Biology subjects

Velazquez-Campoy, A.

Publications and source records attributed to Velazquez-Campoy, A..

11 recordsLinked to original sources

bioRxiv2026

Interlaboratory benchmarking of differential scanning fluorimetry reveals sources of variability in protein thermal stability parameters

Differential scanning fluorimetry (DSF) is widely used to assess protein thermal stability and to compare protein variants, ligands, or formulations with limited sample consumption. Yet, the reproducibility of DSF derived parameters across laboratories, instruments and analysis workflows remains poorly defined. Here, we report a two phase interlaboratory benchmark using a shared VHH sample across 26 laboratories, 28 instruments and seven DSF instrument models. In Phase 1, participants applied their routine acquisition and analysis workflows, whereas in Phase 2, acquisition conditions were harmonised. Datasets were analysed locally and then centrally reprocessed with MoltenProt. Participant reported apparent melting temperatures (Tm) showed good within instrument repeatability but moderate inter instrument dispersion. Harmonisation of acquisition conditions did not substantially reduce this dispersion. Conversely, centralised reprocessing narrowed the distribution among retained datasets, indicating contributions from analysis workflows and data acceptance criteria in addition to instrument dependent factors. Comparison with differential scanning calorimetry (DSC) further showed that apparent Tm depended strongly on the fluorescence readout. For this VHH, single wavelength fluorescence at 350 nm showed the closest agreement with the DSC transition temperature, whereas the 350/330 nm ratio and barycentric mean generally yielded higher values. Furthermore, DSF derived apparent unfolding enthalpies were more variable than apparent Tm. Together, these findings show that DSF can be highly repeatable within a defined instrument and workflow, but that quantitative comparability across instruments depends on instrument specific effects, the choice of fluorescence readout and analysis strategy. Improved comparability will require transparent reporting of acquisition and analysis conditions, explicit data quality criteria, and well characterised reference materials to monitor instrument dependent offsets.

biophysics↗

bioRxiv2026

Functional plasticity of AIF revealed by dimerization and CHCHD4 interaction states

Apoptosis-inducing factor is a mitochondrial flavoprotein that links redox metabolism to mitochondrial homeostasis through its interaction with the disulfide relay protein CHCHD4. Although NADH-dependent AIF dimerization has been proposed as the activated state mediating CHCHD4 engagement, whether it is strictly required for productive AIF-CHCHD4 function remains unclear. Here, combining cellular, biochemical and biophysical approaches, we show that disruption of the AIF dimer interface compromises oxidative phosphorylation, respiratory-chain organization and CHCHD4-dependent mitochondrial homeostasis, yet preserves partial AIF function. Our data reveal that the AIF-CHCHD4 system operates as a conformational dynamic redox module in which distinct AIF oligomeric and redox states sustain CHCHD4 activity with different efficiencies. Mechanistically, dimerization is coupled to NADH-dependent conformational changes that regulate coenzyme binding, charge-transfer complex stabilization and catalytic efficiency. In turn, CHCHD4 binding remodels AIF conformational and redox properties, partially compensating for defects in dimer stabilization or redox coupling. Consistently, a peptide derived from the CHCHD4 N-terminus partially restores redox function in a pathogenic AIF variant defective in dimer stabilization, supporting partner-assisted allosteric regulation as a potential therapeutic strategy.

biochemistry↗

bioRxiv2025

Dissecting Annexin-A11 into its functional domains revealed calcium as a key regulator for RNA transport and its association with ALS

Recent studies reveal a "hitchhiking" mechanism in neurons, where organelles transported along microtubules carry other cargos via tethering molecules. Annexin A11 (ANXA11), a calcium-dependent phospholipid-binding protein, functions as a tether linking RNA granules to lysosomes, aiding mRNA transport for rapid neuronal responses. Structurally, its N-terminal (Nt) binds RNA, while the C-terminal (Ct) associates with lysosomal membranes. Mutations in ANXA11 linked to Amyotrophic lateral sclerosis (ALS) may disrupt this function. Here, applying a multidisciplinary approach, we revealed that Ca2+ acts as a master regulator of ANXAlls physiological function by modulating its conformational states. Specifically, Ca2+ influences a switch between two conformations: a close state, in which the Nt and Ct interact with each other, and an open state, which occurs in the presence of Ca2+ ions, where this self-interaction is disrupted, allowing the two domains to interact freely with RNA and liposomes. Surprisingly, we observed that both the Ct and Nt are capable of interacting with liposomes and RNA in a Ca2+-dependent manner, and these interactions can occur simultaneously. This dual binding and its calcium-regulated hierarchy finely tunes ANXAlls binding to RNA and lysosomes, promoting a large complex essential for overcoming transport steric hindrance. Moreover, our result showed that the p.D40G mutation, in the Nt domain, associated with ALS, displays destabilized interdomain interactions and bypass Ca2+ regulation, leading to aberrant aggregation. These insights advance our understanding of ANXAlls role in neuronal RNA transport and its disruption in neurodegeneration, highlighting potential targets for therapeutic intervention.

biochemistry↗

bioRxiv2025

Characterization of Mycobacterium tuberculosis rhodanese-like sulfurtransferase SseA and its newly identified activator Rv3284: A new pathway to exploit as a drug target?

Tuberculosis (TB) remains a critical global health challenge, with Mycobacterium tuberculosis (Mtb) causing 10.8 million cases and 1.25 million deaths in 2023. Key issues include the dormant state of Mtb, resistant to drugs and immune responses, and the emergence of multi-drug-resistant strains. This underscores the need for new therapeutic targets and deeper research into Mtb pathogenesis and immunology. A potential drug target is the enzyme thiosulfate-sulfurtransferase SseA, which plays a role in macrophage infection by Mtb and its resistance to oxidative stress. SseA belongs to the rhodanese-like enzyme family, which catalyzes sulfur transfer reactions essential for Mtb survival. In our research, we identified a new protein (Rv3284), hereinafter referred to as SufEMtb due to its high homology with E. coli SufE, that interacts with SseA and modulates its activity. Sequence analysis and AI molecular modelling revealed detailed insights into their interaction that can contribute to the modulation of SseA activity. This research provides a mechanistic explanation to the need of a partner for SseA activation. Indeed, we propose that SufEMtb enhances SseA enzymatic function by binding to its non-catalytic N-terminal domain and bringing the active sites of the two proteins in close proximity, thus preparing for the activation-enhancing conformational change in a regulatory loop of SseA. This interaction is crucial for the effective enzyme activity and the maintenance of redox homeostasis in Mtb, making the SseA-SufEMtb protein complex a potential target for new TB therapies.

biochemistry↗

bioRxiv2023

Small protein mediates inhibition of ammonium transport in Methanosarcina mazei - an ancient mechanism?

In the past decade, small open reading frames (sORFs) coding for proteins less than 70 amino acids (aa) in length have moved into the focus of Science. sORFs and corresponding small proteins have been recently identified in all three domains of life. However, the majority of small proteins remain functionally uncharacterized. While several bacterial small proteins have already been described, the number of identified and functionally characterized small proteins in archaea is still limited. In this study, we have discovered that the small protein 36 (sP36), which consists of only 61 aa, plays a critical role in regulating nitrogen metabolism in Methanosarcina mazei. The absence of sP36 significantly delays the growth of M. mazei when transitioning from nitrogen limitation to nitrogen sufficiency, as compared to the wild type. Through our in vivo experiments, we have observed that during nitrogen limitation, sP36 is dispersed throughout the cytoplasm; however, upon shifting the cells to nitrogen sufficiency, it relocates to the cytoplasmic membrane. Moreover, in vitro biochemical analysis clearly showed that sP36 interacts with high-affinity with the ammonium transporter AmtB1 present in the cytoplasmic membrane during nitrogen limitation, as well as with the PII-like protein GlnK1. Based on our findings, we propose that in response to an ammonium up-shift, sP36 targets the ammonium transporter AmtB1 and inhibits its activity by mediating the interaction with GlnK1. ImportanceSmall proteins containing fewer than 70 aa, which were previously disregarded due to computational prediction and biochemical detection challenges, have gained increased attention in the scientific community in recent years. However, the number of functionally characterized small proteins, especially in archaea, is still limited. Here, by using biochemical and genetic approaches, we demonstrate a crucial role for the small protein sP36 in the nitrogen metabolism of M. mazei, regulating the ammonium transporter AmtB1 according to nitrogen availability. This regulation might represent an ancient archaeal mechanism of AmtB1 inhibition by GlnK, in contrast to the well-studied regulation in bacteria, which depends on covalent modification of GlnK.

molecular biology↗

bioRxiv2022

Allosteric feedback inhibition of deoxy-D-xylulose-5-phosphate synthase involves monomerization of the active dimer.

Isoprenoids are a very large and diverse family of metabolites required by all living organisms. All isoprenoids derive from the double-bond isomers isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are produced by the methylerythritol 4-phosphate (MEP) pathway in bacteria and plant plastids. Understanding the regulation of the MEP pathway, probably the main metabolic pathway elucidated in this century, is a must for the rational design of biotechnological endeavors aimed at increasing isoprenoid contents in microbial and plant systems. It has been reported that IPP and DMAPP feedback regulate the activity of deoxyxylulose 5-phosphate (DXS), a dimeric enzyme catalyzing the main flux-controlling step of the MEP pathway. Here we provide experimental insights on the underlying mechanism. Our data show that direct allosteric binding of IPP and DMAPP to bacterial and plant DXS promotes monomerization of the enzyme. This allows a fast response to a sudden increase or decrease in IPP/DMAPP supply by rapidly shifting the dimer-monomer equilibrium accordingly. DXS monomers expose hydrophobic domains that are hidden in the dimer, resulting in aggregation and eventual degradation. Removal of monomers that would otherwise be available for dimerization and enzyme reactivation appears as a more drastic response in case of persistent IPP/DMAPP overabundance (e.g., by a blockage in their conversion to downstream isoprenoids). Our model provides a mechanistic explanation of how IPP and DMAPP supply can be adapted to changes in their demand and it also explains the changes in DXS protein levels observed after long-term interference of the MEP pathway flux. Significance StatementIsoprenoids are a vast family of organic compounds with essential roles in respiration, photosynthesis, photoprotection, membrane structure, and signaling. Many of them have great economic and nutritional relevance as pigments, aromas, drugs or phytonutrients. Despite their functional and structural diversity, they all derive from the same five-carbon precursors. We show that these precursors feedback-regulate their own synthesis in bacteria and plant plastids by allosterically shifting the dimer:monomer equilibrium of the enzyme that catalyzes the first step of their biosynthetic pathway towards the inactive monomeric form. This evolutionary conserved mechanism allows for both short-term (immediate) and long-term (sustained) control of the pathway flux, and its manipulation could be critical for the rational engineering of high-value isoprenoid products in bacterial and plant systems.

biochemistry↗

bioRxiv2020

Tyrosinase from mushroom Agaricus bisporus as an inhibitor of the Hepatitis C virus

Tyrosinases from both a commercial semi-purified Agaricus bisporus protein extract and directly isolated from white mushroom have been demonstrated to show antiviral activity against the Hepatitis C virus for the first time. The well-known tyrosinase from A. bisporus (TyrAB) of 45kDa and a newly discovered 50-kDa isoform from this tyrosinase (Tyr50kDa) have been tested. Cell toxicity and antiviral activity of tyrosinases in cultured Huh 5-2 liver tumor cells transfected with a replicon system (a plasmid that includes all non-structural Hepatitis C virus proteins and replicates autonomously) was determined. Native TyrAB was able to inhibit the replication of the hepatitis C virus without inducing toxicity in liver cells. In addition, the post-translational isoform of Tyr50kDa showed higher antiviral capacity than the former (up to 10 times greater), also exhibiting 10 times higher activity than the commercial drug Ribavirin(R). This antiviral activity was directly proportional to the enzymatic activity of tyrosinases, since no antiviral capacity was observed for the inactive enzymes. The tyrosinases approach could represent a new antiviral inhibition mechanism, through a catalytic mechanism of selective hydroxylation of key role tyrosine residues in viral proteases. The tyrosinases directly extracted from fresh mushrooms (containing both tyrosinases) showed similar antiviral activity and, therefore, might provide low-cost drugs for the treatment of hepatitis C.

microbiology↗

bioRxiv2020

UMP inhibition and sequential firing in aspartate transcarbamoylase open ways to regulate plant growth

Pyrimidine nucleotides are essential to plant development. We proved that Arabidopsis growth can be inhibited or enhanced by down- or upregulating aspartate transcarbamoylase (ATC), the first committed enzyme for de novo biosynthesis of pyrimidines in plants. To understand the unique mechanism of feedback inhibition of this enzyme by uridine 5-monophosphate (UMP), we determined the crystal structure of the Arabidopsis ATC trimer free and bound to UMP, demonstrating that the nucleotide binds and blocks the active site. The regulatory mechanism relies on a loop exclusively conserved in plants, and a single-point mutation (F161A) turns ATC insensitive to UMP. Moreover, the structures in complex with a transition-state analog or with carbamoyl phosphate proved a mechanism in plant ATCs for sequential firing of the active sites. The disclosure of the unique regulatory and catalytic properties suggests new strategies to modulate ATC activity and to control de novo pyrimidine synthesis and plant growth.

plant biology↗

bioRxiv2020

Mitochondrial cytochrome c liberates the nucleophosmin-sequestered ARF tumor suppressor in the nucleolus

The alternative reading frame (ARF) protein is crucial in the cellular response to oncogenic stress, being likewise the second most frequently inactivated gene in a wide spectrum of human cancers. ARF is usually sequestered in the nucleolus by the well-known oncogenic nucleophosmin (NPM) protein and is liberated in response to cell damage to exhibit its tumor-suppressor ability. However, the mechanism underlying ARF activation is unknown. Here we show that mitochondria-to-nucleus translocation of cytochrome c upon DNA damage leads to the break-off of the NPM-ARF ensemble and subsequent release of ARF from the nucleoli. Our structural and subcellular data support a molecular model in which the hemeprotein triggers the extended-to-compact conformation of NPM and competes with ARF for binding to NPM.

biophysics↗

bioRxiv2019

Mechanism of the allosteric activation of the ClpP protease machinery by substrates and active-site inhibitors

Coordinated conformational transitions in oligomeric enzymatic complexes modulate function in response to substrates and play a crucial role in enzyme inhibition and activation. ClpP protease is a tetradecameric complex that has emerged as a drug target against multiple pathogenic bacteria. During drug development efforts, the activation of different ClpPs by inhibitors was independently reported, but so far, no rationale for inhibitor-induced activation has been proposed. Using an integrated approach that included X-ray crystallography, solid-and solution-state NMR, MD simulations and ITC we show that the proteasome-inhibitor bortezomib binds to the ClpP active site serine mimicking a peptide substrate and induces the concerted allosteric activation of the complex. The bortezomib activated conformation also displays a higher affinity for its cognate unfoldase ClpX. We propose a universal allosteric mechanism where substrate binding to a single subunit locks ClpP into an active conformation optimized for chaperone association as well as protein processive degradation.

biochemistry↗

bioRxiv2018

MeCP2-E1 isoform is a dynamically expressed, weakly DNA-bound protein with different protein and DNA interactions compared to MeCP2-E2

MeCP2 - a chromatin-binding protein associated with Rett syndrome - has two main isoforms, MeCP2-E1 and MeCP2-E2, with 96% amino acid identity differing in a few N-terminal amino acid residues. Previous studies have shown brain region-specific expression of these isoforms which, in addition to their different cellular localization and differential expression during brain development, suggest they may also have non-overlapping molecular mechanisms. However, differential functions of MeCP2-E1 and E2 remain largely unexplored. Here, we show that the N-terminal domains (NTD) of MeCP2-E1 and E2 modulate the ability of the methyl binding domain (MBD) to interact with DNA as well as influencing the turnover rates, binding dynamics, response to nuclear depolarization, and circadian oscillations of the two isoforms. Our proteomics data indicate that both isoforms exhibit unique interacting protein partners. Moreover, genome-wide analysis using ChIP-seq provide evidence for a shared as well as a specific regulation of different sets of genes. Our findings provide insight into the functional complexity of MeCP2 by dissecting differential aspects of its two isoforms.\n\nSignificanceWhether the two E1 and E2 isoforms of MeCP2 have different structural and/or functional implications has been highly controversial and is not well known. Here we show that the relatively short N-terminal sequence variation between the two isoforms impinges them with an important DNA binding difference. Moreover, MeCP2-E1 and E2 exhibit a different cellular dynamic behavior and have some distinctive interacting partners. In addition, while sharing genome occupancy they specifically bind to several distinctive genes.

biochemistry↗