Biology subjects

Caaveiro, J. M. M.

Publications and source records attributed to Caaveiro, J. M. M..

5 recordsLinked to original sources

bioRxiv2024

Malaria parasites require a divergent heme oxygenase for apicoplast gene expression and biogenesis

Malaria parasites have evolved unusual metabolic adaptations that specialize them for growth within heme-rich human erythrocytes. During blood-stage infection, Plasmodium falciparum parasites internalize and digest abundant host hemoglobin within the digestive vacuole. This massive catabolic process generates copious free heme, most of which is biomineralized into inert hemozoin. Parasites also express a divergent heme oxygenase (HO)-like protein (PfHO) that lacks key active-site residues and has lost canonical HO activity. The cellular role of this unusual protein that underpins its retention by parasites has been unknown. To unravel PfHO function, we first determined a 2.8 [A]-resolution X-ray structure that revealed a highly -helical fold indicative of distant HO homology. Localization studies unveiled PfHO targeting to the apicoplast organelle, where it is imported and undergoes N-terminal processing but retains most of the electropositive transit peptide. We observed that conditional knockdown of PfHO was lethal to parasites, which died from defective apicoplast biogenesis and impaired isoprenoid-precursor synthesis. Complementation and molecular-interaction studies revealed an essential role for the electropositive N-terminus of PfHO, which selectively associates with the apicoplast genome and enzymes involved in nucleic acid metabolism and gene expression. PfHO knockdown resulted in a specific deficiency in levels of apicoplast-encoded RNA but not DNA. These studies reveal an essential function for PfHO in apicoplast maintenance and suggest that Plasmodium repurposed the conserved HO scaffold from its canonical heme-degrading function in the ancestral chloroplast to fulfill a critical adaptive role in organelle gene expression.

biochemistry↗

bioRxiv2024

Characterization of high affinity IgM and IgG monoclonal antibodies against norovirus variants GII.4 and GII.17

Human norovirus, a leading cause of viral gastroenteritis, results in significant global health and economic burden, requiring sensitive and accurate diagnosis and effective therapeutics and vaccines. In this study, we immunized mice with the virus like capsid particles of GII.4, a mainstream strain, and GII.17, a modern strain that began to circulate in 2014, and used hybridoma technology to generate hybridoma cells that produce norovirus-binding antibodies against GII.4 and GII.17, respectively. Selection of these hybridoma cells yielded monoclonal IgG and IgM antibodies against these strains. Characterization of these antibodies revealed that avidity effect by multivalent binding is necessary for IgM to bind to norovirus at high efficiency, while IgG achieve high affinity even by monovalent binding. Surface plasmon resonance and ELISA data suggest that the high density of antigen protrusion domain in the norovirus capsid, containing approximately protomers, facilitates IgM to bind to norovirus capsid with high efficiency.

biochemistry↗

bioRxiv2024

Key role of P230 in the hinge region on the IgG architecture and function

Immunoglobulin G (IgG) is a molecule that plays an important role in biological defense; IgG molecules have been applied as drugs due to their high specificity for antigens and their ability to activate immunity via effector molecules on immune cells. On the other hand, the flexibility of the hinge region makes it difficult to apply conventional structural biology approaches due to its dynamic conformational changes, and the mechanism of action of the molecule as a whole has not been elucidated. Here, we introduced a deletion amino acid mutation in the hinge region to elucidate the role of the hinge region and its effect on the structure and function of the IgG molecule. Deletion of Pro230 resulted in the formation of a half-molecular in which the interaction between heavy chains was lost. We elucidated the mechanism of half-IgG formation by structural analysis using nuclear magnetic resonance (NMR) measurements and by disulfide quantification using peptide mapping using LC-MS/MS. For this purpose, a new NMR stable isotope labeling method was introduced. Finally, cell assay revealed that the IgG half-molecules have specific Fc{gamma}RI-mediated activity. This report provides new insights into the higher-order structure formation of IgG molecules and is expected to contribute to the elucidation of the molecular basis of the Fc{gamma} receptor-mediated activation mechanism of the immune system.

biophysics↗

bioRxiv2022

Discovery of Chlorofluoroacetamide-Based Covalent Inhibitors for SARS-CoV-2 3CL Protease

The pandemic of coronavirus disease 2019 (COVID-19) has urgently necessitated the development of antiviral agents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The 3C-like protease (3CLpro) is a promising target for COVID-19 treatment. Here, we report the new class of covalent inhibitors for 3CLpro possessing chlorofluoroacetamide (CFA) as a cysteine reactive warhead. Based on the aza-peptide scaffold, we synthesized the series of CFA derivatives in enantiopure form and evaluated their biochemical efficiencies. The data revealed that 8a (YH-6) with R configuration at the CFA unit strongly blocks the SARS-CoV-2 replication in the infected cells and this potency is comparable to that of nirmatrelvir. The X-ray structural analysis shows that 8a (YH-6) forms a covalent bond with Cys145 at the catalytic center of 3CLpro. The strong antiviral activity and sufficient pharmacokinetics property of 8a (YH-6) suggest its potential as a lead compound for treatment of COVID-19.

biochemistry↗

bioRxiv2020

Dimerization mechanism and structural features of human LI-cadherin

LI-cadherin is a member of the cadherin superfamily, which encompasses a group of Ca2+-dependent cell adhesion proteins. The expression of LI-cadherin is observed on various types of cells in the human body, such as normal small intestine and colon cells, and gastric cancer cells. Because its expression is not observed on normal gastric cells, LI-cadherin is a promising target for gastric cancer imaging. However, since the cell adhesion mechanism of LI-cadherin has remained unknown, rational design of therapeutic molecules targeting this cadherin has been hampered. Here, we have studied the homodimerization mechanism of LI-cadherin. We report the crystal structure of the LI-cadherin EC1-4 homodimer. The EC1-4 homodimer exhibited a unique architecture different from that of other cadherins reported so far. The crystal structure also revealed that LI-cadherin possesses a noncanonical calcium ion-free linker between EC2 and EC3. Various biochemical techniques and molecular dynamics (MD) simulations were employed to elucidate the mechanism of homodimerization. We also showed that the formation of the homodimer observed by the crystal structure is necessary for LI-cadherin-dependent cell adhesion by performing cell aggregation assay.

biochemistry↗