Biology subjects

Mancilla, H.

Publications and source records attributed to Mancilla, H..

4 recordsLinked to original sources

bioRxiv2026

The compartmentalized activity of a legume subtilase SBT12a allows symbiosome stabilization

The stabilization of rhizobia in specialized organelles, called symbiosomes, is an evolutionary hallmark for maintaining high rates of nitrogen fixation in legumes. This is achieved by releasing thousands of bacteria from nodular infection threads within in a single plant cell, which poses a great challenge for the host to keep control over these differentiated bacteria. Considering the importance of proteolytic degradation of antimicrobial proteins or generation of symbiosis-promoting peptides, proteolytic activity may represent a key regulatory system. Indeed, we identified the Medicago truncatula subtilisin-like protease (subtilase, SBT) 12a acting as a novel regulator of symbiosome functionality and maintenance. Loss-of-function mutations in SBT12a led to severe symbiotic defects with nodules of sbt12a being characterized by high level induction of defense/senescence-related genes. Using untargeted proteomic High-efficiency Undecanal-based N-Termini EnRichment (HUNTER) we identified and individually confirmed specific SBT12a target proteins that are involved in plant defense responses and symbiosome maintenance. This positions SBT12a as a central host factor controlling symbiosome performance.

plant biology↗

bioRxiv2026

Biomolecular condensate architecture of an autophagic cargo at molecular resolution in situ

Biomolecular condensates organise cellular biochemistry, yet their molecular architecture in situ remains poorly understood. During selective autophagy, macromolecules frequently accumulate into biomolecular condensates, forming discrete entities for autophagic engulfment and degradation - ideal systems for structural analysis. We employed in situ cryo-electron tomography to determine the near-atomic resolution structure of Aminopeptidase 1 condensates within cells. These condensates form densely packed, spherical assemblies with amorphous organisation and liquid-like properties, elucidating the requirements of a selective autophagic cargo for exclusive targeting. Structural analysis and multiscale simulations reveal that the short, transient -helical structures in the disordered N-terminus of Aminopeptidase 1 enable site-specific, coiled-coil-like interactions required for condensate formation and properties. A single point mutation that increases -helical propensity directly modulates condensate viscosity and dynamics from a liquid-like to a glass-like state, while preserving local molecular packing. Our results demonstrate that disordered regions encode both specificity and material properties through transiently structured motifs, linking sequence specificity to phase behaviour in cells and expanding the molecular logic of phase separation in cells.

cell biology↗

bioRxiv2024

Codon Pair-Specific Translation Defects Trigger Ribosome-Associated Quality Control to Avoid Proteotoxic Stress

tRNA modifications tune translation rates and codon optimality, thereby optimizing co-translational protein folding, but how codon optimality defects trigger cellular phenotypes remains unclear. Here, we show that ribosomes stall at specific modification-dependent codon pairs, triggering ribosome collisions and inducing a coordinated and hierarchical response of cellular quality control pathways. Ribosome profiling reveals an unexpected functional diversity for wobble-uridine (U34) modifications during decoding. The same modification can have different effects at the A and P sites. Furthermore, modification-dependent stalling codon pairs induce ribosome collisions, triggering ribosome-associated quality control (RQC) to prevent protein aggregation by degrading aberrant nascent peptides and mRNAs. RQC inactivation stimulates the expression of molecular chaperones to remove protein aggregates. Our results show that loss of tRNA modifications primarily disrupts translation rates of suboptimal codon pairs and reveal the coordinated regulation and adaptability of cellular surveillance systems to ensure efficient and accurate protein synthesis and maintain protein homeostasis.

biochemistry↗

bioRxiv2020

Fast isolation of sub-nanomolar affinity alpaca nanobody against the Spike RBD of SARS-CoV-2 by combining bacterial display and a simple single-step density gradient selection

Despite unprecedented global efforts to rapidly develop SARS-CoV-2 treatments, in order to reduce the burden placed on health systems, the situation remains critical. Effective diagnosis, treatment, and prophylactic measures are urgently required to meet global demand: recombinant antibodies fulfill these requirements and have marked clinical potential. Here, we describe the fast-tracked development of an alpaca Nanobody specific for the receptor-binding-domain (RBD) of the SARS-CoV-2 Spike protein with therapeutic potential applicability. We present a rapid method for nanobody isolation that includes an optimized immunization regimen coupled with VHH library E. coli surface display, which allows single-step selection of high-affinity nanobodies using a simple density gradient centrifugation of the bacterial library. The selected single and monomeric Nanobody, W25, binds to the SARS-CoV-2 S RBD with sub-nanomolar affinity and efficiently competes with ACE-2 receptor binding. Furthermore, W25 potently neutralizes SARS-CoV-2 wild type and the D614G variant with IC50 values in the nanomolar range, demonstrating its potential as antiviral agent.

molecular biology↗