Journal: Nature Communications

Article Title: Cryo-EM and femtosecond spectroscopic studies provide mechanistic insight into the energy transfer in CpcL-phycobilisomes

doi: 10.1038/s41467-023-39689-7

Figure Lengend Snippet: a Overall distribution of bilins in CpcL-PBS with the bilins 1I β 82 1 , 1I β 82 2 , 1I β 82 3 , and 1II β 82 1 highlighted in pink. b The spatial relationship between the linker proteins (CpcL and CpcC1) and the highlighted bilins in squares. c The conformation of the bilins from either CpcL-PBS or CpcG-PBS of Synechocystis 6803. From top to bottom: 1I β 82 1 , 1I β 82 2 , 1I β 82 3 , and 2II β 82 3 of CpcL, 1I β 82 1G (G indicates that the bilin is from CpcG-PBS) and 1I β 82 2G of the peripheral rod1, and the bilin α 84 of the terminal emitter ApcD of the CpcG-PBS core. The coordinates of bilins from CpcG-PBS of Synechocystis 6803 were from a recent cryo-EM study . d Schematic presentation of bilins from rings A to D in panel ( c ). The angles between the adjacent pyrrole planes are shown.

Article Snippet: Recently determined cryo-EM PBS structures from red algae , and the cyanobacteria – revealed how linker proteins and PBP are organized into a highly ordered light harvesting architectures.

Techniques: Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a Schematic depiction of plasma EV ultracentrifugation and RBD-bead based depletion. b Cryo-EM images of human EV pellets isolated from acute phase COVID-19 plasma (bar = 100 nm). c Immunoblots of plasma EV pellets (sero-negative and COVID-19 acute phase patients CBB-005 and -013) for ACE2 and loading control of protein staining with Ponceau). Laemmli buffer was used for lysis ( N = 1 experiment). d ACE2 + EV pellets from acute phase patients 007, 008, 009, 012, and 013 (CBB) ( n = 2 biological replicates each) blocked SARS-CoV-2 infection-induced death of Vero-6 cells whereas the sero-negative control ( n = 2 biological replicates) and CBB-005 (no detectable ACE2) ( n = 2 biological replicates) did not show neutralization effects. One-tail t test, **** p = 2.24E−08 shown as compared to sero-negative. e , f Levels of ACE2 + EV counts ( n = 3 biological replicates) in plasma EVs (green) and bead-depleted EVs (light blue). One-tail paired t test, * p = 0.011 and ** p = 0.0063 (data are presented as mean values ± SD) ( e ) and altered neutralization effects on RBD–host cell binding ( f ) of the COVID-19 plasma EV pellets prior to and after RBD-bead depletion (convalescent phase CSB-012 and -024; acute phase CBB-008, 009, and 013). One-tail paired t test **** p = 5.11E−05.

Article Snippet: Human plasma and plasma-derived EV samples (resuspended in PBS) were lysed with Laemmli buffer (Bio-Rad, 1610747) for 30 min on ice and processed as mentioned above.

Techniques: Clinical Proteomics, Cryo-EM Sample Prep, Isolation, Western Blot, Control, Staining, Lysis, Infection, Negative Control, Neutralization, Binding Assay

Journal: Journal of Extracellular Vesicles

Article Title: Proteomics analysis of vesicles isolated from plasma and urine of prostate cancer patients using a multiplex, aptamer-based protein array

doi: 10.3402/jev.v5.31209

Figure Lengend Snippet: Analysis of plasma fractionated by size-exclusion chromatography. Pre-cleared plasma was subject to separation on a 12-cm long Sepharose CL2B size-exclusion column, and 30× serial 500-µl fractions were collected and analysed. The protein concentration was estimated by NanoDrop™ (absorbance at 280 nm), and the particle concentration was measured by nanoparticle tracking analysis (NanoSight™). Individual fractions where NanoSight™ analysis was not performed are indicated (with an X). The ratio of particles to protein (particles/µg) was calculated and plotted (left axis: blue bars) with total protein on the right axis (red line) (a). A proportion of the same fraction series was immobilized onto high-protein-binding microplate strips and allowed to couple overnight. After blocking, wells were stained with primary antibodies against CD9, CD81, ApoB or HSA, and binding detected using a time-resolved fluorometric readout (arbitrary TRF units shown) (b). Selected fractions (F10 to F15), identified as vesicle rich but protein low by the aforementioned assays, were pooled and concentrated by ultracentrifugation. After re-suspending the pellet, a proportion was examined by cryo-EM (scale bar=100 nm), and representative fields are shown (c). A proportion was also analysed by nanoparticle tracking to examine the size distribution of particles in the final sample, and the histogram mean and mode is shown (based on triplicate measurements) (d).

Article Snippet: After overnight coupling and blocking (with 1% (w/v) BSA in PBS for 2 h at room temperature (RT)), the bound material was labelled with primary antibodies including CD9, ApoB, THP (Tamm–Horsfall protein) (at concentrations of 1 μg/ml) or HSA (human serum albumin) (at 250 ng/ml) (R&D Systems) all for 2 h at RT on a plate shaker.

Techniques: Clinical Proteomics, Size-exclusion Chromatography, Protein Concentration, Concentration Assay, Protein Binding, Blocking Assay, Staining, Binding Assay, Cryo-EM Sample Prep

Journal: Journal of Extracellular Vesicles

Article Title: Proteomics analysis of vesicles isolated from plasma and urine of prostate cancer patients using a multiplex, aptamer-based protein array

doi: 10.3402/jev.v5.31209

Figure Lengend Snippet: Analysis of urine fractionated by ultracentrifugation and size-exclusion chromatography. Urine was concentrated by pelleting (200,000× g , 2 h, 4°C) and after re-suspending, the material was fractionated using a Sepharose CL-2B size-exclusion chromatography column. Thirty fractions were collected. Equal volumes of fraction 4 to fraction 25 were examined by western blotting; staining for the endo/lysosomal-related proteins including TSG101, ALIX, LAMP2 or for serum albumin (HSA). For this number of samples, 2 gels were required, and the position of the divide between the gels is indicated by a dotted line (a). In addition, the protein and particle concentrations were determined (for the latter, those that were not measured are denoted by an X). The particle-to-protein ratio was calculated and is plotted (blue bars) together with total protein estimation (red line) (b). A proportion of the same fraction series was immobilized onto high-protein-binding microplate strips and allowed to couple overnight. After blocking, wells were stained with primary antibodies against CD9, THP (uromodulin), HSA or isotype control, and binding detected using a time-resolved fluorometric readout (arbitrary TRF units shown) (c). Selected fractions (F5 to F12), identified as vesicle rich but protein low by the aforementioned assays, were pooled and concentrated by ultracentrifugation. After re-suspending the pellet, a proportion was examined by cryo-EM (scale bar=100 nm), and representative fields are shown (d). A proportion was also analysed by nanoparticle tracking to examine the size distribution of particles in the final sample, and the histogram mean and mode is shown (based on triplicate measurements) (e).

Article Snippet: After overnight coupling and blocking (with 1% (w/v) BSA in PBS for 2 h at room temperature (RT)), the bound material was labelled with primary antibodies including CD9, ApoB, THP (Tamm–Horsfall protein) (at concentrations of 1 μg/ml) or HSA (human serum albumin) (at 250 ng/ml) (R&D Systems) all for 2 h at RT on a plate shaker.

Techniques: Size-exclusion Chromatography, Western Blot, Staining, Protein Binding, Blocking Assay, Control, Binding Assay, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structural landscape of AAA+ ATPase motor states in the substrate-degrading human 26S proteasome reveals conformation-specific binding of TXNL1

doi: 10.1101/2024.11.08.622731

Figure Lengend Snippet: A) Purification of TXNL1-bound and TXNL1-free human 26S proteasomes from HEK293 cells. Top: Western blots showing aliquots from the washing (W1-W4) and elution (E) steps in low-salt or high-salt buffer for HTBH-tagged proteasomes that were immobilized on streptavidin agarose. Bottom left: Coomassie-stained SDS-PAGE gel showing the separation of 1 μg human 26S proteasomes purified by size-exclusion chromatography after previous low salt or high salt washes and compared to specific concentrations of recombinant FLAG-tagged TXNL1 purified from E. coli. Bottom right: Western blot of the SDS-PAGE samples on the left, showing TXNL1 levels that co-purified with low-salt or high-salt washed proteasomes in comparison to recombinant His-FLAG-tagged TXNL1. Low-salt washed proteasomes contain sub-stoichiometric amounts of TXNL1, whereas TXNL1 levels for high-salt washed proteasomes are almost undetectable. B) Left: Elution profile for the size-exclusion chromatography (SD75 16/600) of recombinant human TXNL1 that was expressed in E. coli and Ni-NTA affinity purified using its His-(TEV)-FLAG tag. Right: Coomassie-stained SDS-PAGE gel with aliquots from individual stages of recombinant TXNL1 purification. C) Redox activity of recombinant TXNL1 (15 μM) measured by the increase in turbidity (absorbance at 600 nm) that results from the reduction and consequent aggregation of insulin (30 μM). Activities are compared to different concentrations of DTT (top) and TXNL1 mutants that contained only the N-terminal catalytic TRX domain, the C-terminal PITH domain, or the C34S mutation in the catalytic CXXC motif (bottom). D) Degradation of Eos-titin V15P -tail (5 μM) substrate by human 26S proteasome (200 nM) in the absence or presence of excess TXNL1 (15 μM), monitored by the loss of Eos fluorescence. E) FAM fluorescence detection (top) and Coomassie staining (bottom) of the SDS-PAGE gel with samples from the degradation of N-terminally FAM-labeled and ubiquitinated FAM Eos-titin V15P -tail substrate (2.5 μM) by human 26S proteasomes (200 nM) in the absence and presence of excess TXNL1 (15 μM). The right 2 lanes show a negative control with non-ubiquitinated substrate (no E1, E2, E3 enzymes).

Article Snippet: Samples were separated by SDS-PAGE and transferred to PVDF membranes (Thermo Scientific), before blocking in 5% milk TBS-T. TXNL1 primary antibodies (15289-1-AP, Thermo Scientific) were incubated at room temperature with the membranes for 90 min in 5% milk TBS-T, before 5x 5 min washing (∼10 mL in TBS-T) and incubation with secondary Rabbit-HRP antibodies (ab79773, Abcam) for 60 min. After more washes, blots were visualized using HRP after 2 min of incubation.

Techniques: Purification, Western Blot, Staining, SDS Page, Size-exclusion Chromatography, Recombinant, Comparison, Affinity Purification, FLAG-tag, Activity Assay, Mutagenesis, Fluorescence, Labeling, Negative Control

Journal: bioRxiv

Article Title: Structural landscape of AAA+ ATPase motor states in the substrate-degrading human 26S proteasome reveals conformation-specific binding of TXNL1

doi: 10.1101/2024.11.08.622731

Figure Lengend Snippet: Shown are the sequential steps taken to process cryo-EM data from movies to final maps for the human 26S proteasome at ∼ 2 min after incubation with excess Txnl1, FAT10-Eos substrate, and the NUB1 cofactor. From step 13 the workflow focuses on proteasomes in the processing states (PS), while the subsequent workflow for proteasomes in the resting state (RS) is shown in and .

Article Snippet: Samples were separated by SDS-PAGE and transferred to PVDF membranes (Thermo Scientific), before blocking in 5% milk TBS-T. TXNL1 primary antibodies (15289-1-AP, Thermo Scientific) were incubated at room temperature with the membranes for 90 min in 5% milk TBS-T, before 5x 5 min washing (∼10 mL in TBS-T) and incubation with secondary Rabbit-HRP antibodies (ab79773, Abcam) for 60 min. After more washes, blots were visualized using HRP after 2 min of incubation.

Techniques: Cryo-EM Sample Prep, Incubation

Journal: bioRxiv

Article Title: Structural landscape of AAA+ ATPase motor states in the substrate-degrading human 26S proteasome reveals conformation-specific binding of TXNL1

doi: 10.1101/2024.11.08.622731

Figure Lengend Snippet: A) Density of TXNL1’s PITH domain bound to the human 26S proteasome in the processing state PS Rpt5 with Rpt5 at the top of the AAA+ ATPase spiral staircase and engaged with a translocating peptide during degradation of FAT10-Eos substrate delivered by NUB1. TXNL1’s PITH domain and C-terminal tail are shown in orange, the DUB Rpn11 in petrol, Rpn2 in salmon, Rpn1 and Rpn10’s VWA domain in tomato, the AAA+ ATPase hexamer in alternating dark blue and cyan, lid subunits (except Rpn11) in light grey, and the 20S CP in dark grey. B) Top: Schematic of TXNL1’s domain organization. Bottom: AlphaFold model of full length TXNL1. The N-terminal TRX domain is flexibly attached to the C-terminal PITH domain and therefore not resolved in our EM maps. C) Atomic model of PS Rpt5 with bound TXNL1. Zoom-in views on the right depict the interfaces of TXNL1 with Rpn2 and Rpn10, as well as the Rpt pore-1 loops forming a spiral staircase around the translocating substrate polypeptide. D) Left: Density for the PITH domain and its C-terminal tail bound to Rpn11, with Rpn11’s Ins-1 region shown in purple. Right: Zoom-in view of TXNL1’s C-terminal tail, with hydrophobic residues (M275 and F278) pointing toward Rpn11’s catalytic groove and the C-terminal H289 coordinating the active-site Zn 2+ .

Article Snippet: Samples were separated by SDS-PAGE and transferred to PVDF membranes (Thermo Scientific), before blocking in 5% milk TBS-T. TXNL1 primary antibodies (15289-1-AP, Thermo Scientific) were incubated at room temperature with the membranes for 90 min in 5% milk TBS-T, before 5x 5 min washing (∼10 mL in TBS-T) and incubation with secondary Rabbit-HRP antibodies (ab79773, Abcam) for 60 min. After more washes, blots were visualized using HRP after 2 min of incubation.

Techniques:

Journal: bioRxiv

Article Title: Structural landscape of AAA+ ATPase motor states in the substrate-degrading human 26S proteasome reveals conformation-specific binding of TXNL1

doi: 10.1101/2024.11.08.622731

Figure Lengend Snippet: A) B) Sequence alignment of TXNL1 from uniport sequences using ENDscript at default settings: Homo sapiens (Q43396), Mus musculus (Q8CDN6), Gallus gallus (A0A1D5NUH1), Xenopus tropicalis (F6RTD9), Danio rerio (F6NTA0), Drosophila melanogaster (Q9VRP3), Caenorhabditis elegans (G5EES9) and Schizosaccharomyces pombe (Q9USR1). Red underlay shows conserved residues and blue boxes indicate similarity in amino acid identity. Residues in the C-terminal tail that mediate the interaction with Rpn11, including the terminal His, are highly conserved, except for fission yeast. B) Atomic models of Rpn11 with the Insert-1 (Ins-1) region in three conformations. The open loop conformation is found in resting-state proteasomes, the deubiquitination conformation with a β-hairpin is adopted upon ubiquitin binding to Rpn11, and the inhibitory, closed conformation is observed substrate-processing proteasomes. The three models are derived from our structure for the resting state RS.1 with TXNL1 in the forward orientation, the crystal structure of ubiquitin-bound Rpn11-Rpn8 (PDB ID: 5U4P), and our structure of the substrate-processing state PS Rpt5 , respectively. C) Space filling atomic model of PS Rpt5 with the ribbon-represented PITH domain of TXNL1 in orange. The AlphaFold model for full-length TXNL1 is aligned by its PITH domain (light green) with the PITH domain in our structure and shows the TRX domain (dark green) with its catalytic CXXC motif (yellow spheres) close to the entrance of the ATPase motor. D) Overlay of the atomic models for PS Rpt5 (Rpts in cyan) and PS Rpt2 (Rpts in dark blue), aligned by Rpn11 (petrol), shows a movement of the Rpt4/Rpt5 coiled coil that significantly increases the gap to the Ins-1 loop (pink for PS Rpt5, red for PS Rpt2 ).

Article Snippet: Samples were separated by SDS-PAGE and transferred to PVDF membranes (Thermo Scientific), before blocking in 5% milk TBS-T. TXNL1 primary antibodies (15289-1-AP, Thermo Scientific) were incubated at room temperature with the membranes for 90 min in 5% milk TBS-T, before 5x 5 min washing (∼10 mL in TBS-T) and incubation with secondary Rabbit-HRP antibodies (ab79773, Abcam) for 60 min. After more washes, blots were visualized using HRP after 2 min of incubation.

Techniques: Sequencing, Binding Assay, Derivative Assay

Journal: bioRxiv

Article Title: Structural landscape of AAA+ ATPase motor states in the substrate-degrading human 26S proteasome reveals conformation-specific binding of TXNL1

doi: 10.1101/2024.11.08.622731

Figure Lengend Snippet: A) Densities of the resting-state (RS.1) 26S proteasome bound to TXNL1’s PITH domain in the forward (left, PITH shown in orange) and backward (right, PITH shown in cyan) conformations. The forward position is identical to the location of the PITH domain on processing-state proteasomes, whereas the backward conformation is rotated by ∼ 90 °. RS.1 refers to a particular class of resting state proteasomes that differ from RS.2 through a slight shift in the regulatory particle (see ). B) Particle distribution of resting-state proteasomes with TXNL1 bound in the forward, backward, or mixed conformations. Mixed conformations represent intermediate states due to PITH domain motions or particles with ambiguous probabilities of belonging to either conformation. C) Top left: Atomic model of the RS.1 proteasome with the PITH domain (cyan) is the backward conformation. Right and bottom: Zoom-in views of specific interactions between the PITH domain and Rpn2, Rpn10’s VWA domain, and Rpn8. The contacts with Rpn2 and Rpn10 strongly rely on ion pairs, while a polar-ν interaction is at the center of the interface with Rpn8.

Article Snippet: Samples were separated by SDS-PAGE and transferred to PVDF membranes (Thermo Scientific), before blocking in 5% milk TBS-T. TXNL1 primary antibodies (15289-1-AP, Thermo Scientific) were incubated at room temperature with the membranes for 90 min in 5% milk TBS-T, before 5x 5 min washing (∼10 mL in TBS-T) and incubation with secondary Rabbit-HRP antibodies (ab79773, Abcam) for 60 min. After more washes, blots were visualized using HRP after 2 min of incubation.

Techniques:

Journal: bioRxiv

Article Title: Structural landscape of AAA+ ATPase motor states in the substrate-degrading human 26S proteasome reveals conformation-specific binding of TXNL1

doi: 10.1101/2024.11.08.622731

Figure Lengend Snippet: A) ‘Slight left’ and ‘Slight right’ particles were processed separately. Particle stacks were aligned (local refinement in CryoSparc) with a local mask surrounding the PITH domain, Rpn2, Rpn10, Rpn11, and the N-ring of the Rpt hexamer. CryoSparc 3D variability analysis of aligned particles with the local mask, followed by 3D cluster analysis, allowed grouping of particles into two distinct conformations, forward and backward with respect to the PITH domain position. A third group of particles showed the PITH domain in variable positions and could not be assigned to a particular state, suggesting that the PITH domain has continuous motion between forward and backward orientations. B) Local refinement with a mask surrounding the whole RP resulted in 4 distinct conformations defined by a slight shift in the RP and the two orientations of the PITH domain: RS.1 TXNL1 Forward (RS.1 state 1), RS.1 TXNL1 Backward (RS.1 state 2), RS.2 TXNL1 Forward (RS.2 state 1), and RS.2 TXNL1 Backward (RS.2 state 2). Each structure was resolved to high resolution, as demonstrated by the maps colored according to estimations of local resolutions, calculated with CryoSparc using a 0.143 FSC cutoff. C) Alignments of the atomic models for RS.1 and RS.2, states 1 and 2, highlight the shift in the RP, in addition to variations in Rpn1’s position. Right: Particle numbers for each conformational state that were used to calculate the percentages of proteasomes with TXNL1 in the forward, backward, or mixed orientations shown in .

Article Snippet: Samples were separated by SDS-PAGE and transferred to PVDF membranes (Thermo Scientific), before blocking in 5% milk TBS-T. TXNL1 primary antibodies (15289-1-AP, Thermo Scientific) were incubated at room temperature with the membranes for 90 min in 5% milk TBS-T, before 5x 5 min washing (∼10 mL in TBS-T) and incubation with secondary Rabbit-HRP antibodies (ab79773, Abcam) for 60 min. After more washes, blots were visualized using HRP after 2 min of incubation.

Techniques:

Journal: bioRxiv

Article Title: Structural landscape of AAA+ ATPase motor states in the substrate-degrading human 26S proteasome reveals conformation-specific binding of TXNL1

doi: 10.1101/2024.11.08.622731

Figure Lengend Snippet: A) Proteasome binding and dissociation of N-terminally fluoresceine-amidite (FAM) labeled TXNL1 (50 nM) was measured by changes in fluorescence polarization after incubation with human 26S proteasome (500 nM) in the absence or presence of FAT10 substrate (10 μM) and the NUB1 cofactor (12 μM). B) Time-resolved cryo-EM of actively degrading proteasomes with sub-stoichiometric amounts of TXNL1 reveals TXNL1’s binding preference for processing states. Left: EM density of the proteasome in the PS Rpt5 conformation shows partial occupancy with TXNL1’s PITH domain (orange). Middle: Zoom-in views of example density maps from particles that were sorted into TXNL1-bound (top) and TXNL1-unbound (bottom) after local 3D classification and refinement of the PITH-domain density. C) Fractions of proteasome particles with and without bound TXNL1 as a function of the processing-state conformation.

Article Snippet: Samples were separated by SDS-PAGE and transferred to PVDF membranes (Thermo Scientific), before blocking in 5% milk TBS-T. TXNL1 primary antibodies (15289-1-AP, Thermo Scientific) were incubated at room temperature with the membranes for 90 min in 5% milk TBS-T, before 5x 5 min washing (∼10 mL in TBS-T) and incubation with secondary Rabbit-HRP antibodies (ab79773, Abcam) for 60 min. After more washes, blots were visualized using HRP after 2 min of incubation.

Techniques: Binding Assay, Labeling, Fluorescence, Incubation, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structural landscape of AAA+ ATPase motor states in the substrate-degrading human 26S proteasome reveals conformation-specific binding of TXNL1

doi: 10.1101/2024.11.08.622731

Figure Lengend Snippet: A) Left: TXNL1’s PITH domain is necessary and sufficient for the conformational selective binding to actively degrading 26S proteasomes. Shown are representative traces for the fluorescence polarization of FAM-labeled TRX domain (50 nM) or PITH domain (50 nM) that was incubated in isolation or with human 26S proteasome (500 nM) in the presence of 10 μM FAT10 substrate and 12 μM NUB1 cofactor. Right: Proteasomes whose proteolytic cleavage of the FAT10 substrate was inhibited by MG132 and which were therefore stalled during substrate translocation interact stably with TXNL1. Shown are the fluorescence polarization time courses for N-terminally FAM-labeled full-length FAM TXNL1 (50 nM) that was incubated with FAT10 (5 μM), NUB1 (6 μM), and human 26S proteasome (500 nM) in the absence or presence of MG132 (20 μM), as indicated. B) Titration of proteasomes that were stalled during substrate degradation through MG132 inhibition of proteolysis reveals the TXNL1 binding affinity for processing-state proteasomes. Left: Shown are the fluorescence polarization traces for FAM TXNL1 (50 nM) incubated with human 26S proteasome at indicated concentrations in the presence of FAT10 substrate (5 μM) and NUB1 cofactor (6 μM). Right: FAM TXNL1 binding curve derived from the 900 s time points of the polarization traces shown on the left indicates an affinity of K D ∼ 35 nM.

Article Snippet: Samples were separated by SDS-PAGE and transferred to PVDF membranes (Thermo Scientific), before blocking in 5% milk TBS-T. TXNL1 primary antibodies (15289-1-AP, Thermo Scientific) were incubated at room temperature with the membranes for 90 min in 5% milk TBS-T, before 5x 5 min washing (∼10 mL in TBS-T) and incubation with secondary Rabbit-HRP antibodies (ab79773, Abcam) for 60 min. After more washes, blots were visualized using HRP after 2 min of incubation.

Techniques: Binding Assay, Fluorescence, Labeling, Incubation, Isolation, Translocation Assay, Stable Transfection, Titration, Inhibition, Derivative Assay

Journal: bioRxiv

Article Title: Structural landscape of AAA+ ATPase motor states in the substrate-degrading human 26S proteasome reveals conformation-specific binding of TXNL1

doi: 10.1101/2024.11.08.622731

Figure Lengend Snippet: Cryo-EM data processing for the human 26S proteasomes 30 s after incubation with FAT10 substrate and NUB1 cofactor. A) Data were processed as shown in the workflow in . Top: Shown are the unsharpened maps and particle numbers for each state. Bottom: To improve resolution of the AAA+ ATPase domains a local mask was generated for the Rpt hexamer and Rpn11 and local refinement was used. Maps show local resolutions of the Rpt subunits and Rpn11 for each processing state. B) Human proteasomes used in this dataset were purified from HEK293 cells under low-salt conditions and therefore contain sub-stoichiometric amounts of bound TXNL1. Extensive local 3D classification focused on TXNL1 for each processing state allowed separation of particles with and without bound TXNL1. Briefly particles were locally aligned by a mask surrounding TXNL1’s PITH domain and subjected to 3D classification with a filtered resolution of 10 Å. Each class was reconstructed and refined to group particles based on the presence or absence of PITH density, and the particles numbers for PITH-bound and -unbound proteasomes are plotted for each processing state (right). C) Local resolution of locally refined PS Rpt5 after removing unbound TXNL1 particles.

Article Snippet: Samples were separated by SDS-PAGE and transferred to PVDF membranes (Thermo Scientific), before blocking in 5% milk TBS-T. TXNL1 primary antibodies (15289-1-AP, Thermo Scientific) were incubated at room temperature with the membranes for 90 min in 5% milk TBS-T, before 5x 5 min washing (∼10 mL in TBS-T) and incubation with secondary Rabbit-HRP antibodies (ab79773, Abcam) for 60 min. After more washes, blots were visualized using HRP after 2 min of incubation.

Techniques: Cryo-EM Sample Prep, Incubation, Generated, Purification

Journal: bioRxiv

Article Title: Structural landscape of AAA+ ATPase motor states in the substrate-degrading human 26S proteasome reveals conformation-specific binding of TXNL1

doi: 10.1101/2024.11.08.622731

Figure Lengend Snippet: A) Atomic model of the human proteasome in the PS Rpt2 state during degradation of the FAT10-Eos model substrate. EM density (green) is shown for the substrate, with the partially unraveled beta-barrel of Eos pulled against Rpn11 and a translocating polypeptide spanning the central channel of the ATPase motor. B) Focus on the Rpt hexamer with the EM density and atomic model (green) shown for the translocating substrate, which is engaged by a staircase of ATPase domains. The substrate density is at high enough resolution to identify the C-terminal portion of FAT10 inside the ATPase ring. C) Comparison between the partially unfolded Eos intermediate (left, green) and the crystal structure of folded Eos (right, cyan; PDB ID: 3S05), with the chromophore shown in yellow and dark blue, respectively. The intermediate has β1 as well as the β2-β3 and β5-β6 hairpins partially pulled off from the beta barrel. D) EM density and atomic model for the chromophore in partially unfolded Eos. E) Overlay of the folded (cyan) and partially unfolded Eos (green) show the disruption of the chromophore environment that likely lead to a loss of fluorescence. F) Hydrophobic residues in β1 and the β5-β6 hairpin that normally face the Eos hydrophobic core interact with Rpn11 near the catalytic groove and the Ins-1 loop. G) EM density for proteasomes with bound TXNL1 and the Eos unfolding intermediate show an overlap of binding sites for Eos and TXNL1’s C-terminal tail on Rpn11.

Article Snippet: Samples were separated by SDS-PAGE and transferred to PVDF membranes (Thermo Scientific), before blocking in 5% milk TBS-T. TXNL1 primary antibodies (15289-1-AP, Thermo Scientific) were incubated at room temperature with the membranes for 90 min in 5% milk TBS-T, before 5x 5 min washing (∼10 mL in TBS-T) and incubation with secondary Rabbit-HRP antibodies (ab79773, Abcam) for 60 min. After more washes, blots were visualized using HRP after 2 min of incubation.

Techniques: Comparison, Disruption, Fluorescence, Binding Assay

Journal: bioRxiv

Article Title: Structural landscape of AAA+ ATPase motor states in the substrate-degrading human 26S proteasome reveals conformation-specific binding of TXNL1

doi: 10.1101/2024.11.08.622731

Figure Lengend Snippet: A) Top: Cryo-EM densities of the AAA+ motor for all processing-state (PS) conformation, colored by Rpt subunit. Substrate-disengaged seam subunits are at lower resolution likely due to higher mobility and various vertical positions between the bottom and top of the spiral staircases. PS Rpt3 , PS Rpt6, and PS Rpt2 show larger density gaps between subunits at the seam, likely because they contain two substrate-disengaged seam subunits with potentially high continuous motions. Bottom: Spiral staircase arrangements of pore-1-loop Tyr residues (Phe for Rpt5) for each processing state. Distances between the substrate backbone and the backbone Cα of the pore-1 loop aromatic residue are indicated for the disengaged seam subunits. Interestingly, some seam subunits reside at the bottom, while others are observed toward the top of the staircase. Positions of the pore-loop residues for these seam subunits are approximate due to the higher mobility and consequently lower resolution. B) Cryo-EM densities and atomic models, including the specific nucleotide (ATP or ADP) for all Rpts of the six processing states. C) Particle numbers observed for each processing state in the separate cryo-EM data sets for the 26S proteasome in the presence of sub-stoichiometric TXNL1, 30 s after incubations with the FAT10 substrate (left) or in the presence of excess TXNL1, 2 min after incubation with FAT10 (right). D) Plotted distances between the substrate backbone and the backbone backbone pore loop interactions with substrate backbone and the backbone Cα of the pore-1 loop aromatic residue for Rpt1-6 in each substrate-processing state of the proteasome.

Article Snippet: Samples were separated by SDS-PAGE and transferred to PVDF membranes (Thermo Scientific), before blocking in 5% milk TBS-T. TXNL1 primary antibodies (15289-1-AP, Thermo Scientific) were incubated at room temperature with the membranes for 90 min in 5% milk TBS-T, before 5x 5 min washing (∼10 mL in TBS-T) and incubation with secondary Rabbit-HRP antibodies (ab79773, Abcam) for 60 min. After more washes, blots were visualized using HRP after 2 min of incubation.

Techniques: Cryo-EM Sample Prep, Residue, Incubation

Journal: bioRxiv

Article Title: Structural landscape of AAA+ ATPase motor states in the substrate-degrading human 26S proteasome reveals conformation-specific binding of TXNL1

doi: 10.1101/2024.11.08.622731

Figure Lengend Snippet: A) TXNL1 loosely interacts with Rpn2, Rpn10, and Rpn11 of the resting-state proteasome and tightly binds processing states during substrate unfolding and translocation through additional contacts of its C-terminal tail with Rpn11’s catalytic groove, allowing a potential do-degradational reduction of oxidized substrates. B) The conformationally selective binding of TXNL1 prevents interference with deubiquitination during the turnover of ubiquitin-tagged substrates. After ubiquitin binding to a receptor and Rpn11 in the resting-state proteasome, the insertion of the substrate’s flexible initiation region into the ATPase motor induces the conformational switch to processing states and subsequent or simultaneous cleavage of the affinity-conferring ubiquitin chain by Rpn11. TXNL1 then binds with high affinity during substrate unfolding and translocation, yet lower TXNL1 affinity and the exclusion of its C-terminal tail from Rpn11 in the PS Rpt2 state would allow a co-translocational removal of any additional ubiquitin chains.

Article Snippet: Samples were separated by SDS-PAGE and transferred to PVDF membranes (Thermo Scientific), before blocking in 5% milk TBS-T. TXNL1 primary antibodies (15289-1-AP, Thermo Scientific) were incubated at room temperature with the membranes for 90 min in 5% milk TBS-T, before 5x 5 min washing (∼10 mL in TBS-T) and incubation with secondary Rabbit-HRP antibodies (ab79773, Abcam) for 60 min. After more washes, blots were visualized using HRP after 2 min of incubation.

Techniques: Translocation Assay, Binding Assay

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a ACE2+ EVs detected in human plasma samples of sero-negative controls (light blue), acute phase (dark green), and convalescent COVID-19 patients (green). One-tail t test (* p = 0.038, ** p = 0.0061 and ** p = 0.0016). Data are presented as mean values ± SEM. b Representative microflow vesiclometry (MFV) plots with gated ACE2+ EVs from sero-negative, acute phase and convalescent COVID-19 patients. c MFV detection of circulating ACE2 + EVs with CD63 + EVs in human plasma of convalescent COVID-19 patient samples (CSB-029 and CSB-023) (green line). Blue line is isotype IgG-negative control. d Flow profiles of ACE2 expression in HEK and HeLa parental control cells (Con, light blue line, ACE2 − ) and with ACE2 overexpression (ACE2, green line). e NanoSight NTA analysis of the sizes of HEK-derived ACE2 − (ev1Con) and ACE2 + (ev1ACE2) and HeLa-derived ACE2 − (ev2Con) and ACE2 + (ev2ACE2). f Immunoblots of HEK and HeLa (ACE2 − and ACE2 + ) EVs and cell lysates for ACE2, TSG101, CD63, CD81, GRP94 and loading control of the membrane proteins upon Ponceau staining. RIPA buffer and Bradford protein assay were used for cells/EVs lysis and protein measurement, respectively ( N = 1 experiment). g Cryo-EM images of HEK-derived EVs, ACE2 − (evCon, left) and ACE2 + (evACE2, right), stained with ACE2 (top) and CD81 (bottom). Scale bars = 100 nm. h Quantified counts of Apogee MFV-based total extracellular vesicles (EVs) and ACE2 + EVs ( N = 2 experiments with n = 6 technical replicates for total EV particles and n = 3 technical replicates for ACE2 + counts). Control EVs are in light blue and ACE2 + EVs in green. Data are presented as mean values +/− SD. i Overlay flow profiles of ACE2 positivity within CD63 + (left column) and CD81+ (right column) EVs isolated from HEK-ACE2 (top row) and HeLa-ACE2 (bottom row) cells, respectively ( n = 3 technical replicates). Light blue line for Control EVs and green line for ACE2 + EVs.

Article Snippet: RIPA buffer and Bradford protein assay were used for cells/EVs lysis and protein measurement, respectively ( N = 1 experiment). g Cryo-EM images of HEK-derived EVs, ACE2 − (evCon, left) and ACE2 + (evACE2, right), stained with ACE2 (top) and CD81 (bottom).

Techniques: Negative Control, Expressing, Over Expression, Derivative Assay, Western Blot, Staining, Bradford Protein Assay, Lysis, Cryo-EM Sample Prep, Isolation