Biology subjects

Garcia-Murria, M. J.

Publications and source records attributed to Garcia-Murria, M. J..

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↗

bioRxiv2025

Cholesterol promotes the formation of dimers and oligomers of the receptor tyrosine kinase ROR1

ROR1 is a member of the receptor tyrosine kinase (RTK) family that plays a crucial role during organogenesis of bone and neural systems by regulating non-canonical Wnt signaling. Misregulation of ROR1 is additionally a causative factor for carcinogenesis in solid and liquid tumors. However, we have a poor understanding of how ROR1 activity is regulated. We employed a recently developed single-molecule method termed SiMPull-POP to study the oligomeric state of ROR1. RTK function is typically triggered by ligand binding, which promotes self-assembly of RTKs to form dimers and in some cases oligomers. However, our data indicate that ROR1 does not follow this paradigm. Instead, ROR1 forms dimers and oligomers in a process that is not affected by the presence of the ROR1 ligand Wnt5a. Additional experiments indicate that the transmembrane domain of ROR1 has a strong tendency to self-assemble, suggesting that this domain modulates ROR1 dimerization. Investigation into a regulatory mechanism for ROR1 self-assembly led to evaluation of the role of the lipid cholesterol, which plays pleiotropic roles in Wnt signaling. Cholesterol was found to promote the assembly of ROR1, and our results point to the transmembrane domain as the region where cholesterol exerts the regulatory effect. Taken together, our results indicate that ROR1 self-assembles in human cells; however, unlike other RTKs, this process is not stabilized by ligand binding but is instead facilitated by membrane cholesterol.

biochemistry↗

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↗

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↗