic downregulation by its ability to access the endosomal recycling pathway through its C-terminal recycling motif, LENL. MOR sorting towards the lysosome results in downregulation of opioid signaling while recycling of MOR to the plasma membrane preserves signaling function. However, the mechanisms by which LENL promotes MOR recycling are unknown, and this sequence does not match any known consensus recycling motif. Here we took a functional genomics approach with a comparative genome-wide screen design to identify genes which control opioid receptor expression and downregulation. We identified 146 hits including all three subunits of the endosomal Retromer complex. We show that the LENL motif in MOR is a novel Retromer recycling motif and that LENL is a necessary, sufficient, and conserved mechanism to give MOR access to the Retromer recycling pathway and protect MOR from agonist-induced downregulation to multiple clinically relevant opioids including fentanyl and methadone.

cell biology↗

bioRxiv2024

A genetically encoded sensor for real-time monitoring of poly-ADP-ribosylation dynamics in-vitro and in cells

ADP-ribosylation, the transfer of ADP-ribose (ADPr) from nicotinamide adenine dinucleotide (NAD+) groups to proteins, is a conserved post-translational modification (PTM) that occurs most prominently in response to DNA damage. ADP-ribosylation is a dynamic PTM regulated by writers (PARPs), erasers (ADPr hydrolases), and readers (ADPR binders). PARP1 is the primary DNA damage-response writer responsible for adding a polymer of ADPR to proteins (PARylation). Real-time monitoring of PARP1-mediated PARylation, especially in live cells, is critical for understanding the spatial and temporal regulation of this unique PTM. Here, we describe a genetically encoded FRET probe (pARS) for semi-quantitative monitoring of PARylation dynamics. pARS feature a PAR-binding WWE domain flanked with turquoise and Venus. With a ratiometric readout and excellent signal-to-noise characteristics, we show that pARS can monitor PARP1-dependent PARylation temporally and spatially in real-time. pARS provided unique insights into PARP1-mediated PARylation kinetics in vitro and high-sensitivity detection of PARylation in live cells, even under mild DNA damage. We also show that pARS can be used to determine the potency of PARP inhibitors in vitro and, for the first time, in live cells in response to DNA damage. The robustness and ease of use of pARS make it an important tool for the PARP field.

biochemistry↗

bioRxiv2022

A genome-wide CRISPR interference screen using an engineered trafficking biosensor reveals a role for RME-8 in opioid receptor regulation

G protein-coupled receptors (GPCRs) are the largest family of membrane-bound signaling molecules. Activity of these receptors is critically regulated by their trafficking through the endo-lysosomal pathway. Identifying the genes involved in GPCR trafficking is challenging due the complexity of sorting operations and low affinity protein-protein interactions. Here we present a chemical biology fluorescence-based technique to interrogate GPCR trafficking. We show that the engineered enzyme APEX2 is a highly sensitive biosensor for GPCR trafficking to the lysosome, and this trafficking can be monitored through APEX-based activation of fluorogenic substrates such as Amplex UltraRed (AUR). We used this approach to perform a genome-wide CRISPR interference screen focused on the delta type opioid receptor (DOR), a GPCR which modulates anxiety, depression, and pain. The screen identified 492 genes including known- and novel-regulators of DOR expression and trafficking. We demonstrate that one of the novel genes, RME-8, localizes to early endosomes and plays a critical role in regulating DOR trafficking to the lysosome. Together, our data demonstrate that GPCR-APEX2/AUR is a flexible and highly sensitive chemical biology platform for genetic interrogation of receptor trafficking.

cell biology↗