Biology subjects

Garfagnini, T.

Publications and source records attributed to Garfagnini, T..

4 recordsLinked to original sources

bioRxiv2026

Molecular organization of the Chlorella sorokiniana pyrenoid

To overcome the enzymatic limitations of Rubisco, algae operate CO2-concentrating mechanisms (CCMs) that deliver concentrated CO2 to Rubisco tightly packaged in a specialized microcompartment called a pyrenoid. Pyrenoids are globally important biomolecular condensates, but their convergent evolution means that their molecular composition and emergent architecture cannot be inferred across clades. Here we characterize the pyrenoid of the Trebouxiophyceae alga, Chlorella sorokiniana. Using cryo-electron tomography, we provide an architectural overview of the pyrenoid and visualize pyrenoid-specific protein complexes. Quantitative proteomics and Rubisco co-immunoprecipitation followed by mass spectrometry demonstrate that inorganic carbon delivery machinery is conserved across green algae but the pyrenoid structural components are not. In vitro reconstitution supports the role of two previously undescribed proteins, one in assembly of pyrenoid traversing thylakoids (putative matrix thylakoid tether; PMTT) and another in starch tethering to the Rubisco matrix (putative matrix starch tether; PMST). In Nicotiana benthamiana, PMTT localized to the thylakoid stromal lamellae and PMST to chloroplast starch granules. Our findings provide insights into the molecular logic of pyrenoid assembly; how proteins mediate condensate-membrane and condensate-starch interactions; and expands the pyrenoid plant engineering toolkit, setting the stage for engineering a Chlorella pyrenoid into plants.

plant biology↗

bioRxiv2024

Fibril Paint: a class of amyloid-targeting peptides

Amyloid fibrils are a common pathological hallmark in multiple neurodegenerative diseases, yet molecular tools to selectively recognise and manipulate them remain scarce. We report FibrilPaints, a family of modular peptides designed for selective amyloid binding and adaptable chemical functionality. The degenerative amyloid-targeting unit of FibrilPaints, W5P4H3R2, has a high content of {pi}-stacking and aromatic side chains. Systematic sequence variation, altering charge, termini, and residue order, revealed the importance of the composition of the amyloid-targeting unit for high-affinity binding across Tau and Huntingtin fibrils. Importantly, sequence changes outside this unit do not preclude fibril binding, which permits attachment of fluorophores or E3-recruiting motifs for targeted protein degradation. This work establishes FibrilPaint as a modular peptide system for the detection and modulation of amyloids. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/609586v4_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1e9cadborg.highwire.dtl.DTLVardef@50f9eorg.highwire.dtl.DTLVardef@446ee8org.highwire.dtl.DTLVardef@cd47a2_HPS_FORMAT_FIGEXP M_FIG Figure of content The modular design of FibrilPaints enables systematic evaluation of their functionality by testing the binding capacity of each variant (FibrilPaintX) to distinct amyloid fibrils. Successful binding results in visible painting of the fibrils, facilitating their detection and downstream research. C_FIG

biochemistry↗

bioRxiv2023

Fibril Paint to detect Amyloids and determine Fibril Length

Tau aggregation into amyloid fibrils is linked to the development of neurodegenerative diseases, including Alzheimers Disease. The molecular processes underlying aggregation in disease are poorly understood. Here, we introduce FibrilPaint1 as a tool to measure the size of Tau amyloid fibrils in fluids, from early aggregation stages to mature fibrils. FibrilPaint1 is a 22mer peptide with many exciting properties, which makes it a tool for diagnostics and an attractive start point for developing a class of effective fibril targeting degraders: (i) FibrilPaint1 binds fibrils with nanomolar affinity; (ii) it does also bind to oligomeric precursors, down to a size of only 4 layers; (iii) it does not bind to monomers (KD > 100 {micro}M); (iv) it is fluorescently labelled, which allows monitoring and localising interactions. (v) FibrilPaint1 recognises various Tau fibrils, including patient derived fibrils from Alzheimer, Corticobasal degeneration and Frontotemporal dementia; (vi) FibrilPaint1 is selective for the amyloid state and does not have background binding to amorphous aggregates, blood serum or cell lysate. In combination with Flow Induced Dispersion Analysis (FIDA), a microfluidics technology, we determined the molecular size of amyloid fibrils with sub-microliter sample volumes. This set-up acts as a molecular ruler at layer resolution - we determined Tau fibril length from 4 to 1100 layers in solution. This is an interesting parameter that can be used for diagnostic applications and biochemical research in dementia.

biochemistry↗

bioRxiv2022

A peptide strategy for inhibiting different protein aggregation pathways in disease

Protein aggregation correlates with many human diseases. Protein aggregates differ in shape, ranging from amorphous aggregates to amyloid fibrils. Possibly for such heterogeneity, strategies to develop effective aggregation inhibitors that reach the clinic failed so far. Here, we present a new strategy by which we developed a family of peptides targeting early aggregation stages for both amorphous and fibrillar aggregates of proteins unrelated in sequence and structure. Thus, they act on dynamic precursors before a mechanistic differentiation takes place. Using a peptide array approach, we first identified peptides inhibiting the predominantly amorphous aggregation of a molten globular, aggregation-prone protein, a thermolabile mutant of the Axin tumor suppressor. A series of optimization steps revealed that the peptides activity did not depend on their sequences but rather on their molecular determinants. The key properties that made a peptide active were a composition of 20-30% flexible, 30-40% aliphatic and 20-30% aromatic residues, a hydrophobicity/hydrophilicity ratio close to 1 and an even distribution of residues of different nature throughout the sequence. Remarkably, the optimized peptides also suppressed fibrillation of Tau, a disordered protein that forms amyloids in Alzheimers disease, and entirely unrelated to Axin. Our compounds thus target early aggregation stages, independent of the aggregation mechanism, inhibiting both amorphous and amyloid aggregation. Such cross-mechanistic, multi-targeting aggregation inhibitors may be attractive lead compounds against multiple protein aggregation diseases.

biochemistry↗