Piere Rodriguez Aliaga

All Publications

• In situ analysis reveals the TRiC duty cycle and PDCD5 as an open-state cofactor. Nature Xing, H., Rosenkranz, R. R., Rodriguez-Aliaga, P., Lee, T. T., Majtner, T., Böhm, S., Turoňová, B., Frydman, J., Beck, M. 2024

  Abstract

  The ring-shaped chaperonin T-complex protein ring complex (TRiC; also known as chaperonin containing TCP-1, CCT) is an ATP-driven protein-folding machine that is essential for maintenance of cellular homeostasis1,2. Its dysfunction is related to cancer and neurodegenerative disease3,4. Despite its importance, how TRiC works in the cell remains unclear. Here we structurally analysed the architecture, conformational dynamics and spatial organization of the chaperonin TRiC in human cells using cryo-electron tomography. We resolved distinctive open, closed, substrate-bound and prefoldin-associated states of TRiC, and reconstructed its duty cycle in situ. The substrate-bound open and symmetrically closed TRiC states were equally abundant. Closed TRiC containing substrate forms distinctive clusters, indicative of spatial organization. Translation inhibition did not fundamentally change the distribution of duty cycle intermediates, but reduced substrate binding for all states as well as cluster formation. From our in-cell structures, we identified the programmed cell death protein 5 (PDCD5) as an interactor that specifically binds to almost all open but not closed TRiC, in a position that is compatible with both substrate and prefoldin binding. Our data support a model in which TRiC functions at near full occupancy to fold newly synthesized proteins inside cells. Defining the TRiC cycle and function inside cells lays the foundation to understand its dysfunction during cancer and neurodegeneration.

  View details for DOI 10.1038/s41586-024-08321-z

  View details for PubMedID 39663456

  View details for PubMedCentralID 6776438

• Brain malformations and seizures by impaired chaperonin function of TRiC. Science (New York, N.Y.) Kraft, F., Rodriguez-Aliaga, P., Yuan, W., Franken, L., Zajt, K., Hasan, D., Lee, T. T., Flex, E., Hentschel, A., Innes, A. M., Zheng, B., Julia Suh, D. S., Knopp, C., Lausberg, E., Krause, J., Zhang, X., Trapane, P., Carroll, R., McClatchey, M., Fry, A. E., Wang, L., Giesselmann, S., Hoang, H., Baldridge, D., Silverman, G. A., Radio, F. C., Bertini, E., Ciolfi, A., Blood, K. A., de Sainte Agathe, J. M., Charles, P., Bergant, G., Čuturilo, G., Peterlin, B., Diderich, K., Streff, H., Robak, L., Oegema, R., van Binsbergen, E., Herriges, J., Saunders, C. J., Maier, A., Wolking, S., Weber, Y., Lochmüller, H., Meyer, S., Aleman, A., Polavarapu, K., Nicolas, G., Goldenberg, A., Guyant, L., Pope, K., Hehmeyer, K. N., Monaghan, K. G., Quade, A., Smol, T., Caumes, R., Duerinckx, S., Depondt, C., Van Paesschen, W., Rieubland, C., Poloni, C., Guipponi, M., Arcioni, S., Meuwissen, M., Jansen, A. C., Rosenblum, J., Haack, T. B., Bertrand, M., Gerstner, L., Magg, J., Riess, O., Schulz, J. B., Wagner, N., Wiesmann, M., Weis, J., Eggermann, T., Begemann, M., Roos, A., Häusler, M., Schedl, T., Tartaglia, M., Bremer, J., Pak, S. C., Frydman, J., Elbracht, M., Kurth, I. 2024; 386 (6721): 516-525

  Abstract

  Malformations of the brain are common and vary in severity, from negligible to potentially fatal. Their causes have not been fully elucidated. Here, we report pathogenic variants in the core protein-folding machinery TRiC/CCT in individuals with brain malformations, intellectual disability, and seizures. The chaperonin TRiC is an obligate hetero-oligomer, and we identify variants in seven of its eight subunits, all of which impair function or assembly through different mechanisms. Transcriptome and proteome analyses of patient-derived fibroblasts demonstrate the various consequences of TRiC impairment. The results reveal an unexpected and potentially widespread role for protein folding in the development of the central nervous system and define a disease spectrum of "TRiCopathies."

  View details for DOI 10.1126/science.adp8721

  View details for PubMedID 39480921

• Clinical validation of RCSMS: A rapid and sensitive CRISPR-Cas12a test for the molecular detection of SARS-CoV-2 from saliva. PloS one Abugattas-Núñez Del Prado, J., Quintana Reyes, A., Leon, J., Blume La Torre, J., Gutiérrez Loli, R., Pinzón Olejua, A., Chamorro Chirinos, E. R., Loza Mauricio, F. A., Maguiña, J. L., Rodriguez-Aliaga, P., Málaga-Trillo, E. 2024; 19 (3): e0290466

  Abstract

  Peru's holds the highest COVID death rate per capita worldwide. Key to this outcome is the lack of robust, rapid, and accurate molecular tests to circumvent the elevated costs and logistics of SARS-CoV-2 detection via RT-qPCR. To facilitate massive and timely COVID-19 testing in rural and socioeconomically deprived contexts, we implemented and validated RCSMS, a rapid and sensitive CRISPR-Cas12a test for the molecular detection of SARS-CoV-2 from saliva. RCSMS uses the power of CRISPR-Cas technology and lateral flow strips to easily visualize the presence of SARS-CoV-2 even in laboratories with limited equipment. We show that a low-cost thermochemical treatment with TCEP/EDTA is sufficient to inactivate viral particles and cellular nucleases in saliva, eliminating the need to extract viral RNA with commercial kits, as well as the cumbersome nasopharyngeal swab procedure and the requirement of biosafety level 2 laboratories for molecular analyses. Notably, RCSMS performed outstandingly in a clinical validation done with 352 patients from two hospitals in Lima, detecting as low as 50 viral copies per 10 μl reaction in 40 min, with sensitivity and specificity of 96.5% and 99.0%, respectively, relative to RT-qPCR. The negative and positive predicted values obtained from this field validation indicate that RCSMS can be confidently deployed in both high and low prevalence settings. Like other CRISPR-Cas-based biosensors, RCSMS can be easily reprogrammed for the detection of new SARS-CoV-2 variants. We conclude that RCSMS is a fast, efficient and inexpensive alternative to RT-qPCR for expanding COVID-19 testing capacity in Peru and other low- and middle-income countries with precarious healthcare systems.

  View details for DOI 10.1371/journal.pone.0290466

  View details for PubMedID 38527001

• Diversity, Equity and Inclusion in the Laboratory: Strategies to Enhance Inclusive Laboratory Culture. Molecular cell Marshall, A. G., Vue, Z., Beasley, H. K., Neikirk, K., Stephens, D., Wanjalla, C. N., Damo, S. M., Trejo, J., Rodriguez-Aliaga, P., Headley, C. A., Shuler, H., Liu, K., Smith, N., Garza-Lopez, E., Barongan, T., Scudese, E., Spencer, E., Heemstra, J., Vazquez, A. D., Murray, S. A., Hinton, A. J. 2023; 83 (21): 3766-3772

  Abstract

  Building a diverse laboratory that is equitable is critical for the retention of talent and the growth of trainees professionally and personally. Here, we outline several strategies including enhancing understanding of cultural competency and humility, establishing laboratory values, and developing equitable laboratory structures to create an inclusive laboratory environment to enable trainees to achieve their highest success.

  View details for DOI 10.1016/j.molcel.2023.09.011

  View details for PubMedID 37922871

• A Practical Guide to Graduate School Interviewing for Historically Excluded Individuals. American journal of physiology. Heart and circulatory physiology Ransey, E., Brookens, S., Beasley, H. K., Marshall, A., Marlin, B. J., Rodriguez-Aliaga, P., Headley, C. A., Wanjalla, C., Vazquez, A. D., Murray, S., Damo, S., Taabazuing, C. Y., Hinton, A. J. 2023

  View details for DOI 10.1152/ajpheart.00123.2023

  View details for PubMedID 37027327

• Dissecting the structural basis of Huntingtin pathogenesis: one molecule at the time Rodriguez-Aliaga, P., Sosa, R. P., Bustamante, C., Frydman, J. CELL PRESS. 2022: 22

  View details for Web of Science ID 000759523000103

• Knots can impair protein degradation by ATP-dependent proteases. Proceedings of the National Academy of Sciences of the United States of America San Martín, Á. n., Rodriguez-Aliaga, P. n., Molina, J. A., Martin, A. n., Bustamante, C. n., Baez, M. n. 2017; 114 (37): 9864–69

  Abstract

  ATP-dependent proteases translocate proteins through a narrow pore for their controlled destruction. However, how a protein substrate containing a knotted topology affects this process remains unknown. Here, we characterized the effects of the trefoil-knotted protein MJ0366 from Methanocaldococcus jannaschii on the operation of the ClpXP protease from Escherichia coli ClpXP completely degrades MJ0366 when pulling from the C-terminal ssrA-tag. However, when a GFP moiety is appended to the N terminus of MJ0366, ClpXP releases intact GFP with a 47-residue tail. The extended length of this tail suggests that ClpXP tightens the trefoil knot against GFP, which prevents GFP unfolding. Interestingly, if the linker between the knot core of MJ0366 and GFP is longer than 36 residues, ClpXP tightens and translocates the knot before it reaches GFP, enabling the complete unfolding and degradation of the substrate. These observations suggest that a knot-induced stall during degradation of multidomain proteins by AAA proteases may constitute a novel mechanism to produce partially degraded products with potentially new functions.

  View details for DOI 10.1073/pnas.1705916114

  View details for PubMedID 28847957

  View details for PubMedCentralID PMC5604015

• Substrate-translocating loops regulate mechanochemical coupling and power production in AAA plus protease C1pXP NATURE STRUCTURAL & MOLECULAR BIOLOGY Rodriguez-Aliaga, P., Ramirez, L., Kim, F., Bustamante, C., Martin, A. 2016; 23 (11): 974-981

  Abstract

  ATP-dependent proteases of the AAA+ family, including Escherichia coli ClpXP and the eukaryotic proteasome, contribute to maintenance of cellular proteostasis. ClpXP unfolds and translocates substrates into an internal degradation chamber, using cycles of alternating dwell and burst phases. The ClpX motor performs chemical transformations during the dwell and translocates the substrate in increments of 1-4 nm during the burst, but the processes occurring during these phases remain unknown. Here we characterized the complete mechanochemical cycle of ClpXP, showing that ADP release and ATP binding occur nonsequentially during the dwell, whereas ATP hydrolysis and phosphate release occur during the burst. The highly conserved translocating loops within the ClpX pore are optimized to maximize motor power generation, the coupling between chemical and mechanical tasks, and the efficiency of protein processing. Conformational resetting of these loops between consecutive bursts appears to determine ADP release from individual ATPase subunits and the overall duration of the motor's cycle.

  View details for DOI 10.1038/nsmb.3298

  View details for Web of Science ID 000386992700007

  View details for PubMedID 27669037

• Key Roles of Translocating Loops in the Mechanochemical Coupling and Power Production of a AAA(+) Protease Machine Rodriguez-Aliaga, P., Ramirez, L., Kim, F., Bustamante, C., Martin, A. CELL PRESS. 2016: 390A

  View details for DOI 10.1016/j.bpj.2015.11.2109

  View details for Web of Science ID 000375142200396

• New insights into the regulatory mechanisms of ppGpp and DksA on Escherichia coli RNA polymerase–promoter complex Nucleic Acids Research Doniselli*, N., Rodriguez-Aliaga*, P., Amidani, D., Bardales, J. A., Bustamante, C., Guerra, D. G., Rivetti, C. 2015; 43 (10): 5249–5262

  Abstract

  The stringent response modulators, guanosine tetraphosphate (ppGpp) and protein DksA, bind RNA polymerase (RNAP) and regulate gene expression to adapt bacteria to different environmental conditions. Here, we use Atomic Force Microscopy and in vitro transcription assays to study the effects of these modulators on the conformation and stability of the open promoter complex (RPo) formed at the rrnA P1, rrnB P1, its discriminator (dis) variant and λ pR promoters. In the absence of modulators, RPo formed at these promoters show different extents of DNA wrapping which correlate with the position of UP elements. Addition of the modulators affects both DNA wrapping and RPo stability in a promoter-dependent manner. Overall, the results obtained under different conditions of ppGpp, DksA and initiating nucleotides (iNTPs) indicate that ppGpp allosterically prevents the conformational changes associated with an extended DNA wrapping that leads to RPo stabilization, while DksA interferes directly with nucleotide positioning into the RNAP active site. At the iNTPs-sensitive rRNA promoters ppGpp and DksA display an independent inhibitory effect, while at the iNTPs-insensitive pR promoter DksA reduces the effect of ppGpp in accordance with their antagonistic role.

  View details for DOI 10.1093/nar/gkv391

  View details for PubMedCentralID PMC4446441

• Protein denaturation at a single-molecule level: the effect of nonpolar environments and its implications on the unfolding mechanism by proteases. Nanoscale Cheng, B., Wu, S., Liu, S., Rodriguez-Aliaga, P., Yu, J., Cui, S. 2015; 7 (7): 2970-7

  Abstract

  Most proteins are typically folded into predetermined three-dimensional structures in the aqueous cellular environment. However, proteins can be exposed to a nonpolar environment under certain conditions, such as inside the central cavity of chaperones and unfoldases during protein degradation. It remains unclear how folded proteins behave when moved from an aqueous solvent to a nonpolar one. Here, we employed single-molecule atomic force microscopy and molecular dynamics (MD) simulations to investigate the structural and mechanical variations of a polyprotein, I278, during the change from a polar to a nonpolar environment. We found that the polyprotein was unfolded into an unstructured polypeptide spontaneously when pulled into nonpolar solvents. This finding was corroborated by MD simulations where I27 was dragged from water into a nonpolar solvent, revealing details of the unfolding process at the water/nonpolar solvent interface. These results highlight the importance of water in maintaining folding stability, and provide insights into the response of folded proteins to local hydrophobic environments.

  View details for DOI 10.1039/c4nr07140a

  View details for PubMedID 25597693

• The ClpXP Protease Unfolds Substrates Using a Constant Rate of Pulling but Different Gears CELL Sen, M., Maillard, R. A., Nyquist, K., Rodriguez-Aliaga, P., Presse, S., Martin, A., Bustamante, C. 2013; 155 (3): 636-646

  Abstract

  ATP-dependent proteases are vital to maintain cellular protein homeostasis. Here, we study the mechanisms of force generation and intersubunit coordination in the ClpXP protease from E. coli to understand how these machines couple ATP hydrolysis to mechanical protein unfolding. Single-molecule analyses reveal that phosphate release is the force-generating step in the ATP-hydrolysis cycle and that ClpXP translocates substrate polypeptides in bursts resulting from highly coordinated conformational changes in two to four ATPase subunits. ClpXP must use its maximum successive firing capacity of four subunits to unfold stable substrates like GFP. The average dwell duration between individual bursts of translocation is constant, regardless of the number of translocating subunits, implying that ClpXP operates with constant "rpm" but uses different "gears."

  View details for DOI 10.1016/j.cell.2013.09.022

  View details for Web of Science ID 000326571800017

  View details for PubMedID 24243020