Biology subjects

Laman, H.

Publications and source records attributed to Laman, H..

8 recordsLinked to original sources

bioRxiv2026

Combinatorial screening of biological degrader modules identifies heterobifunctional molecules that efficiently destroy MYC, leading to rapid tumour cell death in vitro and in vivo

Targeted protein degradation is a potent strategy against intracellular proteins impervious to traditional drugs. We describe a platform reliant on high-throughput cloning and mRNA-based delivery to quickly screen 100s-1000s of modular biological degraders, which we challenge against the high-turnover c-Myc oncoprotein. We uncover critical principles to drive degrader discovery against any target, free from tagging or prior cell line engineering, with direct applications in research and potentially therapy. We demonstrate the feasibility of targeted protein degradation of c-Myc across in vitro cancer models and in vivo xenograft model, uncovering distinct cellular responses between c-Myc degradation and inhibition.

bioengineering↗

bioRxiv2026

Potent substoichiometric inhibition of alpha-synuclein aggregation by de novo oligomer-binding proteins

The intrinsically disordered, non-amyloid-{beta} component domain of -synuclein (Syn) drives aggregation in Parkinson's disease but lacks a stable epitope for structure-based drug design. We used deep learning-based protein design to generate de novo binders capturing this region in a {beta}-strand conformation. Three designs bound Syn with nanomolar affinities, with solution NMR spectroscopy supporting the intended fold. All three suppressed fibril formation at substoichiometric ratios, and fitting of aggregation kinetics resolved the microscopic steps inhibited by each. Notably, one binder preferentially engaged oligomers and inhibited secondary nucleation, both closely linked to toxicity. In cells, all three suppressed seeded Syn assembly, with the oligomer binder being the most effective. Together, these findings establish a set of molecules that target distinct aggregation intermediates and mechanistically reshape Syn assembly.

biophysics↗

bioRxiv2024

Restoration of Fbxo7 expression in dopaminergic neurons restores tyrosine hydroxylase in a mouse model of Parkinson's disease

Mutations in FBXO7 are linked to an atypical parkinsonism. Conditional knock out (KO) of Fbxo7 in dopaminergic neurons in a mouse model caused a neurodegenerative phenotype, including a significant reduction in striatal TH staining at 6 weeks of age and a significant loss of dopaminergic neurons in the SNpc. To test whether re-expression of Fbxo7 could act as a treatment to prevent or restore TH expression in the striatum in this model, we used a rAAV vector to deliver murine Fbxo7 and a mRuby fluorescent marker to dopaminergic neurons. We found that Fbxo7 expression, both before and after the TH loss, restored its expression in the striatum and nucleus accumbens in the mouse. This study therefore highlights that Fbxo7 is important for the integrity and persistence of the dopaminergic nigrostriatal pathway in the mammalian brain, which could be of relevance to Parkinsons disease with therapeutic implications.

neuroscience↗

bioRxiv2023

Development of Constrained Peptide Inhibitors Targeting an Oncogenic E3 Ubiquitin Ligase

SCFSkp2/Cks1 is an E3 ubiquitin ligase, whose substrate specificity is determined by the oncogenic F-box protein Skp2 and the adaptor protein Cks1. A principal target of SCFSkp2/Cks1 is the cyclin-dependent kinase inhibitor p27. Elevated levels of Skp2 and reduced levels of p27 are common in a variety of cancers, and there is consequently a need to develop effective inhibitors of the Skp2-p27 interaction. However, conventional small-molecule approaches are challenging due to the extended bi-molecular interface that spans both Skp2 and Cks1, the lack of suitable binding pockets on this surface, and the intrinsically disordered nature of p27. Here, we develop macrocyclic peptides capable of binding to SCFSkp2/Cks1 with nanomolar affinities, an enhancement of almost two orders of magnitude over the natural p27 peptide. We show that these macrocyclic peptides inhibit p27 ubiquitination in vitro, restore p27 levels in a breast cancer cell line, and reduce cell proliferation.

cancer biology↗

bioRxiv2021

Study of an FBXO7 patient mutation reveals Fbxo7 and PI31 co-regulate proteasomes and mitochondria

Mutations in FBXO7 have been discovered associated with an atypical parkinsonism. We report here a new homozygous missense mutation in a paediatric patient that causes an L250P substitution in the dimerization domain of Fbxo7. This alteration selectively ablates the Fbxo7-PI31 interaction and causes a significant reduction in Fbxo7 and PI31 levels in patient cells. Consistent with their association with proteasomes, L250P patient fibroblasts have reduced proteasome activity and proteasome subunits. We also show PI31 interacts directly with the MiD49/51 fission adaptor proteins, and unexpectedly, PI31 acts as an adaptor enabling SCFFbxo7 ligase to ubiquitinate MiD49. Thus, the L250P mutation changes the function of Fbxo7 by altering its substrate repertoire. Although MiD49/51 expression was reduced in L250P patient cells, there was no effect on the mitochondrial network. However, patient cells had higher levels of ROS and reduced viability under stress. Our study shows that Fbxo7 and PI31 affect each others functions in regulating both proteasomal and mitochondrial function and demonstrate a new function for PI31, as an adaptor for the SCFFbxo7 E3 ubiquitin ligase.

neuroscience↗

bioRxiv2021

Fbxo7 promotes Cdk6 activity to inhibit PFKP and glycolysis in T cells.

Deregulated Fbxo7 expression is associated with many pathologies, including anaemia, male sterility, cancer, and Parkinsons disease, demonstrating its critical role in a variety of cell types. Although Fbxo7 is an F-box protein that recruits substrates for SCF-type E3 ubiquitin ligases, it also promotes the formation of cyclin D/Cdk6/p27 complexes in an E3-ligase independent fashion. We discovered PFKP, the major gatekeeper of glycolysis, in a screen for Fbxo7 substrates. PFKP has been previously shown to be a critical substrate of Cdk6 for the viability of T-ALL cells. We investigated the molecular relationships between Fbxo7, Cdk6 and PFKP, and the functional effect Fbxo7 has on T cell metabolism, viability, and activation. Fbxo7 promotes Cdk6-independent ubiquitination and Cdk6-dependent phosphorylation of PFKP. Importantly Fbxo7-deficient cells have reduced Cdk6 activity, and haematopoietic and lymphocytic cell lines show a significant dependency on Fbxo7. Compared to WT cells, CD4+ T cells with reduced Fbxo7 expression show increased glycolysis, despite lower cell viability and activation levels. Metabolomic studies of activated CD4+ T cells confirm increased glycolytic flux in Fbxo7-deficient cells, as well as altered nucleotide biosynthesis and arginine metabolism. We show Fbxo7 expression is glucose-responsive at the mRNA and protein level, and we propose Fbxo7 inhibits PFKP and glycolysis via its activation of Cdk6.

cell biology↗

bioRxiv2019

Analysis of the FBXO7 promoter reveals overlapping Pax5 and c-Myb binding sites functioning in B cells

Fbxo7 is a key player in the differentiation and function of numerous blood cell types, and in neurons, oligodendrocytes and spermatocytes. In an effort to gain insight into the physiological and pathological settings where Fbxo7 is likely to play a key role, we sought to define the transcription factors which direct FBXO7 expression. Using sequence alignments across 28 species, we defined the human FBXO7 promoter and found that it contains two conserved regions enriched for multiple transcription factor binding sites. Many of these have roles in either neuronal or haematopoietic development. Using various FBXO7 promoter reporters, we found ELF4, Pax5 and c-Myb have functional binding sites that activate transcription. Overlap of Pax5 and c-Myb binding sites suggest that these factors bind cooperatively to transactivate the FBXO7 promoter. Although endogenous Pax5 is bound to the FBXO7 promoter in B cells, c-Myb is also required for FBXO7 expression. Our data suggest the interplay of multiple transcription factors regulate the FBXO7 promoter.

molecular biology↗

bioRxiv2019

A conserved requirement for Fbxo7 during male germ cell cytoplasmic remodelling

Summary statementFbxo7 is the substrate-recognition subunit of an SCF-type ubiquitin E3 ligase complex. It has physiologically important functions in regulating mitophagy, proteasome activity and the cell cycle in multiple cell types, like neurons, lymphocytes and erythrocytes. Here we show that in addition to the previously-known Parkinsonian and haematopoietic phenotypes, Fbxo7-deficient male mice are completely sterile. In these males, despite successful meiosis, nuclear elongation and eviction of histones from chromatin, the developing spermatids are phagocytosed by Sertoli cells during late spermiogenesis, as the cells undergo cytoplasmic remodelling. Surprisingly, despite the loss of all germ cells, there was no evidence of the symplast formation and cell sloughing that is typically associated with spermatid death in other mouse sterility models, suggesting that novel cell death and/or cell disposal mechanisms may be engaged in Fbxo7-deficient males. Mutation of the Drosophila Fbxo7 orthologue, nutcracker (ntc) was previously shown to cause sterility at a similar stage of germ cell development, indicating that the requirement for Fbxo7 is conserved. The ntc phenotype was attributed to proteasome mis-regulation via an interaction with the proteasome regulator, DmPI31. Our data suggest rather that in mice, the requirement for Fbxo7 is either independent of its interaction with PI31, or relates specifically to cytoplasmic proteasome activity during spermiogenesis.

developmental biology↗