Biology subjects

Lange, S. M.

Publications and source records attributed to Lange, S. M..

4 recordsLinked to original sources

bioRxiv2023

Comprehensive approach to study branched ubiquitinchains reveals roles for K48-K63 branches inVCP/p97-related processes

Branched ubiquitin (Ub) chains make up a significant proportion of Ub polymers in human cells and are formed when two or more sites on a single Ub molecule are modified with Ub creating bifurcated architectures. Despite their abundance, we have a poor understanding of the cellular functions of branched Ub signals that stems from a lack of facile tools and methods to study them. Here we develop a comprehensive pipeline to define branched Ub function, using K48-K63-branched chains as a case study. We discover branch-specific binders and, by developing a method that monitors cleavage of linkages within complex polyUb, we discover the VCP/p97-associated ATXN3, and MINDY family deubiquitinases to act as debranching enzymes. By engineering and utilizing a branched K48-K63-Ub chain-specific nanobody, we reveal roles for these chains in VCP/p97-related processes. In summary, we provide a blueprint to investigate branched Ub function that can be readily applied to study other branched chain types. HighlightsO_LIAssembly of defined branched ubiquitin chains enables identification of specific binding proteins C_LIO_LIDevelopment of quantitative DUB assay monitoring cleavage of individual Ub linkages within complex ubiquitin chains identifies debranching enzymes C_LIO_LIEngineering specific, high-affinity nanobody against branched K48-K63 ubiquitin reveals roles in VCP/p97 related processes and DNA damage responses C_LIO_LIGeneral blueprint of new methods and tools for in-depth characterization of branched ubiquitin chains and their underlying biology C_LI

biochemistry↗

bioRxiv2022

Impact of 98 LRRK2 variants linked to Parkinson's Disease on kinase activity and microtubule binding

Mutations enhancing the kinase activity of LRRK2 cause Parkinsons disease (PD) and therapies that reduce LRRK2 kinase activity are being tested in clinical trials. Numerous rare variants of unknown clinical significance have been reported, but how the vast majority impact on LRRK2 function is unknown. Here, we investigate 100 LRRK2 variants linked to PD, including previously described pathogenic mutations. We identify 23 LRRK2 variants that robustly stimulate kinase activity, including variants within the N-terminal non-catalytic regions [ARM (E334K, A419V), ANK(R767H), LRR (R1067Q, R1325Q)], as well as variants predicted to destabilise the ROC:CORB interface [ROC (A1442P, V1447M), CORA (R1628P) CORB (S1761R, L1795F)] and COR:COR dimer interface [CORB (R1728H/L)]. Most activating variants decrease LRRK2 biomarker site phosphorylation (pSer935/pSer955/pSer973), consistent with the notion that the active kinase conformation blocks their phosphorylation. We conclude that the impact of variants on kinase activity is best evaluated by deploying a cellular assay of LRRK2-dependent Rab10 substrate phosphorylation, compared to a biochemical kinase assay, as only a minority of activating variants [CORB (Y1699C, R1728H/L, S1761R) and kinase (G2019S, I2020T, T2031S)], enhance in vitro kinase activity of immunoprecipitated LRRK2. Twelve variants including several that activate LRRK2 and have been linked to PD, suppressed microtubule association in the presence of a Type I kinase inhibitor [ARM(M712V), LRR(R1320S), ROC (A1442P, K1468E, S1508R), CORA(A1589S), CORB (Y1699C, R1728H/L) and WD40(R2143M, S2350I, G2385R)]. Our findings will stimulate work to better understand the mechanisms by which variants impact biology and provide rationale for variant carrier inclusion or exclusion in ongoing and future LRRK2 inhibitor clinical trials.

neuroscience↗

bioRxiv2021

Mechanism of activation and regulation of Deubiquitinase activity in MINDY1 and MINDY2

Of the eight distinct polyubiquitin chains that can be assembled, K48-linked ubiquitin is the most well-understood linkage and modification of proteins with K48 chains targets the modified protein for degradation. By removing ubiquitin from substrates or trimming ubiquitin chains, deubiquitinases (DUBs) can modulate the outcome of ubiquitylation. MINDY1 and MINDY2 are members of the MINDY family of DUBs that have exquisite specificity for cleaving K48-linked polyubiquitin. Being recently discovered DUBs, we have a poor understanding of their catalytic mechanism. By analysing crystal structures of MINDY1 alone and in complex with monoubiquitin or K48-linked ubiquitin chains, we here reveal how substrate interaction relieves autoinhibition and activates the DUB. Further, our analyses reveal a non-canonical catalytic triad composed of Cys-His-Thr and explain how these DUBs sense both ubiquitin chain length and linkage type to trim K48-linked ubiquitin chains. Our findings highlight the multiple layers of regulation modulating DUB activity in MINDY1 and MINDY2. SynopsisO_LIStructure of MINDY1 in complex with K48-linked diUb reveals how K48-linked polyUb is recognized and cleaved C_LIO_LIThe Cys loop mediates autoinhibition of the DUB and substrate binding at the S1 and S1 sites relieves autoinhibition and activates the enzyme for catalysis C_LIO_LIMINDY1 uses a non-canonical catalytic triad composed of Cys-His-Thr C_LIO_LIMINDY1 has five ubiquitin binding sites within its catalytic domain and switches from exo to endo cleavage in a ubiquitin chain length-dependent manner C_LI

biochemistry↗

bioRxiv2020

Characterization of protease activity of Nsp3 from SARS-CoV-2 and its in vitro inhibition by nanobodies

Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease PLpro domain that cleaves not only the viral protein but also polyubiquitin and the ubiquitin-like modifier ISG15 from host cells. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.

biochemistry↗