Biology subjects

Martinez-Gil, L.

Publications and source records attributed to Martinez-Gil, L..

7 recordsLinked to original sources

bioRxiv2026

SARS-CoV-2 membrane protein biogenesis

Viral protein biogenesis underpins every viral life cycle stage, and elucidating these processes could reveal fundamental principles of virus-host interaction, and vulnerabilities amenable to therapeutic targeting. Here we apply biophysical, molecular, and cell biology techniques to investigate the insertion, folding, and oligomerization of the SARS-CoV-2 M protein. We describe the sequential co-translational insertion of the hydrophobic core, and demonstrate that the cytosolic C-terminal domain undergoes slower adoption of its tertiary structure. Additionally, we characterize how the transmembrane domain bundle facilitates M-protein oligomerization. Our results reveal a hydrophobic residue cluster that is essential for protein folding and co-translational dimerization. Additionally, we identify the cellular machinery responsible for targeting and inserting the M protein into the ER membrane, and chaperones and cofactors that may contribute to proper folding.

molecular biology↗

bioRxiv2024

The sequence and structural integrity of the SARS-CoV-2 Spike protein transmembrane domain is crucial for viral entry

The Spike (S) protein of SARS-CoV-2 is a type I membrane protein that mediates target cell recognition and membrane fusion. While its transmembrane domain (TMD) is traditionally viewed as a passive anchor to the viral envelope, emerging evidence suggests that TMDs often play active roles in the biogenesis and function of membrane proteins. Here, we investigated the functional role of the SARS-CoV-2 S protein TMD during viral entry. To this end, we introduced a series of amino acid substitutions and insertions within the hydrophobic core of the TMD and assessed their impact on S protein activity. Our findings reveal that the SARS-CoV-2 S protein is susceptible to alterations in its TMD. Functional determinants, including sequence features and structural parameters critical for viral entry, are distributed throughout the TMD, with a more pronounced contribution from its N-terminal region. We also demonstrate that the relative orientation of the regions flanking the TMD influences viral entry. Finally, our data suggest that the TMD mediates homo-oligomerization through a motif enriched in small residues, underscoring its functional importance beyond membrane anchoring.

molecular biology↗

bioRxiv2024

Sequence-dependent scale for translocon-mediated insertion of interfacial helices in membranes

Biological membranes consist of a lipid bilayer studded with integral and peripheral membrane proteins. Most -helical membrane proteins require protein-conducting insertases known as translocons to assist in their membrane insertion and folding. While the sequence-dependent propensities for a helix to either translocate through the translocon or insert into the membrane have been codified into numerical hydrophobicity scales, the corresponding propensity to partition into the membrane interface remains unraveled. By engineering diagnostic glycosylation sites around test peptide sequences inserted into a host protein, we devised a system that can differentiate between water-soluble, surface-bound, and transmembrane (TM) states of the sequence based on its glycosylation pattern. Using this system, we determined the sequence-dependent propensities for transfer from the translocon to a TM, interfacial or extramembrane space. UMAP analysis of a large collection of TM and water-soluble helices provide useful embeddings for analysis of these propensities and aid in understanding the physical properties and functions of antimicrobial, lytic, and fusogenic peptides.

molecular biology↗

bioRxiv2022

Identification of small molecules with virus growth enhancement properties

The novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused the pandemic disease known as coronavirus disease 2019 (COVID-19). COVID-19 vaccines were developed at record speed and were authorized approximately a year after the original outbreak. This fast response saved the lives of countless individuals and reduced the disease burden of many more. The experience has served as a reminder of the necessity to implement solid vaccine development platforms and fast production pipelines. Manufacturing vaccines for enveloped viruses, including some SARS-CoV-2 vaccines, often relies on the production of large quantities of viruses in vitro. Thus, speeding up or increasing virus production would expedite vaccine development. With this objective in mind, we established a high throughput screening (HTS) to identify small molecules that enhance or speed up host-virus membrane fusion. Among the HTS hits, we identified that ethynylestradiol augments SARS-CoV-2 fusion activity in both the absence and presence of TMPRSS2. Furthermore, we confirmed that ethynylestradiol can boost the growth of not only SARS-CoV-2 but also Influenza A virus in vitro. A small molecule with these characteristics could be implemented to improve vaccines production. ImportanceThe (COVID-19) pandemic had a tremendous impact on our healthcare systems and the global economy. The rapid development of effective vaccines saved the lives of countless individuals and reduced the disease burden of many more. Intending to increase vaccine production, we developed and performed a high-throughput screening (HTS) to identify small molecules that enhance viral and cellular membrane fusion. Among the HTS hits, we confirmed that Ethynylestradiol can boost the growth of SARS-CoV-2 and Influenza A virus in vitro.

microbiology↗

bioRxiv2022

Computational design of BclxL inhibitors that target transmembrane domain interactions

Several methods have been developed to explore interactions among water-soluble proteins or regions of proteins. However, techniques to target transmembrane domains have not been examined thoroughly. Here we developed a novel computational approach to design transmembrane sequences that specifically modulate protein-protein interactions in the membrane. To illustrate this method we demonstrated that BclxL can interact with other members of the Bcl2 family through the transmembrane domain and that these interactions are necessary for BclxL control of cell death. Next, we designed sequences that specifically recognize and sequester the transmembrane domain of BclxL. Hence, we were able to prevent BclxL intra-membrane interactions and cancel its anti-apoptotic effect. These results advance our understanding of protein-protein interactions in membranes and provide new means to modulate them. Moreover, the success of our approach may trigger the development of a new generation of inhibitors targeting interactions between transmembrane domains.

biochemistry↗

bioRxiv2020

SARS-CoV-2 envelope protein topology in eukaryotic membranes

Coronavirus E protein is a small membrane protein found in the virus envelope. Different coronavirus E proteins share striking biochemical and functional similarities, but sequence conservation is limited. In this report, we studied the E protein topology from the new SARS-CoV-2 virus both in microsomal membranes and in mammalian cells. Experimental data reveal that E protein is a single-spanning membrane protein with the N-terminus being translocated across the membrane, while the C-terminus is exposed to the cytoplasmic side (Ntlum/Ctcyt). The defined membrane protein topology of SARS-CoV-2 E protein may provide a useful framework to understand its interaction with other viral and host components and establish the basis to tackle the pathogenesis of SARS-CoV-2.

biochemistry↗

bioRxiv2019

A Bimolecular Multicelular complementation system for the detection of syncytium formation: A new methodology for the identification of entry inhibitors

Fusion of viral and cellular membranes is a key step during the viral life cycle. Enveloped viruses trigger this process by means of specialized viral proteins expressed on their surface, the so called viral fusion proteins. There are multiple assays to analyze the viral entry including those that focus on the cell-cell fusion induced by some viral proteins. These methods often rely on the identification of multinucleated cells (syncytium) as a result of cell membrane fusions. In this manuscript, we describe a novel methodology for the study of cell-cell fusion. Our approach, named Bimolecular Multicelular Complementation (BiMuC), provides an adjustable platform to investigate qualitatively and quantitatively the formation of a syncytium. Furthermore, we demonstrated that our procedure meets the requirements of a drug discovery approach and performed a proof of concept small molecule high-throughput screening to identify compounds that could block the entry of the emerging Nipah virus.

microbiology↗