Biology subjects

Santiago, U.

Publications and source records attributed to Santiago, U..

4 recordsLinked to original sources

bioRxiv2021

Heterotypic Assembly Mechanism Regulates CHIP E3 Ligase Activity

The E3 ubiquitin ligases CHIP/CHN-1 and UFD-2 team up to accelerate ubiquitin chain formation. However, it remained largely unclear how the high processivity of this E3 set is achieved. Here we studied the molecular mechanism and function of the CHN-1/UFD-2 complex in Caenorhabditis elegans. Our data show that UFD-2 binding promotes the cooperation between CHN-1 and ubiquitin-conjugating E2 enzymes by stabilizing the CHN-1 U-box dimer. The HSP-1 chaperone outcompetes UFD-2 for CHN-1 binding and promotes the auto-inhibited CHN-1 state by acting on the conserved position of the U-box domain. The interaction with UFD-2 enables CHN-1 to efficiently ubiquitinate S-Adenosylhomocysteinase (AHCY-1), an enzyme crucial for lipid metabolism. Our results define the molecular mechanism underlying the synergistic cooperation of CHN-1 and UFD-2 in substrate ubiquitylation. HIGHLIGHTSO_LIE3 ligase UFD-2 stimulates ubiquitylation activity of CHIP/CHN-1 C_LIO_LIUFD-2 binding promotes dimerization of CHIP/CHN-1 U-box domains and utilization of E2 enzymes C_LIO_LIHSP70/HSP-1 by latching the U-box and TPR domains stabilizes the autoinhibitory state of CHIP/CHN-1, limiting interactions with E2s and UFD-2 C_LIO_LIAssembly with UFD-2 enables CHIP/CHN-1 to regulate lipid metabolism by ubiquitylation of S-Adenosylhomocysteinase C_LI

molecular biology↗

bioRxiv2020

A robust and versatile nanobody platform for drug delivery

Therapeutic and diagnostic efficacies of numerous small biomolecules and chemical compounds are hampered by the short half-lives. Here we report the development of a repertoire of diverse, high-affinity albumin-nanobodies (NbHSA) to facilitate drug delivery. By integrating biophysics, and hybrid structural biology, we have systematically characterized the NbHSA for albumin binding, mapped the epitopes, and resolved the architecture of a tetrameric Nb-albumin complex. We employed quantitative proteomics for accurate, multiplex Nb pharmacokinetic analysis. Using a humanized albumin mouse model, we found that the NbHSA has outstanding pharmacokinetics; the most stable NbHSA has a 771-fold T1/2 improvement compared with a control Nb. Interestingly, the pharmacokinetics of NbHSA is related to their biophysical and structural properties. To demonstrate the utility of NbHSA, we developed a highly stable NbHSA-hIL-2 cytokine conjugate "Duraleukin" and confirmed its improved anticancer properties than hIL-2 alone. We envision that this high-quality Nb resource will advance research into novel biotherapeutics.

bioengineering↗

bioRxiv2020

On the molecular mechanism of SARS-CoV-2 retention in the upper respiratory tract

Cell surface receptor engagement is a critical aspect of viral infection. At low pH, binding of SARS-CoV and its ACE2 receptor has a tight interaction that catalyzes the fusion of the spike and endosomal membranes followed by genome release. Largely overlooked has been the role of neutral pH in the respiratory tract, where we find that SARS-CoV stabilizes a transition state that enhances the off-rate from its receptor. An alternative pH-switch is found in CoV-2-like coronaviruses of tropical pangolins, but with a reversed phenotype where the tight interaction with ACE2 is at neutral pH. We show that a single point mutation in pangolin-CoV, unique to CoV-2, that deletes the last His residue in their receptor binding domain perpetuates this tight interaction independent of pH. This tight bond, not present in previous respiratory syndromes, implies that CoV-2 stays bound to the highly expressed ACE2 receptors in the nasal cavity about 100 times longer than CoV. This finding supports the unfamiliar pathology of CoV-2, observed virus retention in upper respiratory tract1, longer incubation times and extended periods of shedding. Implications to combat pandemics that, like SARS-CoV-2, export evolutionarily successful strains via higher transmission rates due to retention in nasal epithelium and their evolutionary origin are discussed.

biophysics↗

bioRxiv2020

Loss of pH switch unique to SARS-CoV2 supports unfamiliar virus pathology

Cell surface receptor engagement is a critical aspect of viral infection. This paper compares the dynamics of virus-receptor interactions for SARS-CoV (CoV1) and CoV2. At low (endosomal) pH, the binding free energy landscape of CoV1 and CoV2 interactions with the angiotensin-converting enzyme 2 (ACE2) receptor is almost the same. However, at neutral pH the landscape is different due to the loss of a pH-switch (His445Lys) in the receptor binding domain (RBD) of CoV2 relative to CoV1. Namely, CoV1 stabilizes a transition state above the bound state. In situations where small external strains are applied by, say, shear flow in the respiratory system, the off rate of the viral particle is enhanced. As a result, CoV1 virions are expected to detach from cell surfaces in time scales that are much faster than the time needed for other receptors to reach out and stabilize virus attachment. On the other hand, the loss of this pH-switch, which sequence alignments show is unique to CoV2, eliminates the transition state and allows the virus to stay bound to the ACE2 receptor for time scales compatible with the recruitment of additional ACE2 receptors diffusing in the cell membrane. This has important implications for viral infection and its pathology. CoV1 does not trigger high infectivity in the nasal area because it either rapidly drifts down the respiratory tract or is exhaled. By contrast, this novel mutation in CoV2 should not only retain the infection in the nasal cavity until ACE2-rich cells are sufficiently depleted, but also require fewer particles for infection. This mechanism explains observed longer incubation times, extended period of viral shedding, and higher rate of transmission. These considerations governing viral entry suggest that number of ACE2-rich cells in human nasal mucosa, which should be significantly smaller for children (and females relative to males), should also correlate with onset of viral load that could be a determinant of higher virus susceptibility. Critical implications for the development of new vaccines to combat current and future pandemics that, like SARS-CoV2, export evolutionarily successful strains via higher transmission rates by viral retention in nasal epithelium are also discussed.

biophysics↗