Biology subjects

Micheletti, C.

Publications and source records attributed to Micheletti, C..

10 recordsLinked to original sources

bioRxiv2026

Pufferfish inflation: drivers of axial and hoop skin strain and regional variation across the body

Pufferfish skin undergoes large multi-axial extensions during defensive inflation behaviour, as water is pumped into the stomach and the fish increases its body volume threefold. This remarkable capacity for deformation thus raises questions on how skin extension is spatially distributed across the body, and how skin extension relates to changes in body volume and stomach pressure. However, directly measuring skin extension remains experimentally challenging, particularly when attempting to preserve the bodys complex 3D geometry and natural multiaxial strain during ex vivo testing. We used biplanar x-ray videography to measure skin strain (fluoromicrometry) and body volume (dynamic digital endocast) simultaneously with stomach pressure recordings during natural and artificial inflations in porcupinefish ( Diodon holocanthus ). We found no significant difference in maximum body volumes (2.66 {+/-} 0.65 times their initial body volume), body dimensions (e.g., dorsoventral height 87.31 {+/-} 0.23 mm), peak pressures (2.45 {+/-} 1.10 kPa), and skin extension at maximum inflation between natural and artificial inflation conditions. In all inflations, volume increased steadily while stomach pressure only increased during the last 25-50% of inflation. Skin extension varied biaxially, with mean strains reaching up to 102% in the axial direction and 146% in the hoop direction as the fish became nearly spherical. The greatest skin extensions occurred most dorsocranially (axial) and ventrolaterally (hoop). Strains in the hoop direction were only significantly different between body regions (defined as dorsal, lateral and ventral), with the greatest hoop strains occurring in the ventral body region. Our findings may provide insight into existing hypotheses regarding the relationship between volume, pressure, and skin structure and behaviour, while also providing direct in vivo measurements to inform biomimetic design.

zoology↗

bioRxiv2024

Mapping Cryptic Phosphorylation Sites in the Human Proteome

Advances in computational and experimental methods have revealed the existence of transient, non-native protein folding intermediates that could play roles in disparate biological processes, from regulation of protein expression to disease-relevant misfolding mechanisms. Here, we tested the possibility that specific post-translational modifications may involve residues exposed during the folding process by assessing the solvent accessibility of 87,138 post-translationally modified amino acids in the human proteome. Unexpectedly, we found that one-third of phosphorylated proteins present at least one phosphosite completely buried within the proteins inner core. Computational and experimental analyses suggest that these cryptic phosphosites may become exposed during the folding process, where their modification could destabilize native structures and trigger protein degradation. Phylogenetic investigation also reveals that cryptic phosphosites are more conserved than surface-exposed phosphorylated residues. Finally, cross-referencing with cancer mutation databases suggests that phosphomimetic mutations in cryptic phosphosites can increase tumor fitness by inactivating specific onco-suppressors. These findings define a novel role for co-translational phosphorylation in shaping protein folding and expression, laying the groundwork for exploring the implications of cryptic phosphosites in health and disease.

biochemistry↗

bioRxiv2023

Shedding light (... electrons) on human bone ultrastructure with correlative on-axis electron tomography and energy dispersive X-ray spectroscopy tomography

Mineralized collagen fibrils are the building block units of bone at the nanoscale. While it is known that collagen fibrils are mineralized both inside their gap zones (intra-fibrillar mineralization) and on their outer surfaces (extra-fibrillar mineralization), a clear visualization of this architecture in three dimensions (3D), combining structural and compositional information over large volumes, but without compromising the resolution, remains challenging. In this study, we demonstrate the use of on-axis Z-contrast electron tomography (ET) with correlative energy-dispersive X-ray spectroscopy (EDX) tomography to examine rod-shaped samples with a diameter up to 700 nm prepared from individual osteonal lamellae in the human femur. Our work mainly focuses on two aspects: i) low-contrast nanosized circular spaces ("holes") observed in sections of bone oriented perpendicular to the long axis of a long bone; and ii) extra-fibrillar mineral, especially in terms of morphology and spatial relationship with respect to intra-fibrillar mineral and collagen fibrils. From our analyses, it emerges quite clearly that most "holes" are cross-sectional views of collagen fibrils. While this had been postulated before, our 3D reconstructions and reslicing along meaningful two-dimensional (2D) cross-sections provide a direct visual confirmation. Extra-fibrillar mineral appears composed of thin plates that are interconnected and span over several collagen fibrils, confirming that mineralization is cross-fibrillar, at least for the extra-fibrillar phase. EDX tomography shows mineral signature (Ca and P) within the gap zones, but the signal appears weaker than that associated to the extra-fibrillar mineral, pointing towards the existence of dissimilarities between the two types of mineralization.

bioengineering↗

bioRxiv2021

Unfolding and Translocation of Knotted Proteins by Clp Biological Nanomachines: Synergistic Contribution of Primary Sequence and Topology Revealed by Molecular Dynamics Simulations

We use Langevin dynamics simulations to model, at atomistic resolution, how various natively-knotted proteins are unfolded in repeated allosteric translocating cycles of the ClpY ATPase. We consider proteins representative of different topologies, from the simplest knot (trefoil 31), to the three-twist 52 knot, to the most complex stevedore, 61, knot. We harness the atomistic detail of the simulations to address aspects that have so far remained largely unexplored, such as sequence-dependent effects on the ruggedness of the landscape traversed during knot sliding. Our simulations reveal the combined effect on translocation of the knotted protein structure, i.e. backbone topology and geometry, and primary sequence, i.e. side chain size and interactions, and show that the latter can even dominate translocation hindrance. In addition, we observe that, due to the interplay between the knotted topology and intramolecular contacts, the transmission of tension along the peptide chain occurs very differently from homopolymers. Finally, by considering native and non-native interactions, we examine how the disruption or formation of such contacts can affect the translocation processivity and concomitantly create multiple unfolding pathways with very different activation barriers.

biophysics↗

bioRxiv2021

RNA Pore Translocation with Static and Periodic Forces: Effect of Secondary and Tertiary Elements on Process Activation and Duration

We use MD simulations to study the pore translocation properties of a pseudoknotted viral RNA. We consider the 71-nucleotide long xrRNA from Zika virus and establish how it responds when driven through a narrow pore by static or periodic forces applied to either one of the two termini. Unlike the case of fluctuating homopolymers, the onset of translocation is significantly delayed with respect to the application of static driving forces. Because of the peculiar xrRNA architecture, activation times can differ by orders of magnitude at the two ends. Instead, translocation duration is much smaller than activation times and occurs on timescales comparable at the two ends. Periodic forces amplify significantly the differences at the two ends, both for activation times and translocation duration. Finally, we use a waiting-times analysis to examine the systematic slowing-downs in xrRNA translocations and associate them to the hindrance of specific secondary and tertiary elements of xrRNA. The findings ought to be useful as a reference to interpret and design future theoretical and experimental studies of RNA translocation.

biophysics↗

bioRxiv2020

Directional translocation resistance of Zika xrRNA

xrRNAs from flaviviruses survive in host cells for their exceptional dichotomic response to the unfolding action of different enzymes. They can be unwound, and hence copied, by replicases, and yet can resist degradation by exonucleases. How the same stretch of xrRNA can encode such diverse responses is an open question. Here, by using atomistic models and translocation simulations, we uncover an elaborate and directional mechanism for how stress propagates when the two xrRNA ends, 5' and 3', are driven through a pore. Pulling the 3' end, as done by replicases, elicits a progressive unfolding; pulling the 5' end, as done by exonucleases, triggers a counterintuitive molecular tightening. Thus, in what appears to be a remarkable instance of intra-molecular tensegrity, the very pulling of the 5' end is what boosts resistance to translocation and consequently to degradation. The uncovered mechanistic principle might be co-opted to design molecular meta-materials.

biophysics↗

bioRxiv2018

Dynamics of supercoiled DNA with complex knots: large-scale rearrangements and persistent multi-strand interlocking

Knots and supercoiling are both introduced in bacterial plasmids by catalytic processes involving DNA strand passages. While the effects on plasmid organization has been extensively studied for knotting and supercoiling taken separately, much less is known about their concurrent action. Here, we use molecular dynamics simulations and oxDNA, an accurate mesoscopic DNA model, to study the kinetic and metric changes introduced by complex (five-crossing) knots and supercoiling in 2kbp-long DNA rings. We find several unexpected results. First, the conformational ensemble is dominated by two distinct states, differing in branchedness and knot size. Secondly, fluctuations between these states are as fast as the metric relaxation of unknotted rings. In spite of this, certain boundaries of knotted and plectonemically-wound regions can persist over much longer timescales. These pinned regions involve multiple strands that are interlocked by the cooperative action of topological and supercoiling constraints. Their long-lived character may be relevant for the simplifying action of topoisomerases.

biophysics↗

bioRxiv2017

From Sequence to Function: Coevolving Amino Acids Encode Structural and Functional Domains

Amino acids interactions within protein families are so optimized that the sole analysis of evolutionary co-mutations can identify pairs of contacting residues. It is also known that evolution conserves functional dynamics, i.e., the concerted motion or displacement of large protein regions or domains. Is it, therefore, possible to use a pure sequence-based analysis to identify these dynamical domains? To address this question, we introduce here a general co-evolutionary coupling analysis strategy and apply it to a curated sequence database of hundreds of protein families. For most families, the sequence-based method partitions amino acids into few clusters. When viewed in the context of the native structure, these clusters have the signature characteristics of viable protein domains: they are spatially separated but individually compact. They have a direct functional bearings too, as shown for various reference cases. We conclude that even large-scale structural and functionally-related properties can be recovered from inference methods applied to evolutionary-related sequences. The method introduced here is available as a software package and web server (http://spectrus.sissa.it/spectrus-evo_webserver).

bioinformatics↗

bioRxiv2016

How to fold intricately: using theory and experiments to unravel the properties of knotted proteins

Over the years, advances in experimental and computational methods have helped us to understand the role of thermodynamic, kinetic and active (chaperone-aided) effects in coordinating the folding steps required to achieving a knotted native state. Here, we review such developments by paying particular attention to the complementarity of experimental and computational studies. Key open issues that could be tackled with either or both approaches are finally pointed out.

biophysics↗

bioRxiv2016

Hi-C-constrained physical models of human chromosomes recover functionally-related properties of genome organization

Combining genome-wide structural models with phenomenological data is at the forefront of efforts to understand the organizational principles regulating the human genome. Here, we use chromosome-chromosome contact data as knowledge- based constraints for large-scale three-dimensional models of the human diploid genome. The resulting models remain minimally entangled and acquire several functional features that are observed in vivo and that were never used as input for the model. We find, for instance, that gene-rich, active regions are drawn towards the nuclear center, while gene poor and lamina-associated domains are pushed to the periphery. These and other properties persist upon adding local contact constraints, suggesting their compatibility with non-local constraints for the genome organization. The results show that suitable combinations of data analysis and physical modelling can expose the unexpectedly rich functionally-related properties implicit in chromosome-chromosome contact data. Specific directions are suggested for further developments based on combining experimental data analysis and genomic structural modelling.

biophysics↗