Single Molecule Mass Photometry Reveals Dynamic Oligomerization of Plant and Human Peroxiredoxins for Functional Conservation and Diversification

AbstractSingle molecule mass photometry was used to study the dynamic equilibria of the ubiquitous and highly abundant 2-Cysteine peroxiredoxins (2-CysPRX). 2-CysPRXs adopt distinct functions in all cells dependent on their oligomeric conformation ranging from dimers to decamers and high molecular weight aggregates (HMW). The oligomeric state depends on the redox state of their catalytic cysteinyl residues. To which degree they interconvert, how the interconversion is regulated, and how the oligomerisation propensity is organism specific remains, however, poorly understood. The dynamics differs between wild-type and single point mutants affecting the oligomerization interfaces, with concomitant changes to function. Titrating concentration and redox state of Arabidopsis thaliana and human 2-CysPRXs revealed features conserved among all 2-CysPRX and clear differences concerning oligomer transitions, the occurrence of transition states and the formation of HMW which are associated with chaperone activity or storage. The results indicate functional differentiation of human 2-CysPRXs. Our results point to a diversified functionality of oligomerization for 2-CysPRXs and illustrate the power of mass photometry to non-invasively quantify oligomer distributions in a redox environment. This knowledge is important to fully address and model PRX function in cell redox signaling e.g., in photosynthesis, cardiovascular and neurological diseases or carcinogenesis.

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Genetic perturbation enhances functional heterogeneity in alkaline phosphatase

10.1101/2021.11.25.470051 ◽

2021 ◽

Author(s):

Morito Sakuma ◽

Shingo Honda ◽

Hiroshi Ueno ◽

Kentaro Miyazaki ◽

Nobuhiko Tokuriki ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Catalytic Activity ◽

Single Molecule ◽

Single Point ◽

Point Mutations ◽

Clear Correlation ◽

Wild Type ◽

Functional Heterogeneity ◽

Genetic Perturbations ◽

In The Wild

Enzymes inherently exhibit molecule-to-molecule heterogeneity in catalytic activity or function, which underlies the acquisition of new functions in evolutionary processes. However, correlations between the functional heterogeneity of an enzyme and its multi-functionality or promiscuity remain elusive. In addition, the modulation of functional heterogeneity upon genetic perturbation is currently unexplored. Here, we quantitatively analyzed functional heterogeneity in the wild-type and 69 single-point mutants of Escherichia coli alkaline phosphatase (AP) by employing single-molecule assay with a femtoliter reactor array device. Most mutant enzymes exhibited higher functional heterogeneity than the wild-type enzyme, irrespective of catalytic activity. These results indicated that the wild-type AP minimizes functional heterogeneity, and single-point mutations can significantly expand the span of functional heterogeneity in AP. Moreover, we identified a clear correlation between functional heterogeneity and promiscuous activities. These findings suggest that enzymes can acquire greater functional heterogeneity following marginal genetic perturbations that concomitantly promote catalytic promiscuity.

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Single-molecule imaging reveals the oligomeric state of functional TNFα-induced plasma membrane TNFR1 clusters in cells

Science Signaling ◽

10.1126/scisignal.aax5647 ◽

2020 ◽

Vol 13 (614) ◽

pp. eaax5647 ◽

Cited By ~ 8

Author(s):

Christos Karathanasis ◽

Juliane Medler ◽

Franziska Fricke ◽

Sonja Smith ◽

Sebastian Malkusch ◽

...

Keyword(s):

Ligand Binding ◽

Cancer Progression ◽

Single Molecule ◽

Tumor Necrosis Factor Receptor ◽

Wild Type ◽

Oligomeric State ◽

Superresolution Microscopy ◽

Ligand Free ◽

Light Chain Enhancer ◽

Necrosis Factor

Ligand-induced tumor necrosis factor receptor 1 (TNFR1) activation controls nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling, cell proliferation, programmed cell death, and survival and is crucially involved in inflammation, autoimmune disorders, and cancer progression. Despite the relevance of TNFR1 clustering for signaling, oligomerization of ligand-free and ligand-activated TNFR1 remains controversial. At present, models range from ligand-independent receptor predimerization to ligand-induced oligomerization. Here, we used quantitative, single-molecule superresolution microscopy to study TNFR1 assembly directly in native cellular settings and at physiological cell surface abundance. In the absence of its ligand TNFα, TNFR1 assembled into monomeric and dimeric receptor units. Upon binding of TNFα, TNFR1 clustered predominantly not only into trimers but also into higher-order oligomers. A functional mutation in the preligand assembly domain of TNFR1 resulted in only monomeric TNFR1, which exhibited impaired ligand binding. In contrast, a form of TNFR1 with a mutation in the ligand-binding CRD2 subdomain retained the monomer-to-dimer ratio of the unliganded wild-type TNFR1 but exhibited no ligand binding. These results underscore the importance of ligand-independent TNFR1 dimerization in NF-κB signaling.

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Emerin oligomerization and nucleoskeletal coupling at the nuclear envelope regulate nuclear mechanics against stress

10.1101/2021.02.12.429834 ◽

2021 ◽

Author(s):

Anthony Fernandez ◽

Markville Bautista ◽

Fabien Pinaud

Keyword(s):

Nuclear Envelope ◽

Mechanical Stress ◽

Single Molecule ◽

Structural Integrity ◽

Super Resolution ◽

Integral Membrane Protein ◽

Nuclear Shape ◽

Wild Type ◽

Oligomeric State ◽

Nuclear Mechanics

ABSTRACTEmerin is an integral membrane protein of the nuclear envelope that binds to various nucleoskeletal partners and participates in the maintenance of nuclear shape. When mutated or absent, emerin causes X-linked Emery-Dreifuss muscular dystrophy (EDMD). To define how emerin takes parts in molecular scaffolding at the nuclear envelope and helps protect the nucleus against mechanical stress, we established the nanoscale spatial organizations of wild-type emerin and EDMD-inducing emerin mutants in human cells using single molecule tracking and super-resolution microscopy. We show that emerin is distributed as monomers and oligomeric clusters at the inner nuclear membrane and that its interactions with lamin A/C, nuclear actin and BAF differentially modulate its mobility and its ability to oligomerize. In nuclei under stress, the mechanotransducing functions of emerin are coupled to its oligomeric state and the oligomerization of wild-type emerin or that of emerin mutants determine normal or aberrant nuclear deformations against increasing forces. These findings place emerin at the center of the molecular processes that regulate nuclear shape remodeling and maintain the structural integrity of the nuclear envelope in response to mechanical stress.

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Targeting the DNA replication stress phenotype of KRAS mutant cancer cells

Scientific Reports ◽

10.1038/s41598-021-83142-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tara Al Zubaidi ◽

O. H. Fiete Gehrisch ◽

Marie-Michelle Genois ◽

Qi Liu ◽

Shan Lu ◽

...

Keyword(s):

Dna Replication ◽

Single Molecule ◽

Replication Stress ◽

Proton Radiation ◽

Wild Type ◽

Cellular Effects ◽

Proton Treatment ◽

Dna Replication Stress ◽

Fork Stalling ◽

Mutant Cells

AbstractMutant KRAS is a common tumor driver and frequently confers resistance to anti-cancer treatments such as radiation. DNA replication stress in these tumors may constitute a therapeutic liability but is poorly understood. Here, using single-molecule DNA fiber analysis, we first characterized baseline replication stress in a panel of unperturbed isogenic and non-isogenic cancer cell lines. Correlating with the observed enhanced replication stress we found increased levels of cytosolic double-stranded DNA in KRAS mutant compared to wild-type cells. Yet, despite this phenotype replication stress-inducing agents failed to selectively impact KRAS mutant cells, which were protected by CHK1. Similarly, most exogenous stressors studied did not differentially augment cytosolic DNA accumulation in KRAS mutant compared to wild-type cells. However, we found that proton radiation was able to slow fork progression and preferentially induce fork stalling in KRAS mutant cells. Proton treatment also partly reversed the radioresistance associated with mutant KRAS. The cellular effects of protons in the presence of KRAS mutation clearly contrasted that of other drugs affecting replication, highlighting the unique nature of the underlying DNA damage caused by protons. Taken together, our findings provide insight into the replication stress response associated with mutated KRAS, which may ultimately yield novel therapeutic opportunities.

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Breaking Down the Supramolecular Ensemble: Single-Molecule Studies of the Concentration Dependence of Main-Chain Supramolecular Polymer Molecular Weight Distributions on Surfaces

Australian Journal of Chemistry ◽

10.1071/ch09615 ◽

2010 ◽

Vol 63 (4) ◽

pp. 624

Author(s):

Michael J. Serpe ◽

Jason R. Whitehead ◽

Stephen L. Craig

Keyword(s):

Molecular Weight ◽

Single Molecule ◽

Monomer Concentration ◽

Supramolecular Polymer ◽

Force Microscopy ◽

Molecular Weight Distributions ◽

Atomic Force ◽

Multi Stage ◽

Single Molecule Studies ◽

Supramolecular Ensemble

Single molecule atomic force microscopy (AFM) studies of oligonucleotide-based supramolecular polymers on surfaces are used to examine the molecular weight distribution of the polymers formed between a functionalized surface and an AFM tip as a function of monomer concentration. For the concentrations examined here, excellent agreement with a multi-stage open association model of polymerization is obtained, without the need to invoke additional contributions from secondary steric interactions at the surface.

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THE MOLECULAR WEIGHT OF RHODOPSIN AND THE NATURE OF THE RHODOPSIN-DIGITONIN COMPLEX

The Journal of General Physiology ◽

10.1085/jgp.37.3.381 ◽

1954 ◽

Vol 37 (3) ◽

pp. 381-399 ◽

Cited By ~ 143

Author(s):

Ruth Hubbard

Keyword(s):

Molecular Weight ◽

Single Molecule ◽

Extinction Coefficient ◽

Dry Weight ◽

Weight Basis ◽

Sedimentation Behavior ◽

A Value ◽

Wet Weight ◽

Single Boundary ◽

Stoichiometric Complex

The sedimentation behavior of aqueous solutions of digitonin and of cattle rhodopsin in digitonin has been examined in the ultracentrifuge. In confirmation of earlier work, digitonin was found to sediment as a micelle (D-1) with an s20 of about 6.35 Svedberg units, and containing at least 60 molecules. The rhodopsin solutions sediment as a stoichiometric complex of rhodopsin with digitonin (RD-1) with an s20 of about 9.77 Svedberg units. The s20 of the RD-1 micelle is constant between pH 6.3 and 9.6, and in the presence of excess digitonin. RD-1 travels as a single boundary also in the electrophoresis apparatus at pH 8.5, and on filter paper at pH 8.0. The molecular weight of the RD-1 micelle lies between 260,000 and 290,000. Of this, only about 40,000 gm. are due to rhodopsin; the rest is digitonin (180 to 200 moles). Comparison of the relative concentrations of RD-1 and retinene in solutions of rhodopsin-digitonin shows that RD-1 contains only one retinene equivalent. It can therefore contain only one molecule of rhodopsin with a molecular weight of about 40,000. Cattle rhodopsin therefore contains only one chromophore consisting of a single molecule of retinene. It is likely that frog rhodopsin has a similar molecular weight and also contains only one chromophore per molecule. The molar extinction coefficient of rhodopsin is therefore identical with the extinction coefficient per mole of retinene (40,600 cm.2 per mole) and the E(1 per cent, 1 cm., 500 mµ) has a value of about 10. Rhodopsin constitutes about 14 per cent of the dry weight, and 3.7 per cent of the wet weight of cattle outer limbs. This corresponds to about 4.2 x 106 molecules of rhodopsin per outer limb. The rhodopsin content of frog outer limbs is considerably higher: about 35 per cent of the dry weight, and 10 per cent of the wet weight, corresponding to about 2.1 x 109 molecules per outer limb. Thus the frog outer limb contains about five hundred times as much rhodopsin as the cattle outer limb. But the relative volumes of these structures are such that the ratio of concentrations is only about 2.5 to 1 on a weight basis. Rhodopsin accounts for at least one-fifth of the total protein of the cattle outer limb; for the frog, this value must be higher. The extinction (K500) along its axis is about 0.037 cm.2 for the cattle outer limb, and about 0.50 cm.2 for the frog outer limb.

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Neisseria gonorrhoeaePBP3 and PBP4 Facilitate NOD1 Agonist Peptidoglycan Fragment Release and Survival in Stationary Phase

Infection and Immunity ◽

10.1128/iai.00833-18 ◽

2018 ◽

Vol 87 (2) ◽

Cited By ~ 3

Author(s):

Ryan E. Schaub ◽

Krizia M. Perez-Medina ◽

Kathleen T. Hackett ◽

Daniel L. Garcia ◽

Joseph P. Dillard

Keyword(s):

Molecular Weight ◽

Cell Wall ◽

Inflammatory Response ◽

Neisseria Gonorrhoeae ◽

Wild Type ◽

Cross Link ◽

Content Type ◽

Lytic Transglycosylases ◽

Penicillin Binding Proteins ◽

Proinflammatory Molecules

ABSTRACTNeisseria gonorrhoeaereleases peptidoglycan fragments during growth, and these molecules induce an inflammatory response in the human host. The proinflammatory molecules include peptidoglycan monomers, peptidoglycan dimers, and free peptides. These molecules can be released by the actions of lytic transglycosylases or an amidase. However, >40% of the gonococcal cell wall is cross-linked, where the peptide stem on one peptidoglycan strand is linked to the peptide stem on a neighboring strand, suggesting that endopeptidases may be required for the release of many peptidoglycan fragments. Therefore, we characterized mutants with individual or combined mutations in genes for the low-molecular-mass penicillin-binding proteins PBP3 and PBP4. Mutations in eitherdacB, encoding PBP3, orpbpG, encoding PBP4, did not significantly reduce the release of peptidoglycan monomers or free peptides. A mutation indacBcaused the appearance of a larger-sized peptidoglycan monomer, the pentapeptide monomer, and an increased release of peptidoglycan dimers, suggesting the involvement of this enzyme in both the removal of C-terminald-Ala residues from stem peptides and the cleavage of cross-linked peptidoglycan. Mutation of bothdacBandpbpGeliminated the release of tripeptide-containing peptidoglycan fragments concomitantly with the appearance of pentapeptide and dipeptide peptidoglycan fragments and higher-molecular-weight peptidoglycan dimers. In accord with the loss of tripeptide peptidoglycan fragments, the level of human NOD1 activation by thedacB pbpGmutants was significantly lower than that by the wild type. We conclude that PBP3 and PBP4 overlap in function for cross-link cleavage and that these endopeptidases act in the normal release of peptidoglycan fragments during growth.

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A novel von Willebrand disease–causing mutation (Arg273Trp) in the von Willebrand factor propeptide that results in defective multimerization and secretion

Blood ◽

10.1182/blood.v96.2.560 ◽

2000 ◽

Vol 96 (2) ◽

pp. 560-568 ◽

Cited By ~ 41

Author(s):

Simon Allen ◽

Adel M. Abuzenadah ◽

Joanna Hinks ◽

Joanna L. Blagg ◽

Turkiz Gursel ◽

...

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Von Willebrand Factor ◽

Family Members ◽

Amino Acid Position ◽

Von Willebrand Disease ◽

Molecular Defect ◽

Wild Type ◽

Von Willebrand ◽

Willebrand Factor

Abstract In this report we describe the molecular defect underlying partial and severe quantitative von Willebrand factor (VWF) deficiencies in 3 families previously diagnosed with types 1 and 3 Von Willebrand-disease. Analysis of the VWF gene in affected family members revealed a novel C to T transition at nucleotide 1067 of the VWF complemetary DNA (cDNA), predicting substitution of arginine by tryptophan at amino acid position 273 (R273W) of pre–pro-VWF. Two patients, homozygous for the R273W mutation, had a partial VWF deficiency (VWF:Ag levels of 0.06 IU/mL and 0.09 IU/mL) and lacked high-molecular weight VWF multimers in plasma. A third patient, also homozygous for the R273W mutation, had a severe VWF deficiency (VWF:Ag level of less than 0.01 IU/mL) and undetectable VWF multimers in plasma. Recombinant VWF having the R273W mutation was expressed in COS-7 cells. Pulse-chase experiments showed that secretion of rVWFR273W was severely impaired compared with wild-type rVWF. However, the mutation did not affect the ability of VWF to form dimers in the endoplasmic reticulum (ER). Multimer analysis showed that rVWFR273W failed to form high-molecular-weight multimers present in wild-type rVWF. We concluded that the R273W mutation is responsible for the quantitative VWF deficiencies and aberrant multimer patterns observed in the affected family members. To identify factors that may function in the intracellular retention of rVWFR273W, we investigated the interactions of VWF expressed in COS-7 cells with molecular chaperones of the ER. The R273W mutation did not affect the ability of VWF to bind to BiP, Grp94, ERp72, calnexin, and calreticulin in COS-7 cells.

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An Autism-Associated Neuroligin-3 Mutation Affects Developmental Synapse Elimination in the Cerebellum

Frontiers in Neural Circuits ◽

10.3389/fncir.2021.676891 ◽

2021 ◽

Vol 15 ◽

Author(s):

Esther Suk King Lai ◽

Hisako Nakayama ◽

Taisuke Miyazaki ◽

Takanobu Nakazawa ◽

Katsuhiko Tabuchi ◽

...

Keyword(s):

Cell Adhesion Molecule ◽

Synapse Formation ◽

Single Point ◽

Autism Spectrum ◽

Mutant Mice ◽

Single Point Mutation ◽

Synapse Elimination ◽

Wild Type ◽

Post Mortem Brain ◽

And Function

Neuroligin is a postsynaptic cell-adhesion molecule that is involved in synapse formation and maturation by interacting with presynaptic neurexin. Mutations in neuroligin genes, including the arginine to cystein substitution at the 451st amino acid residue (R451C) of neuroligin-3 (NLGN3), have been identified in patients with autism spectrum disorder (ASD). Functional magnetic resonance imaging and examination of post-mortem brain in ASD patients implicate alteration of cerebellar morphology and Purkinje cell (PC) loss. In the present study, we examined possible association between the R451C mutation in NLGN3 and synaptic development and function in the mouse cerebellum. In NLGN3-R451C mutant mice, the expression of NLGN3 protein in the cerebellum was reduced to about 10% of the level of wild-type mice. Elimination of redundant climbing fiber (CF) to PC synapses was impaired from postnatal day 10–15 (P10–15) in NLGN3-R451C mutant mice, but majority of PCs became mono-innervated as in wild-type mice after P16. In NLGN3-R451C mutant mice, selective strengthening of a single CF relative to the other CFs in each PC was impaired from P16, which persisted into juvenile stage. Furthermore, the inhibition to excitation (I/E) balance of synaptic inputs to PCs was elevated, and calcium transients in the soma induced by strong and weak CF inputs were reduced in NLGN3-R451C mutant mice. These results suggest that a single point mutation in NLGN3 significantly influences the synapse development and refinement in cerebellar circuitry, which might be related to the pathogenesis of ASD.

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KIF18A's neck linker permits navigation of microtubule-bound obstacles within the mitotic spindle

Life Science Alliance ◽

10.26508/lsa.201800169 ◽

2019 ◽

Vol 2 (1) ◽

pp. e201800169 ◽

Cited By ~ 4

Author(s):

Heidi LH Malaby ◽

Dominique V Lessard ◽

Christopher L Berger ◽

Jason Stumpff

Keyword(s):

Single Molecule ◽

Mitotic Spindle ◽

Binding Proteins ◽

Mitotic Chromosome ◽

Chromosome Movement ◽

Wild Type ◽

Microtubule Associated Proteins ◽

Chromosome Alignment ◽

Associated Proteins ◽

Fiber Dynamics

KIF18A (kinesin-8) is required for mammalian mitotic chromosome alignment. KIF18A confines chromosome movement to the mitotic spindle equator by accumulating at the plus-ends of kinetochore microtubule bundles (K-fibers), where it functions to suppress K-fiber dynamics. It is not understood how the motor accumulates at K-fiber plus-ends, a difficult feat requiring the motor to navigate protein dense microtubule tracks. Our data indicate that KIF18A's relatively long neck linker is required for the motor's accumulation at K-fiber plus-ends. Shorter neck linker (sNL) variants of KIF18A display a deficiency in accumulation at the ends of K-fibers at the center of the spindle. Depletion of K-fiber–binding proteins reduces the KIF18A sNL localization defect, whereas their overexpression reduces wild-type KIF18A's ability to accumulate on this same K-fiber subset. Furthermore, single-molecule assays indicate that KIF18A sNL motors are less proficient in navigating microtubules coated with microtubule-associated proteins. Taken together, these results support a model in which KIF18A's neck linker length permits efficient navigation of obstacles to reach K-fiber ends during mitosis.

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