Faculty Bibliography

The efficacy of ALVAC-based HIV and SIV vaccines in humans and macaques correlates with antibodies to envelope variable region 2 (V2). We show here that vaccine-induced antibodies to SIV variable region 1 (V1) inhibit anti-V2 antibody-mediated cytotoxicity and reverse their ability to block V2 peptide interaction with the α₄β₇ integrin. SIV vaccines engineered to delete V1 and favor an α helix, rather than a β sheet V2 conformation, induced V2-specific ADCC correlating with decreased risk of SIV acquisition. Removal of V1 from the HIV-1 clade A/E A244 envelope resulted in decreased binding to antibodies recognizing V2 in the β sheet conformation. Thus, deletion of V1 in HIV envelope immunogens may improve antibody responses to V2 virus vulnerability sites and increase the efficacy of HIV vaccine candidates.

Tyrosinase (TYR) is a copper-containing monooxygenase central to the function of melanocytes. Alterations in its expression or activity contribute to variations in skin, hair and eye color, and underlie a variety of pathogenic pigmentary phenotypes, including several forms of oculocutaneous albinism (OCA). Many of these phenotypes are linked to individual missense mutations causing single nucleotide variants and polymorphisms (SNVs) in TYR. We previously showed that two TYR homologues, TYRP1 and TYRP2, modulate TYR activity and stabilize the TYR protein. Accordingly, to investigate whether TYR, TYRP1, and TYRP2 are biophysically compatible with various heterocomplexes, we computationally docked a high-quality 3D model of TYR to the crystal structure of TYRP1 and to a high-quality 3D model of TYRP2. Remarkably, the resulting TYR-TYRP1 heterodimer was complementary in structure and energy with the TYR-TYRP2 heterodimer, with TYRP1 and TYRP2 docking to different adjacent surfaces on TYR that apposed a third realistic protein interface between TYRP1-TYRP2. Hence, the 3D models are compatible with a heterotrimeric TYR-TYRP1-TYRP2 complex. In addition, this heterotrimeric TYR-TYRP1-TYRP2 positioned the C-terminus of each folded enzymatic domain in an ideal position to allow their C-terminal transmembrane helices to form a putative membrane embedded three-helix bundle. Finally, pathogenic TYR mutations causing OCA1A, which also destabilize TYR biochemically, cluster on an unoccupied protein interface at the periphery of the heterotrimeric complex, suggesting that this may be a docking site for OCA2, an anion channel. Pathogenic OCA2 mutations result in similar phenotypes to those produced by OCA1A TYR mutations. While this complex may be difficult to detect in vitro, due to the complex environment of the vertebrate cellular membranous system, our results support the existence of a heterotrimeric complex in melanogenesis.

AIMS OF THE STUDY/OBJECTIVE:Patient comprehension is a critical part of meeting medical ethics standards of informed consent in study designs. The aim of the study was to determine if sufficient literature exists to require clinicians to disclose the specific risk that COVID-19 vaccines could worsen disease upon exposure to challenge or circulating virus. METHODS USED TO CONDUCT THE STUDY/UNASSIGNED:Published literature was reviewed to identify preclinical and clinical evidence that COVID-19 vaccines could worsen disease upon exposure to challenge or circulating virus. Clinical trial protocols for COVID-19 vaccines were reviewed to determine if risks were properly disclosed. RESULTS OF THE STUDY/UNASSIGNED:COVID-19 vaccines designed to elicit neutralizing antibodies may sensitize vaccine recipients to more severe disease than if they were not vaccinated. Vaccines for SARS, MERS and RSV have never been approved, and the data generated in the development and testing of these vaccines suggest a serious mechanistic concern: that vaccines designed empirically using the traditional approach (consisting of the unmodified or minimally modified coronavirus viral spike to elicit neutralizing antibodies), be they composed of protein, viral vector, DNA or RNA and irrespective of delivery method, may worsen COVID-19 disease via antibody-dependent enhancement (ADE). This risk is sufficiently obscured in clinical trial protocols and consent forms for ongoing COVID-19 vaccine trials that adequate patient comprehension of this risk is unlikely to occur, obviating truly informed consent by subjects in these trials. CONCLUSIONS DRAWN FROM THE STUDY AND CLINICAL IMPLICATIONS/UNASSIGNED:The specific and significant COVID-19 risk of ADE should have been and should be prominently and independently disclosed to research subjects currently in vaccine trials, as well as those being recruited for the trials and future patients after vaccine approval, in order to meet the medical ethics standard of patient comprehension for informed consent.

The Integrated Stress Response (ISR) helps metazoan cells adapt to cellular stress by limiting the availability of initiator methionyl-tRNA for translation. Such limiting conditions paradoxically stimulate the translation of ATF4 mRNA through a regulatory 5' leader sequence with multiple upstream Open Reading Frames (uORFs), thereby activating stress-responsive gene expression. Here, we report the identification of two critical regulators of such ATF4 induction, the noncanonical initiation factors eIF2D and DENR. Loss of eIF2D and DENR in Drosophila results in increased vulnerability to amino acid deprivation, susceptibility to retinal degeneration caused by endoplasmic reticulum (ER) stress, and developmental defects similar to ATF4 mutants. eIF2D requires its RNA-binding motif for regulation of 5' leader-mediated ATF4 translation. Consistently, eIF2D and DENR deficient human cells show impaired ATF4 protein induction in response to ER stress. Altogether, our findings indicate that eIF2D and DENR are critical mediators of ATF4 translational induction and stress responses in vivo.

Activation of dual-specificity tyrosine-phosphorylation-regulated kinases 1A and 1B (DYRK1A and DYRK1B) requires prolyl hydroxylation by PHD1 prolyl hydroxylase. Prolyl hydroxylation of DYRK1 initiates a cascade of events leading to the release of molecular constraints on von Hippel-Lindau (VHL) ubiquitin ligase tumor suppressor function. However, the proline residue of DYRK1 targeted by hydroxylation and the role of prolyl hydroxylation in tyrosine autophosphorylation of DYRK1 are unknown. We found that a highly conserved proline in the CMGC insert of the DYRK1 kinase domain is hydroxylated by PHD1, and this event precedes tyrosine autophosphorylation. Mutation of the hydroxylation acceptor proline precludes tyrosine autophosphorylation and folding of DYRK1, resulting in a kinase unable to preserve VHL function and lacking glioma suppression activity. The consensus proline sequence is shared by most CMGC kinases, and prolyl hydroxylation is essential for catalytic activation. Thus, formation of prolyl-hydroxylated intermediates is a novel mechanism of kinase maturation and likely a general mechanism of regulation of CMGC kinases in eukaryotes.

Staphylococcus aureus infection is a major public health threat in part due to the spread of antibiotic resistance and repeated failures to develop a protective vaccine. Infection is associated with production of virulence factors that include exotoxins that attack host barriers and cellular defenses, such as the leukocidin (Luk) family of bicomponent pore-forming toxins. To investigate the structural basis of antibody-mediated functional inactivation of Luk toxins, we generated a panel of murine monoclonal antibodies (MAbs) that neutralize host cell killing by the γ-hemolysin HlgCB. By biopanning these MAbs against a phage-display library of random Luk peptide fragments, we identified a small subregion within the rim domain of HlgC as the epitope for all the MAbs. Within the native holotoxin, this subregion folds into a conserved β-hairpin structure, with exposed key residues, His252 and Tyr253, required for antibody binding. On the basis of the phage-display results and molecular modeling, a 15-amino-acid synthetic peptide representing the minimal epitope on HlgC (HlgC241-255) was designed, and preincubation with this peptide blocked antibody-mediated HIgCB neutralization. Immunization of mice with HlgC241-255 or the homologous LukS246-260 subregion peptide elicited serum antibodies that specifically recognized the native holotoxin subunits. Furthermore, serum IgG from patients who were convalescent for invasive S. aureus infection showed neutralization of HlgCB toxin activity ex vivo, which recognized the immunodominant HlgC241-255 peptide and was dependent on His252 and Tyr253 residues. We have thus validated an efficient, rapid, and scalable experimental workflow for identification of immunodominant and immunogenic leukotoxin-neutralizing B-cell epitopes that can be exploited for new S. aureus-protective vaccines and immunotherapies.

Cullin-RING ligases (CRLs) control key cellular processes by promoting ubiquitylation of a multitude of soluble cytosolic and nuclear proteins. Subsets of CRL complexes are recruited and activated locally at cellular membranes; however, few CRL functions and substrates at these distinct cellular compartments are known. Here, we use a proteomic screen to identify proteins that are ubiquitylated at cellular membranes and found that Lunapark, an endoplasmic reticulum (ER)-shaping protein localized to ER three-way junctions, is ubiquitylated by the CRL3^KLHL12 ubiquitin ligase. We demonstrate that Lunapark interacts with mechanistic target of rapamycin complex-1 (mTORC1), a central cellular regulator that coordinates growth and metabolism with environmental conditions. We show that mTORC1 binds Lunapark specifically at three-way junctions, and lysosomes, where mTORC1 is activated, make contact with three-way junctions where Lunapark resides. Inhibition of Lunapark ubiquitylation results in neurodevelopmental defects indicating that KLHL12-dependent ubiquitylation of Lunapark is required for normal growth and development.

A physiological role for long-chain acyl-CoA esters to activate ATP-sensitive K⁺ (K_ATP) channels is well established. Circulating palmitate is transported into cells and converted to palmitoyl-CoA, which is a substrate for palmitoylation. We found that palmitoyl-CoA, but not palmitic acid, activated the channel when applied acutely. We have altered the palmitoylation state by preincubating cells with micromolar concentrations of palmitic acid or by inhibiting protein thioesterases. With acyl-biotin exchange assays we found that Kir6.2, but not sulfonylurea receptor (SUR)1 or SUR2, was palmitoylated. These interventions increased the K_ATP channel mean patch current, increased the open time, and decreased the apparent sensitivity to ATP without affecting surface expression. Similar data were obtained in transfected cells, rat insulin-secreting INS-1 cells, and isolated cardiac myocytes. Kir6.2ΔC36, expressed without SUR, was also positively regulated by palmitoylation. Mutagenesis of Kir6.2 Cys¹⁶⁶ prevented these effects. Clinical variants in KCNJ11 that affect Cys¹⁶⁶ had a similar gain-of-function phenotype, but was more pronounced. Molecular modeling studies suggested that palmitoyl-C166 and selected large hydrophobic mutations make direct hydrophobic contact with Kir6.2-bound PIP₂ Patch-clamp studies confirmed that palmitoylation of Kir6.2 at Cys¹⁶⁶ enhanced the PIP₂ sensitivity of the channel. Physiological relevance is suggested since palmitoylation blunted the regulation of K_ATP channels by α1-adrenoreceptor stimulation. The Cys¹⁶⁶ residue is conserved in some other Kir family members (Kir6.1 and Kir3, but not Kir2), which are also subject to regulated palmitoylation, suggesting a general mechanism to control the open state of certain Kir channels.

AIM/OBJECTIVE:The COVID pandemic is caused by infection with the SARS-CoV-2 virus. The major mutation detected to date in the SARS-CoV-2 viral envelope spike protein, which is responsible for virus attachment to the host and is also the main target for host antibodies, is a mutation of an aspartate (D) at position 614 found frequently in Chinese strains to a glycine (G). We sought to infer health impact of this mutation. RESULT/RESULTS:Increased case fatality rate correlated strongly with the proportion of viruses bearing G614 on a country by country basis. The amino acid at position 614 occurs at an internal protein interface of the viral spike, and the presence of G at this position was calculated to destabilize a specific conformation of the viral spike, within which the key host receptor binding site is more accessible. CONCLUSION/CONCLUSIONS:These results imply that G614 is a more pathogenic strain of SARS-CoV-2, which may influence vaccine design. The prevalence of this form of the virus should also be included in epidemiologic models predicting the COVID-19 health burden and fatality over time in specific regions. Physicians should be aware of this characteristic of the virus to anticipate the clinical course of infection.

T cell recognition of antigen and resulting proximal signaling are key steps in the initiation of the adaptive immune response. Previous studies targeting antigen binding site for enhancing T-cell responses to tumor antigens often lead to off-target effects and toxicity. Recently, we used nuclear magnetic resonance (NMR) spectroscopy, mutational analysis and computational docking to derive a 3D structure of the extracellular TCRCD3 assembly. Further, biomolecular force probe (BFP) measurements allowed us to determine how 2D affinity and force-modulated TCR-pMHC kinetics depend on TCR-CD3 interaction sites and affect transduction of extracellular pMHC-TCR ligation into T cell function. Based on our TCR-CD3 structural model and binding data, we generated TCR libraries for a melanoma-specific TCR (DMF5) using site-specific mutagenesis in the Cbhelix 3 and helix 4-F strand regions of the TCR to optimize the TCR-CD3 interaction and to select for mutants with enhanced T-cell effector function. One Cb helix 4-F strand mutant, NP202203AA showed increased T cell response to antigen and showed enhanced TCR-pMHC bond lifetime (catch-bonds) in BFP assays leading to prolonged T cell signaling. In the future, DMF5 TCR with reengineered CD3 binding regions will be used in tumor rejection in pre-clinical mouse melanoma models for eficacy and toxicity to develop more effective T cell therapies for human targets