An amphipathic Bax core dimer forms part of the apoptotic pore wall in the mitochondrial␣membrane.

Bax proteins form pores in the mitochondrial outer membrane to initiate apoptosis. This might involve their embedding in the cytosolic leaflet of the lipid bilayer, thus generating tension to induce a lipid pore with radially arranged lipids forming the wall. Alternatively, Bax proteins might comprise part of the pore wall. However, there is no unambiguous structural evidence for either hypothesis. Using NMR, we determined a high-resolution structure of the Bax core region, revealing a dimer with the nonpolar surface covering the lipid bilayer edge and the polar surface exposed to water. The dimer tilts from the bilayer normal, not only maximizing nonpolar interactions with lipid tails but also creating polar interactions between charged residues and lipid heads. Structure-guided mutations demonstrate the importance of both types of protein-lipid interactions in Bax pore assembly and core dimer configuration. Therefore, the Bax core dimer forms part of the proteolipid pore wall to permeabilize mitochondria.

Expansion of KRAS hotspot mutations reactive T cells from human pancreatic tumors using autologous T cells as the antigen-presenting cells.

Adoptive cell therapy (ACT) with expanded tumor-infiltrating lymphocytes (TIL) or TCR gene-modified T cells (TCR-T) that recognize mutant KRAS neo-antigens can mediate tumor regression in patients with advanced pancreatic ductal adenocarcinoma (PDAC) (Tran et al in N Engl J Med, 375:2255-2262, 2016; Leidner et al in N Engl J Med, 386:2112-2119, 2022). The mutant KRAS-targeted ACT holds great potential to achieve durable clinical responses for PDAC, which has had no meaningful improvement over 40 years. However, the wide application of mutant KRAS-centric ACT is currently limited by the rarity of TIL that recognize the mutant KRAS. In addition, PDAC is generally recognized as a poorly immunogenic tumor, and TILs in PDAC are less abundant than in immunogenic tumors such as melanoma. To increase the success rate of TIL production, we adopted a well-utilized K562-based artificial APC (aAPC) that expresses 4-1BBL as the costimulatory molecules to enhance the TIL production from PDCA. However, stimulation with K562-based aAPC led to a rapid loss of specificity to mutant KRAS. To selectively expand neo-antigen-specific T cells, particularly mKRAS, from the TILs, we used tandem mini gene-modified autologous T cells (TMG-T) as the novel aAPC. Using this modified IVS protocol, we successfully generated TIL cultures specifically reactive to mKRAS (G12V). We believe that autologous TMG-T cells provide a reliable source of autologous APC to expand a rare population of neoantigen-specific T cells in TILs.

Expression, purification and characterization of TMCO1 for structural studies.

Transmembrane and coiled-coil domains 1 (TMCO1) has a highly conserved amino acid sequence among species, indicating a critical role of TMCO1 in cell physiology. The deficiency of TMCO1 in humans is associated with cerebrofaciothoracic dysplasia (CFTD), glaucoma, osteogenesis and the occurrence of cancer. TMCO1 was recently identified as an endoplasmic reticulum (ER) Ca2+ load-activated Ca2+ (CLAC) release channel, which prevents ER Ca2+ overload and maintains calcium homeostasis in the ER. However, the structural basis of the molecular function of TMCO1 channel remains elusive. To determine the structure of TMCO1, we screened the expression of TMCO1 in Escherichia coli and insect cell expression systems. TMCO1 from Dictyostelium discoideum (DdTMCO1) was successfully expressed in Escherichia coli with a high yield. The pure recombinant protein was obtained by affinity chromatography and size exclusion chromatography. The solution NMR of DdTMCO1 in DPC micelles showed three α-helical transmembrane regions.

Deciphering Cholesterol's Role in PD-L2 Stability: A Distinct Regulatory Mechanism From PD-L1.

Programmed cell death 1 ligand 2 (PD-L2), a member of the B7 immune checkpoint protein family, emerges as a crucial player in immune modulation. Despite its functional overlap with programmed cell death 1 ligand 1 (PD-L1) in binding to the programmed cell death protein 1 (PD-1) on T cells, PD-L2 exhibits a divergent expression pattern and a higher affinity for PD-1. However, the regulatory mechanisms of PD-L2 remain under-explored. Here, our investigations illustrate the pivotal role of cholesterol in modulating PD-L2 stability. Using advanced nuclear magnetic resonance (NMR) and biochemical analyses, we demonstrate a direct and specific binding between cholesterol and PD-L2, mediated by an F-xxx-V-xx-LR motif in its transmembrane domain, distinct from that in PD-L1. This interaction stabilizes PD-L2 and prevents its downstream degradation. Disruption of this binding motif compromises PD-L2's cellular stability, underscoring its potential significance in cancer biology. These findings not only deepen our understanding of PD-L2 regulation in the context of tumors, but also open avenues for potential therapeutic interventions.

Structural characterization of a dimerization interface in the CD28 transmembrane domain.

CD28 has a crucial role in regulating immune responses by enhancing T cell activation and differentiation. Recent studies have shown that the transmembrane helix (TMH) of CD28 mediates receptor assembly and activity, but a structural characterization of TMH is still lacking. Here, we determined the dimeric helix-helix packing of CD28-TMH using nuclear magnetic resonance (NMR) technology. Unexpectedly, wild-type CD28-TMH alone forms stable tetramers in lipid bicelles instead of dimers. The NMR structure of the CD28-TMH C165F mutant reveals that a GxxxA motif, which is highly conserved in many dimeric assemblies, is located at the dimerization interface. Mutating G160 and A164 can disrupt the transmembrane helix assembly and reduces CD28 enhancement in cells. In contrast, a previously proposed YxxxxT motif does not affect the dimerization of full-length CD28, but it does affect CD28 activity. These results imply that the transmembrane domain of CD28 regulates the signaling transduction in a complicated manner.

Experimental Investigations on the Structure of Yeast Mitochondrial Pyruvate Carriers.

Mitochondrial pyruvate carrier (MPC) transports pyruvate from the cytoplasm into the mitochondrial matrix to participate in the tricarboxylic acid (TCA) cycle, which further generates the energy for the physiological activities of cells. Two interacting subunits, MPC1 and MPC2 or MPC3, form a heterodimer to conduct transport function. However, the structural basis of how the MPC complex transports pyruvate is still lacking. Here, we described the detailed expression and purification procedures to obtain large amounts of yeast MPC1 and MPC2 for structural characterization. The purified yeast MPC1 and MPC2 were reconstituted in dodecylphosphocholine (DPC) micelles and examined using nuclear magnetic resonance (NMR) spectroscopy, showing that both subunits contain three α-helical transmembrane regions with substantial differences from what was predicted by AlphaFold2. Furthermore, the new protocol producing the recombinant MPC2 using modified maltose-binding protein (MBP) with cyanogen bromide (CNBr) cleavage introduced general way to obtain small membrane proteins. These findings provide a preliminary understanding for the structure of the MPC complex and useful guidance for further studies.

Regulation of PD-L1 through direct binding of cholesterol to CRAC motifs.

Cholesterol, an essential molecule for cell structure, function, and viability, plays crucial roles in the development, progression, and survival of cancer cells. Earlier studies have shown that cholesterol-lowering drugs can inhibit the high expression of programmed-death ligand 1 (PD-L1) that contributes to immunoevasion in cancer cells. However, the regulatory mechanism of cell surface PD-L1 abundance by cholesterol is still controversial. Here, using nuclear magnetic resonance and biochemical techniques, we demonstrated that cholesterol can directly bind to the transmembrane domain of PD-L1 through two cholesterol-recognition amino acid consensus (CRAC) motifs, forming a sandwich-like architecture and stabilizing PD-L1 to prevent downstream degradation. Mutations at key binding residues prohibit PD-L1-cholesterol interactions, decreasing the cellular abundance of PD-L1. Our results reveal a unique regulatory mechanism that controls the stability of PD-L1 in cancer cells, providing an alternative method to overcome PD-L1-mediated immunoevasion in cancers.

Cholesterol Binds in a Reversed Orientation to TCRß-TM in Which Its OH Group is Localized to the Center of the Lipid Bilayer.

T cell receptor (TCR) signaling in response to antigen recognition is essential for the adaptive immune response. Cholesterol keeps TCRs in the resting conformation and mediates TCR clustering by directly binding to the transmembrane domain of the TCRß subunit (TCRß-TM), while cholesterol sulfate (CS) displaces cholesterol from TCRß. However, the atomic interaction of cholesterol or CS with TCRß remains elusive. Here, we determined the cholesterol and CS binding site of TCRß-TM in phospholipid bilayers using solution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulation. Cholesterol binds to the transmembrane residues within a CARC-like cholesterol recognition motif. Surprisingly, the polar OH group of cholesterol is placed in the hydrophobic center of the lipid bilayer stabilized by its polar interaction with K154 of TCRß-TM. An aromatic interaction with Y158 and hydrophobic interactions with V160 and L161 stabilize this reverse orientation. CS binds to the same site, explaining how it competes with cholesterol. Site-directed mutagenesis of the CARC-like motif disrupted the cholesterol/CS binding to TCRß-TM, validating the NMR and MD results.

PD-L1 degradation is regulated by electrostatic membrane association of its cytoplasmic domain.

The cytoplasmic domain of PD-L1 (PD-L1-CD) regulates PD-L1 degradation and stability through various mechanism, making it an attractive target for blocking PD-L1-related cancer signaling. Here, by using NMR and biochemical techniques we find that the membrane association of PD-L1-CD is mediated by electrostatic interactions between acidic phospholipids and basic residues in the N-terminal region. The absence of the acidic phospholipids and replacement of the basic residues with acidic residues abolish the membrane association. Moreover, the basic-to-acidic mutations also decrease the cellular abundance of PD-L1, implicating that the electrostatic interaction with the plasma membrane mediates the cellular levels of PD-L1. Interestingly, distinct from its reported function as an activator of AMPK in tumor cells, the type 2 diabetes drug metformin enhances the membrane dissociation of PD-L1-CD by disrupting the electrostatic interaction, thereby decreasing the cellular abundance of PD-L1. Collectively, our study reveals an unusual regulatory mechanism that controls the PD-L1 level in tumor cells, suggesting an alternative strategy to improve the efficacy of PD-L1-related immunotherapies.

Structural Characterization of the N-Terminal Domain of the Dictyostelium discoideum Mitochondrial Calcium Uniporter.

The mitochondrial calcium uniporter (MCU) plays a critical role in mitochondrial calcium uptake into the matrix. In metazoans, the uniporter is a tightly regulated multicomponent system, including the pore-forming subunit MCU and several regulators (MICU1, MICU2, and Essential MCU REgulator, EMRE). The calcium-conducting activity of metazoan MCU requires the single-transmembrane protein EMRE. Dictyostelium discoideum (Dd), however, developed a simplified uniporter for which the pore-forming MCU (DdMCU) alone is necessary and sufficient for calcium influx. Here, we report a crystal structure of the N-terminal domain (NTD) of DdMCU at 1.7 Å resolution. The DdMCU-NTD contains four helices and two strands arranged in a fold that is completely different from the known structures of other MCU-NTD homologues. Biochemical and biophysical analyses of DdMCU-NTD in solution indicated that the domain exists as high-order oligomers. Mutagenesis showed that the acidic residues Asp60, Glu72, and Glu74, which appeared to mediate the interface II, as observed in the crystal structure, participated in the self-assembly of DdMCU-NTD. Intriguingly, the oligomeric complex was disrupted in the presence of calcium. We propose that the calcium-triggered dissociation of NTD regulates the channel activity of DdMCU by a yet unknown mechanism.

Optimal design and validation of antiviral siRNA for targeting hepatitis B virus.

AIM: Optimal design of antiviral short-interfering RNA (siRNA) targeting highly divergent hepatitis B virus (HBV) was validated by quantitative structure activity relationship (QSAR) analysis. METHODS: The potency of 23 synthetic siRNAs targeting 23 sites throughout HBV pregenomic RNA were evaluated at 10 nmol/L by determining the inhibition on the expression of S/P/pregenomic mRNA and hepatitis B surface antigen (HBsAg) quantitatively in HepG2.2.15 cells. Genotype homology within HBV genomes was identified through plentiful computational analysis and the multiple linear regression analysis was made to validate the relationship between the functional siRNAs and primary characteristics. Based on the preliminary results, relationships between different determined endpoints [S/P mRNA, HBsAg, C/P mRNA, hepatitis B e antigen (HBeAg) and viral DNA load] and siRNA efficacy evaluation were investigated. RESULTS: Genotype homology, open reading frame (ORF) S/P, X and C had tight correlation with the ability of siRNAs on inhibiting the expression of S/P/Pregenomic mRNA and HBsAg (P<0.01), of which, ORF C was negatively correlated with the siRNA potency (P<0.05). Further study showed that siRNA potency evaluation was influenced by different determined endpoints. P-target siRNAs showed significant inhibition on the S mRNA and HBsAg expression. S-target siRNAs inhibited the expression of S mRNA and HBsAg strongly. X-target siRNAs played active roles in inhibiting all 5 determined endpoints. C-target siRNAs blocked the expression of C mRNA, HBeAg and viral DNA load significantly. CONCLUSION: The antiviral potency of siRNA was relevant to its primary characteristics and determined endpoints were important for siRNA efficacy evaluation for complex genome with overlapping ORF, which was helpful for siRNA optimal design.

The AAA protein spastin possesses two levels of basal ATPase activity.

The AAA ATPase spastin is a microtubule-severing enzyme that plays important roles in various cellular events including axon regeneration. Herein, we found that the basal ATPase activity of spastin is negatively regulated by spastin concentration. By determining a spastin crystal structure, we demonstrate the necessity of intersubunit interactions between spastin AAA domains. Neutralization of the positive charges in the microtubule-binding domain (MTBD) of spastin dramatically decreases the ATPase activity at low concentration, although the ATP-hydrolyzing potential is not affected. These results demonstrate that, in addition to the AAA domain, the MTBD region of spastin is also involved in regulating ATPase activity, making interactions between spastin protomers more complicated than expected.

The nucleotide cycle of spastin correlates with its microtubule-binding properties.

Spastin is an AAA (ATPase associated with diverse cellular activities) protein with microtubule (MT)-severing activity. The spastin-encoding gene was identified as the most often mutated gene in the human neurodegenerative disease hereditary spastic paraplegia. Although the structure of the AAA domain of spastin has been determined, the mechanism by which spastin severs MTs remains elusive. Here, we studied the MT-binding and nucleotide-binding properties of spastin, as well as their interplay. The results suggest that ATP-bound spastin interacts strongly and cooperatively with MTs; this interaction stimulates ATP hydrolysis by spastin. After ATP hydrolysis, spastin dissociates from MTs, and then exchanges ADP for ATP in solution for the next round of work. In particular, spastin in the ternary complex of MT-spastin-ATP is the most cooperative state during the working cycle, and is probably the force-generating state that is responsible for MT severing. The results presented in this study establish the nucleotide cycle of spastin in correlation with its MT-binding properties, and provide a biochemical framework for further studies of the working mechanism of spastin.