Biology subjects

Nichols, R. J.

Publications and source records attributed to Nichols, R. J..

10 recordsLinked to original sources

bioRxiv2025

A regulatory loop regulating signaling between LRRK2 and PP2A in cellular models of Parkinsons disease

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of late-onset familial and idiopathic Parkinsons disease (PD), known to date. Importantly, recent data from post-mortem tissue as well as biomarker studies suggest that independent of mutations, increased kinase activity of LRRK2 plays an essential role in idiopathic PD pathogenesis. Despite extensive research on LRRK2, its activation mechanism(s) and how the various mutations result in increased kinase activity and neuronal death is still not completely understood. Accumulating evidence points to LRRK2 phospho-regulation, both auto-phosphorylation and phosphorylation by other kinases, as one potential molecular trigger of its activation. LRRK2 activation and localization is regulated by phosphatases such as Protein phosphatase 1 (PP1) and Protein phosphatase 2A (PP2A), however the exact mechanism of this phospho-regulation is not known. Our data reveal that in vitro PP2A dephosphorylates sites within the RocCOR-GTPase domain of LRRK2 and as a result de-stabilizes LRRK2 dimers, with consequent reduction of its kinase activity. Strikingly, our data further highlight that LRRK2 in turn phosphorylates the catalytic subunit of the PP2A holoenzyme PPP2CA at its critical residue T304. Furthermore, LRRK2-mediated phosphorylation of PP2CA T304 alters the methylation of the C-terminus, which is crucial for both holoenzyme formation and catalytic activity. Importantly, expression of WT-PPP2CA protects from LRRK2-G2019S induced neuronal cell death, while PPP2CA-T304 mutants fail to do so, suggesting that impaired PP2A holoenzyme formation might be detrimental for LRRK2-PD.

neuroscience↗

bioRxiv2022

Dissecting the effects of GTPase and kinase domain mutations on LRRK2 endosomal localization and activity

Parkinsons disease-causing LRRK2 mutations lead to varying degrees of Rab GTPase hyperphosphorylation. Puzzlingly, LRRK2 GTPase-inactivating mutations--which do not affect intrinsic kinase activity--lead to higher levels of cellular Rab phosphorylation than kinase-activating mutations. Here, we investigated whether mutation-dependent differences in LRRK2 cellular localization could explain this discrepancy. We discovered that blocking endosomal maturation leads to the rapid formation of mutant LRRK2+ endosomes on which LRRK2 phosphorylates substrate Rabs. LRRK2+ endosomes are maintained through positive feedback, which mutually reinforces membrane localization of LRRK2 and phosphorylated Rab substrates. Furthermore, across a panel of mutants, cells expressing GTPase-inactivating mutants formed strikingly more LRRK2+ endosomes than cells expressing kinase-activating mutants, resulting in higher total cellular levels of phosphorylated Rabs. Our study suggests that an increased probability of LRRK2 GTPase-inactivating mutants to be retained on intracellular membranes over the kinase-activating mutants leads to higher substrate phosphorylation.

systems biology↗

bioRxiv2022

Evolution of Carbon Isotope Fractionation in Cyanobacteria

The history of Earths carbon cycle reflects trends in atmospheric composition convolved with the evolution of photosynthesis. Fortunately, key parts of the carbon cycle have been recorded in the carbon isotope ratios of sedimentary rocks. The dominant model used to interpret this record as a proxy for ancient atmospheric CO2 is based on carbon isotope fractionations of modern photoautotrophs, and longstanding questions remain about how their evolution might have impacted the record. We tested the intersection of environment and evolution by measuring both biomass ({varepsilon}p) and enzymatic ({varepsilon}Rubisco) carbon isotope fractionations of a cyanobacterial strain (Synechococcus elongatus PCC 7942) solely expressing a putative ancestral Form 1B rubisco dating to >>1 Ga. This strain, nicknamed ANC, grows in ambient pCO2 and displays larger {varepsilon}p values than WT, despite having a much smaller {varepsilon}Rubisco (17.23 {+/-} 0.61{per thousand} vs. 25.18 {+/-} 0.31{per thousand} respectively). Measuring both enzymatic and biomass fractionation revealed a surprising result--ANC {varepsilon}p exceeded ANC {varepsilon}Rubisco in all conditions tested, contradicting prevailing models of cyanobacterial carbon isotope fractionation. However, these models were corrected by accounting for cyanobacterial physiology, notably the CO2 concentrating mechanism (CCM). Our model suggested that additional fractionating processes like powered inorganic carbon uptake systems contribute to {varepsilon}p, and this effect is exacerbated in ANC. Understanding the evolution of rubisco and the CCM is therefore critical for interpreting the carbon isotope record. Large fluctuations in that record may reflect the evolving efficiency of carbon fixing metabolisms in addition to changes in atmospheric CO2. Significance StatementEarth scientists rely on chemical fossils like the carbon isotope record to derive ancient atmospheric CO2 concentrations, but interpretation of this record is calibrated using modern organisms. We tested this assumption by measuring the carbon isotope fractionation of a reconstructed ancestral rubisco enzyme (>1 billion years old) in vivo and in vitro. Our results contradicted prevailing models of carbon flow in Cyanobacteria, but our data could be rationalized if light-driven uptake of CO2 is taken into account. Our study showed that the carbon isotope record tracks both the evolution of photosynthesis physiology as well as changes in atmospheric CO2, highlighting the value of considering both evolution and physiology for comparative biological approaches to understanding Earths history.

evolutionary biology↗

bioRxiv2021

The encapsulin from Thermatoga maritima is a flavoprotein with a symmetry matched ferritin-like cargo protein

Bacterial nanocompartments, also known as encapsulins, are an emerging class of protein-based organelles found in bacteria and archaea. Encapsulins are virus-like icosahedral particles comprising a [~]25-50 nm shell surrounding a specific cargo enzyme. Compartmentalization is thought to create a unique chemical environment to facilitate catalysis and isolate toxic intermediates. Many questions regarding nanocompartment structure-function remain unanswered, including how shell symmetry dictates cargo loading and to what extent the shell facilitates enzymatic activity. Here, we explore these questions using the model T. maritima nanocompartment known to encapsulate a redox-active ferritin-like protein. Biochemical analysis revealed the encapsulin shell to possess a flavin binding site located at the interface between capsomere subunits, suggesting the shell may play a direct and active role in the function of the encapsulated cargo. Furthermore, we used cryoEM to show that cargo proteins use a form of symmetry-matching to facilitate encapsulation and define stoichiometry. In the case of the T. maritima encapsulin, the decameric cargo protein with 5-fold symmetry preferentially binds to the pentameric-axis of the icosahedral shell. Taken together, these observations suggest the shell is not simply a passive barrier--it also plays a significant role in the structure and function of the cargo enzyme.

biochemistry↗

bioRxiv2020

The E3 ligase TRIM1 ubiquitinates LRRK2 and controls its localization, degradation, and toxicity

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinsons Disease (PD); however, pathways regulating LRRK2 subcellular localization, function, and turnover are not fully defined. We performed quantitative mass spectrometry-based interactome studies to identify 48 novel LRRK2 interactors, including the microtubule-associated E3 ubiquitin ligase TRIM1 (Tripartite Motif Family 1). TRIM1 recruits LRRK2 to the microtubule cytoskeleton for ubiquitination and proteasomal degradation by binding LRRK2911-920, a nine amino acid segment within a flexible interdomain region (LRRK2853-981), which we designate the "Regulatory Loop" (RL). Phosphorylation of LRRK2 Ser910/Ser935 within LRRK2 RL serves as a molecular switch controlling LRRK2s association with cytoplasmic 14-3-3 versus microtubule-bound TRIM1. Association with TRIM1 modulates LRRK2s interaction with Rab29 and prevents upregulation of LRRK2 kinase activity by Rab29 in an E3-ligase-dependent manner. Finally, TRIM1 rescues neurite outgrowth deficits caused by PD-driving mutant LRRK2 G2019S. Our data suggest that TRIM1 is a critical regulator of LRRK2, controlling its degradation, localization, binding partners, kinase activity, and cytotoxicity.

molecular biology↗

bioRxiv2020

A nanocompartment containing the peroxidase DypB contributes to defense against oxidative stress in M. tuberculosis

Encapsulin nanocompartments are an emerging class of prokaryotic protein-based organelles consisting of an encapsulin protein shell that encloses a protein cargo1. Genes encoding nanocompartments are widespread in bacteria and archaea, and recent works have characterized the biochemical function of several cargo enzymes2. However, the importance of these organelles to host physiology is poorly understood. Here, we report that the human pathogen Mycobacterium tuberculosis (Mtb) produces a nanocompartment that contains the dye-decolorizing peroxidase DypB. We show that this nanocompartment is important for the ability of Mtb to resist oxidative stress in low pH environments, including during infection of host cells and upon treatment with a clinically relevant antibiotic. Our findings are the first to implicate a nanocompartment in bacterial pathogenesis and reveal a new mechanism that Mtb uses to combat oxidative stress.

microbiology↗

bioRxiv2020

Discovery and characterization of a novel family of prokaryotic nanocompartments involved in sulfur metabolism

Prokaryotic nanocompartments, also known as encapsulins, are a recently discovered proteinaceous organelle in prokaryotes that compartmentalize cargo enzymes. While initial studies have begun to elucidate the structure and physiological roles of encapsulins, bioinformatic evidence suggests that a great diversity of encapsulin nanocompartments remains unexplored. Here, we describe a novel encapsulin in the freshwater cyanobacterium Synechococcus elongatus PCC 7942. This nanocompartment is upregulated upon sulfate starvation and encapsulates a cysteine desulfurase enzyme via an N-terminal targeting sequence. Using cryoelectron microscopy, we have determined the structure of the nanocompartment complex to 2.2 [A] resolution. Lastly, biochemical characterization of the complex demonstrated that the activity of the cysteine desulfurase is enhanced upon encapsulation. Taken together, our discovery, structural analysis, and enzymatic characterization of this prokaryotic nanocompartment provide a foundation for future studies seeking to understand the physiological role of this encapsulin in various bacteria.

biochemistry↗

bioRxiv2019

A revised 1.6 Å structure of the GTPase domain of the Parkinson’s disease-associated protein LRRK2 provides insights into mechanisms

Leucine-rich repeat kinase 2 (LRRK2) is a large 286 kDa multi-domain protein whose mutation is a common cause of Parkinsons disease (PD). One of the common sites of familial PD-associated mutations occurs at residue Arg-1441 in the GTPase domain of LRRK2. Previously, we reported that the PD-associated mutation R1441H impairs the catalytic activity of the GTPase domain thereby traps it in a persistently \"on\" state. More recently, we reported that the GTPase domain of LRRK2 exists in a dynamic dimer-monomer equilibrium where GTP binding shifts it to the monomeric conformation while GDP binding shifts it back to the dimeric state. We also reported that all of the PD-associated mutations at Arg-1441, including R1441H, R1441C, and R1441G, impair the nucleotide-dependent dimer-monomer conformational dynamics of the GTPase domain. However, the mechanism of this nucleotide-dependent conformational dynamics and how it is impaired by the mutations at residue Arg-1441 remained unclear. Here, we report a 1.6 [A] crystal structure of the GTPase domain of LRRK2. Our structure has revealed a dynamic switch region that can be differentially regulated by GTP and GDP binding. This nucleotide-dependent regulation is impaired when residue Arg-1441 is substituted with the PD-associated mutations due to the loss of its exquisite interactions consisting of two hydrogen bonds and a {pi}-stacking interaction at the dimer interface.\n\nSignificance StatementMutations in LRRK2 are associated with familial Parkinsons disease, so understanding its mechanism of actions and how they are changed by the disease-associated mutations is important for developing therapeutic strategies. This paper describes an atomic structure of the G-domain of LRRK2 revealing that the conformational dynamics of the switch regions are potentially important for its normal function. It further shows that a disease-associated mutation could lock the G domain in a persistently active-like conformation, thus perturbing its normal function.

biochemistry↗

bioRxiv2018

Matrix linear models for high-throughput chemical genetic screens

We develop a flexible and computationally efficient approach for analysing high throughput chemical genetic screens. In such screens, a library of genetic mutants is phenotyped in a large number of stresses. The goal is to detect interactions between genes and stresses. Typically, this is achieved by grouping the mutants and stresses into categories, and performing modified t-tests for each combination. This approach does not have a natural extension if mutants or stresses have quantitative or non-overlapping annotations (eg. if conditions have doses, or a mutant falls into more than one category simultaneously). We develop a matrix linear model framework that allows us to model relationships between mutants and conditions in a simple, yet flexible multivariate framework. It encodes both categorical and continuous relationships to enhance detection of associations. To handle large datasets, we develop a fast estimation approach that takes advantage of the structure of matrix linear models. We evaluate our methods performance in simulations and in an E. coli chemical genetic screen, comparing it with an existing univariate approach based on modified t-tests. We show that matrix linear models perform slightly better than the univariate approach when mutants and conditions are classified in non-overlapping categories, and substantially better when conditions can be ordered in dosage categories. Our approach is much faster computationally and is scalable to larger datasets. It is an attractive alternative to current methods, and provides a natural framework extensible to larger, and more complex chemical genetic screens. A Julia implementation of matrix linear models and the code used for the analysis in this paper can be found at https://bitbucket.org/jwliang/mlm_packages and https://bitbucket.org/jwliang/mlm_gs_supplement, respectively.

bioinformatics↗

bioRxiv2017

Efficacy of SHP2 phosphatase inhibition in cancers with nucleotide-cycling oncogenic RAS, RAS-GTP dependent oncogenic BRAF and NF1 loss

Oncogenic alterations in the RAS-RAF-MEK-ERK pathway, including mutant forms of KRAS, BRAF, and loss of the tumor suppressor and RAS GTPase-activating protein (GAP) NF1, drive the growth of a wide spectrum of human cancers. While BRAF and MEK inhibitors are effective in many patients with oncogenic BRAF V600E, there are no effective targeted therapies for individuals with cancers driven by other pathway alterations, including oncogenic KRAS, non-V600E BRAF, and NF1 loss. Here, we show that targeting the PTPN11/SHP2 phosphatase with a novel small molecule allosteric inhibitor is effective against cancers bearing nucleotide-cycling oncogenic RAS (e.g. KRAS G12C), RAS-GTP dependent oncogenic BRAF (e.g. class 3 BRAF mutants), or NF1 loss in multiple preclinical models in vitro and in vivo. SHP2 inhibition suppressed the levels of RAS-GTP and phosphorylated ERK in these models and induced growth inhibition. Expression of a constitutively active mutant of the RAS guanine nucleotide exchange factor (GEF) SOS1 rescued cells from the effects of SHP2 inhibition, suggesting that SHP2 blockade decreases oncogenic RAS-RAF-MEK-ERK signaling by disrupting SOS1-mediated RAS-GTP loading. Our findings illuminate a critical function for SHP2 in promoting oncogenic RAS activation and downstream signaling in cancers with nucleotide-cycling oncogenic RAS, RAS-GTP dependent oncogenic BRAF, and NF1 loss. SHP2 inhibition thus represents a rational, biomarker-driven therapeutic strategy to be tested in patients with cancers of diverse origins bearing these oncogenic drivers and for which current treatments are largely ineffective.

cancer biology↗