Biology subjects

Reitsma, J. M.

Publications and source records attributed to Reitsma, J. M..

4 recordsLinked to original sources

bioRxiv2024

An in-cell approach to evaluate E3 ligases for use in targeted protein degradation.

One of the major challenges in evaluating the suitability of potential [~]700 E3 ligases for target protein degradation (TPD) is the lack of binders specific to each E3 ligase. Here we apply genetic code expansion (GCE) to encode a tetrazine-containing non-canonical amino acid (Tet-ncAA) site-specifically into the E3 ligase, which can be conjugated with strained trans-cyclooctene (sTCO) tethered to a neo-substrate protein binder by click chemistry within living cells. The resulting E3 ligase minimally modified and functionalized in an E3-ligand free (ELF) manner, can be evaluated for TPD of the neo-substrate. We demonstrate that CRBN encoded with clickable Tet-ncAA, either in the known immunomodulatory drug (IMiD)-binding pocket or across surface, can be covalently tethered to sTCO-linker-JQ1 and recruit BRD2/4 for CRBN mediated degradation, indicating the high plasticity of CRBN for TPD. The degradation efficiency is dependent on location of the Tet-ncAA encoding on CRBN as well as the length of the linker, showing the capability of this approach to map the surface of E3 ligase for identifying optimal TPD pockets. This ELF-degrader approach has the advantages of not only maintaining the native state of E3 ligase, but also allowing the interrogation of E3 ligases and target protein partners under intracellular conditions and can be applied to any known E3 ligase.

cell biology↗

bioRxiv2024

Harnessing the FBXW7 somatic mutant R465C for targeted protein degradation

Targeted protein degradation (TPD) is a pharmacological strategy that eliminates specific proteins from cells by harnessing cellular proteolytic degradation machinery. In proteasome-dependent TPD, expanding the repertoire of E3 ligases compatible with this approach could enhance the applicability of this strategy across various biological contexts. In this study, we discovered that a somatic mutant of FBXW7, R465C, can be exploited by heterobifunctional compounds for targeted protein degradation. This work demonstrates the potential of utilizing mutant E3 ligases that occur exclusively in diseased cells for TPD applications.

biochemistry↗

bioRxiv2023

Human Cytomegalovirus UL138 Interaction with USP1 Activates STAT1 in infection

Innate immune responses are crucial for limiting virus infection. However, viruses often hijack our best defenses for viral objectives. Human Cytomegalovirus (HCMV) is a beta herpesvirus which establishes a life-long latent infection. Defining the virus-host interactions controlling latency and reactivation is vital to the control of viral disease risk posed by virus reactivation. We defined an interaction between UL138, a pro-latency HCMV gene, and the host deubiquintase complex, UAF1-USP1. UAF1 is a scaffold protein pivotal for the activity of ubiquitin specific peptidases (USP), including USP1. UAF1-USP1 sustains an innate immune response through the phosphorylation and activation of signal transducer and activator of transcription-1 (pSTAT1), as well as regulates the DNA damage response. After the onset of viral DNA synthesis, pSTAT1 levels are elevated and this depends upon UL138 and USP1. pSTAT1 localizes to viral centers of replication, binds to the viral genome, and influences UL138 expression. Inhibition of USP1 results in a failure to establish latency, marked by increased viral genome replication and production of viral progeny. Inhibition of Jak-STAT signaling also results in increased viral genome synthesis in hematopoietic cells, consistent with a role for USP1-mediated regulation of STAT1 signaling in the establishment of latency. These findings demonstrate the importance of the UL138-UAF1-USP1 virus-host interaction in regulating HCMV latency establishment through the control of innate immune signaling. It will be important going forward to distinguish roles of UAF1-USP1 in regulating pSTAT1 relative to its role in the DNA damage response in HCMV infection. ImportanceHuman cytomegalovirus (HCMV) is one of nine herpesviruses that infect humans. Following a primary infection, HCMV establishes a life-long latent infection that is marked by sporadic, and likely frequent reactivation events. While these reactivation events are asymptomatic in the immune competent host, they pose important disease risks for the immune compromised, including solid organ or stem cell transplant recipients. Its complex interactions with host biology and deep coding capacity make it an excellent model for defining mechanisms important for viral latency and reactivation. Here we define an interaction with host proteins that commandeer typically antiviral innate immune signaling for the establishment of latency.

microbiology↗

bioRxiv2019

Single subunit degradation of WIZ, a lenalidomide- and pomalidomide dependent substrate of E3 ubiquitin ligase CRL4CRBN

Immunomodulators (IMiDs) are an effective class of drugs used to treat blood cancers. IMiDs are believed to work by recruiting protein targets containing a {beta}-hairpin motif for ubiquitination by E3 ubiquitin ligase complexes composed of cereblon (CRBN), Cullin-4a (CUL4a), DNA Damage Binding protein-1 (DDB1), and Ring Box-1 (RBX1). The ubiquitinated protein is subsequently degraded by the proteasome. By characterizing the repertoire of proteins that show an increased physical association with CRBN after IMiD treatment, we identified a novel IMiD substrate, Widely Interspaced Zinc Finger Motifs (WIZ). WIZ contains a C2H2 zinc finger domain, like several other substrates that were previously characterized. We demonstrate that IMiDs stabilize physical association of WIZ with CRBN, deplete WIZ steady state protein levels in a way that is dependent on E3 ligase activity, and enhance the rate of its degradation. Notably, proteins that assemble with WIZ are co-recruited to CRBN by IMiDs but are not degraded, illustrating the potential of targeted protein degradation to eliminate individual subunits of a protein complex. These findings suggest that systematic characterization of the full repertoire of proteins that are targeted for degradation by IMiD compounds will be required to better understand their biological effects.\n\nSynopsisProteolysis Targeting Chimeras (PROTACs) can be used to precisely target a subunit of a transcriptional complex for degradation in subpopulations of cells.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC=\"FIGDIR/small/595389_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (18K):\norg.highwire.dtl.DTLVardef@186fed8org.highwire.dtl.DTLVardef@1c522f9org.highwire.dtl.DTLVardef@17b19baorg.highwire.dtl.DTLVardef@d626ff_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗