Functional Reconstitution Research Articles

Distinct roles for the domains of the mitochondrial aspartate/glutamate carrier citrin in organellar localization and substrate transport

ObjectiveCitrin, the mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), is structurally and mechanistically the most complex SLC25 family member, because it consists of three domains and forms a homo-dimer. Each protomer has an N-terminal calcium-binding domain with EF-hands, followed by a substrate-transporting carrier domain and a C-terminal domain with an amphipathic helix. The absence or dysfunction of citrin leads to citrin deficiency, a highly prevalent pan-ethnic mitochondrial disease. Here, we aim to understand the role of different citrin domains and how they contribute to pathogenic mechanisms in citrin deficiency. MethodsWe have employed structural modeling and functional reconstitution of purified proteins in proteoliposomes to assess the transport activity and calcium regulation of wild-type citrin and pathogenic variants associated with citrin deficiency. We have also developed a double knockout of citrin and aralar (AGC1), the two paralogs of the mitochondrial aspartate/glutamate carrier, in HAP1 cells to perform mitochondrial imaging and to investigate mitochondrial localisation. ResultsUsing 33 pathogenic variants of citrin we clarify determinants of subcellular localization and transport mechanism. We identify crucial elements of the carrier domain that are required for transport, including those involved in substrate binding, network formation and dynamics. We show that the N-terminal domain is not involved in calcium regulation of transport, as previously thought, but when mutated causes a mitochondrial import defect. ConclusionsOur work introduces a new role for the N-terminal domain of citrin and demonstrates that dysfunction of the different domains contributes to distinct pathogenic mechanisms in citrin deficiency.

Structural dynamics of the intrinsically disordered linker region of cardiac troponin T.

The cardiac troponin complex, composed of troponins I, T, and C, plays a central role in regulating the calcium-dependent interactions between myosin and the thin filament. Mutations in troponin can cause cardiomyopathies; however, it is still a major challenge to connect how changes in sequence affect troponin's function. Recent high-resolution structures of the thin filament revealed critical insights into the structure-function relationship of troponin, but there remain large, unresolved segments of troponin, including the troponin-T linker region that is a hotspot for cardiomyopathy mutations. This linker region is predicted to be intrinsically disordered, with behaviors that are not well described by traditional structural approaches; however, this proposal has not been experimentally verified. Here, we used a combination of single-molecule Förster resonance energy transfer (FRET), molecular dynamics simulations, and functional reconstitution assays to investigate the troponin-T linker region. We show that in the context of both isolated troponin and the fully regulated troponin complex, the linker behaves as a dynamic, intrinsically disordered region. This region undergoes polyampholyte expansion in the presence of high salt and distinct conformational changes during the assembly of the troponin complex. We also examine the ΔE160 hypertrophic cardiomyopathy mutation in the linker and demonstrate that it does not affect the conformational dynamics of the linker, rather it allosterically affects interactions with other troponin complex subunits, leading to increased molecular contractility. Taken together, our data clearly demonstrate the importance of disorder within the troponin-T linker and provide new insights into the molecular mechanisms driving the pathogenesis of cardiomyopathies.

An in vitro set-up to study Pdr5-mediated substrate translocation.

Pdr5 is the most abundant ABC transporter in Saccharomyces cerevisiae and plays a major role in the pleiotropic drug resistance (PDR) network, which actively prevents cell entry of a large number of structurally unrelated compounds. Due to a high level of asymmetry in one of its nucleotide binding sites (NBS), Pdr5 serves as a perfect model system for asymmetric ABC transporter such as its medical relevant homologue Cdr1 from Candida albicans. In the past 30 years, this ABC transporter was intensively studied in vivo and in plasma membrane vesicles. Nevertheless, these studies were limited since it was not possible to isolate and reconstitute Pdr5 in a synthetic membrane system while maintaining its activity. Here, the functional reconstitution of Pdr5 in a native-like environment in an almost unidirectional inside-out orientation is described. We demonstrate that reconstituted Pdr5 is capable of translocating short-chain fluorescent NBD lipids from the outer to the inner leaflet of the proteoliposomes. Moreover, this transporter revealed its ability to utilize other nucleotides to accomplish transport of substrates in a reconstituted system. Besides, we were also able to estimate the NTPase activity of reconstituted Pdr5 and determine the kinetic parameters for ATP, GTP, CTP, and UTP.

Potentiation by Protein Synthesis Inducers of Translational Readthrough of Pathogenic Premature Termination Codons in PTEN Isoforms.

The PTEN tumor suppressor is frequently targeted in tumors and patients with PTEN hamartoma tumor syndrome (PHTS) through nonsense mutations generating premature termination codons (PTC) that may cause the translation of truncated non-functional PTEN proteins. We have previously described a global analysis of the readthrough reconstitution of the protein translation and function of the human canonical PTEN isoform by aminoglycosides. Here, we report the efficient functional readthrough reconstitution of the PTEN translational isoform PTEN-L, which displays a minimal number of PTC in its specific N-terminal extension in association with disease. We illustrate the importance of the specific PTC and its nucleotide proximal sequence for optimal readthrough and show that the more frequent human PTEN PTC variants and their mouse PTEN PTC equivalents display similar patterns of readthrough efficiency. The heterogeneous readthrough response of the different PTEN PTC variants was independent of the length of the PTEN protein being reconstituted, and we found a correlation between the amount of PTEN protein being synthesized and the PTEN readthrough efficiency. Furthermore, combination of aminoglycosides and protein synthesis inducers increased the readthrough response of specific PTEN PTC. Our results provide insights with which to improve the functional reconstitution of human-disease-related PTC pathogenic variants from PTEN isoforms by increasing protein synthesis coupled to translational readthrough.

Final outcomes from a phase 2 trial of posoleucel in allogeneic hematopoietic cell transplant recipients

AbstractAllogeneic hematopoietic cell transplantation (allo-HCT) recipients are susceptible to viral infections. We conducted a phase 2 trial evaluating the safety and rate of clinically significant infections (CSIs; viremia requiring treatment or end-organ disease) after infusion of posoleucel, a partially HLA-matched, allogeneic, off-the-shelf, multivirus-specific T-cell investigational product for preventing CSIs with adenovirus, BK virus, cytomegalovirus, Epstein-Barr virus, human herpesvirus-6, or JC virus. This open-label trial enrolled allo-HCT recipients at high risk based on receiving grafts from umbilical cord blood, haploidentical, mismatched, or matched unrelated donors; post-HCT lymphocytes of <180/mm3; or use of T-cell depletion. Posoleucel dosing was initiated within 15 to 49 days of allo-HCT and subsequently every 14 days for up to 7 doses. The primary end point was the number of CSIs due to the 6 target viruses by week 14. Of the 26 patients enrolled, only 3 (12%) had a CSI by week 14, each with a single target virus. In vivo expansion of functional virus-specific T cells detected via interferon-γ enzyme-linked immunosorbent spot assay was associated with viral control. Persistence of posoleucel-derived T-cell clones for up to 14 weeks after the last infusion was confirmed by T-cell–receptor deep sequencing. Five patients (19%) had acute graft-versus-host disease grade 2 to 4. No patient experienced cytokine release syndrome. All 6 deaths were due to relapse or disease progression. allo-HCT recipients at high risk who received posoleucel had low rates of CSIs from 6 targeted viruses. Repeat posoleucel dosing was generally safe and well tolerated and associated with functional immune reconstitution. This trial was registered at www.ClinicalTrials.gov as #NCT04693637.

Abstract LB364: Generation and Application of a Robust Humanized Mouse Tumor Model System for Evaluation of NK-Targeting Therapeutics

Abstract With the new waves of anti-tumor immunotherapy development, novel immunotherapies based on NK cells have emerged and advanced rapidly. Considering the inherent mechanisms of NK cells, various strategies are employed in tumor immunotherapy to maximize the efficacy of NK cells, including the following: 1) immune checkpoint inhibitors to relieve NK cell inhibition signals; 2) activation of NK cell activity; 3) targeted antibody therapy for tumor cell killing via antibody-dependent cellular cytotoxicity (ADCC), and 4) cell therapy, such as CAR-NK. Although numerous NK-targeting immune therapeutics or various NK-modulating modalities have been developed or advanced toward clinical trials, there is a lack of clinically relevant preclinical animal models that can be widely accepted for the evaluation of NK-modulating agents, particularly for models that involve functional human NK cells in the test system. This study generated a humanized mouse tumor model system which involves specific reconstitution of functional and durable human NK cells in a murine system, providing a clinically relevant tumor microenvironment. We first engineered a stable monoclonal feeder cell line in vitro to expand NK cells isolated from human peripheral blood mononuclear cells (hPBMCs) using magnetic bead separation. Using this approach, the human primary NK cells were robustly and consistently amplified (up to hundreds of fold) while maintaining their purity and functionality. When compared with primary NK cells using in vitro immune assays including ADCC and FACs, the expanded NK cells showed comparable results as the parental cells. In vivo experimental results indicated that a durable and functional NK reconstitution can be established in tumor-bearing severely immune deficient mice expressing hIL-15. Furthermore, this NK-humanized tumor model system was proven to respond to standard of care (SOC) NK-targeting drugs. In summary, this novel NK humanized in vivo model may represent an effective preclinical tool for assessing NK-modulating drugs, with the hope of facilitating the development process of novel immune therapeutics employing both adaptive and innate immunity. Citation Format: Feng Lu, Weiqun Ding, Guangliang Yang, Yaoqing Jia, Zhaoxiang Wang, Xusheng Yuan, Ming Yin, Zhijian Yang, WenQing Yang. Generation and Application of a Robust Humanized Mouse Tumor Model System for Evaluation of NK-Targeting Therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB364.

ALVR106, an Off-the-Shelf, Multivirus-Specific T-Cell Therapy, for the Treatment of Respiratory Viral Infections: Results from a Phase 1, First-in-Human, Dose-Ranging Trial

Background: Community-acquired respiratory viruses such as influenza (Flu), human metapneumovirus (hMPV), parainfluenza virus (PIV), and respiratory syncytial virus (RSV) are leading causes of morbidity and mortality after hematopoietic cell transplant (HCT) or solid organ transplant (SOT). While these viruses may begin in the upper respiratory tract, ~50% of cases progress to the lower respiratory tract, potentially leading to more severe conditions such as pneumonia, bronchiolitis, bronchiolitis obliterans, and chronic lung allograft dysfunction. ALVR106 is an allogeneic, off-the-shelf, polyclonal, multi-respiratory virus T-cell therapy specific for Flu, hMPV, PIV, and RSV. ALVR106 is generated by exposing peripheral blood mononuclear cells to peptide mixtures followed by ex vivo expansion in presence of activating cytokines. The expanded T cells are Th1-polarized, polyfunctional, and selectively able to kill viral target cells. ALVR106 cells have not shown alloreactivity, attesting to their selectivity and safety for use in HCT or SOT. Methods: In this first-in-human, placebo (PBO)-controlled, dose-ranging trial, HCT or SOT recipients 17 years or older with upper or mild lower respiratory tract infections (URTI/LRTI) caused by Flu, hMPV, PIV, or RSV were randomized in a 3:1 ratio to receive ALVR106 or PBO. Four doses of ALVR106 ranging from 1 × 10 5 cells/kg to 2 × 10 6 cells/kg were planned to be given to 4 cohorts of 4 patients each. Patients receive up to 2 infusions of ALVR106 or PBO once every 14 days and will be followed for up to 1 year. The primary objective was safety as measured by treatment emergent adverse events (AEs), AEs of special interest (acute and chronic graft vs host disease (GvHD), cytokine release syndrome (CRS), infusion related reactions (IRR), and progressive dyspnea), and clinical laboratory abnormalities from baseline (BL) through 12 weeks after the last infusion. Secondary endpoints included evaluation of antiviral activity by qualitative PCR assay by nasal swab and a composite endpoint through D28 which included change from BL in Radiation Therapy Oncology Group Lung Toxicity Scale Grade and viral detection from nasal swab. Patients with an improvement in symptoms or undetectable virus were considered partial responders (PR); those with improved symptoms and undetectable virus were complete responders (CR), and those whose condition did not improve or worsen from BL were non-responders (NR). To assess functional T-cell immune reconstitution and frequency of circulating virus-specific T cells, IFN-g virus-specific ELISpot analysis was performed on serial blood samples over time. ELISpot results will be presented. Preliminary study results presented herein will be unblinded at the time of data presentation. Results: 16 patients were dosed: four in Cohorts (C) 1 and 2, five in C3, and three in C4. Table shows BL demographics, disease characteristics, and blinded results for safety and antiviral activity by composite endpoint. Cohorts 2, 3, and 4 had at least 1 patient with mild LRTI at BL, no patients in C1 had LRTI. All patients received at least 1 infusion. No patients with Flu were enrolled. In these blinded results, no differences were seen in rate or severity of AEs among cohorts. Four patients (25%) had AEs possibly related to treatment (ALVR106 or PBO); all were Grade 1 (mild) in severity. These related events were reported in 1 patient each: diarrhea, ear pressure, neutrophil count decrease, and 1 patient had pyrexia, cGvHD oral, and aGvHD skin. Eight patients (50%) had serious AEs (SAEs), none related to treatment. One patient (6%) in C3 had G2 progressive dyspnea not related to treatment. One patient (6%) in C1 had Grade I aGVHD skin and cGVHD oral; both were considered possibly related to treatment (ALVR106 or PBO). No patients had Grade II-IV aGVHD, cGVHD, IRR, or CRS. No clinically significant changes from BL or trends over time in lab abnormalities or vital signs were seen. At Day 28, all patients in C1 and C2 had undetectable respiratory virus via nasal swab, except 1 patient in C1 who missed D28 visit due to COVID-19; 4/5 (80%) in C3 were undetectable; and 2/3 (67%) in C4 were undetectable. Conclusion: In this ongoing trial-the first-in-human, double-blind, PBO-controlled trial of virus-specific T cell therapy in transplant recipients with respiratory infection-ALVR106 was safe and well tolerated, supporting its continued evaluation in HCT and SOT recipients.

Roles for diacylglycerol in synaptic vesicle priming and release revealed by complete reconstitution of core protein machinery

Here, we introduce the full functional reconstitution of genetically validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, and Complexin) for synaptic vesicle priming and release in a geometry that enables detailed characterization of the fate of docked vesicles both before and after release is triggered with Ca2+. Using this setup, we identify new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca2+-triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca2+-dependent release, and high concentrations reduce clamping and permit extensive spontaneous release. As expected, DAG also increases the number of docked, release-ready vesicles. Dynamic single-molecule imaging of Complexin binding to release-ready vesicles directly establishes that DAG accelerates the rate of SNAREpin assembly mediated by chaperones, Munc13 and Munc18. The selective effects of physiologically validated mutations confirmed that the Munc18-Syntaxin-VAMP2 "template" complex is a functional intermediate in the production of primed, release-ready vesicles, which requires the coordinated action of Munc13 and Munc18.

Platelet-rich plasma promotes skeletal muscle regeneration and neuromuscular functional reconstitution in a concentration-dependent manner in a rat laceration model

Abnormal function of injured muscle with innervation loss is a challenge in sports medicine. The difficulty of rehabilitation is regenerating and reconstructing the skeletal muscle tissue and the neuromuscular junction (NMJ). Platelet-rich plasma (PRP) releases various growth factors that may provide an appropriate niche for tissue regeneration. However, the specific mechanism of the PRP's efficacy on muscle healing remains unknown. In this study, we injected PRP with different concentration gradients (800, 1200, 1600 × 109 pl/L) or saline into a rat gastrocnemius laceration model. The results of histopathology and neuromyography show that PRP improved myofibers regeneration, facilitated electrophysiological recovery, and reduced fibrosis in a concentration-dependent manner. Furthermore, we found that PRP promotes the activity of satellite cells by upregulating the expression of the myogenic regulatory factor (MyoD, myogenin). Meanwhile, PRP promotes the regeneration and maturation of acetylcholine receptor (AChR) clusters of the Neuromuscular junction (NMJ) on the regenerative myofibers. Finally, we found that the expression of the Agrin, LRP4, and MuSK was upregulated in the PRP-treated groups, which may contribute to AChR cluster regeneration and functional recovery. The conclusions proposed a hypothesis for PRP treatment's efficacy and mechanism in muscle injuries, indicating promising application prospects.

Molecular Characterization of the ClpC AAA+ ATPase in the Biology of Chlamydia trachomatis.

Bacterial AAA+ unfoldases are crucial for bacterial physiology by recognizing specific substrates and, typically, unfolding them for degradation by a proteolytic component. The caseinolytic protease (Clp) system is one example where a hexameric unfoldase (e.g., ClpC) interacts with the tetradecameric proteolytic core ClpP. Unfoldases can have both ClpP-dependent and ClpP-independent roles in protein homeostasis, development, virulence, and cell differentiation. ClpC is an unfoldase predominantly found in Gram-positive bacteria and mycobacteria. Intriguingly, the obligate intracellular Gram-negative pathogen Chlamydia, an organism with a highly reduced genome, also encodes a ClpC ortholog, implying an important function for ClpC in chlamydial physiology. Here, we used a combination of in vitro and cell culture approaches to gain insight into the function of chlamydial ClpC. ClpC exhibits intrinsic ATPase and chaperone activities, with a primary role for the Walker B motif in the first nucleotide binding domain (NBD1). Furthermore, ClpC binds ClpP1P2 complexes via ClpP2 to form the functional protease ClpCP2P1 in vitro, which degraded arginine-phosphorylated β-casein. Cell culture experiments confirmed that higher order complexes of ClpC are present in chlamydial cells. Importantly, these data further revealed severe negative effects of both overexpression and depletion of ClpC in Chlamydia as revealed by a significant reduction in chlamydial growth. Here, again, NBD1 was critical for ClpC function. Hence, we provide the first mechanistic insight into the molecular and cellular function of chlamydial ClpC, which supports its essentiality in Chlamydia. ClpC is, therefore, a potential novel target for the development of antichlamydial agents. IMPORTANCE Chlamydia trachomatis is an obligate intracellular pathogen and the world's leading cause of preventable infectious blindness and bacterial sexually transmitted infections. Due to the high prevalence of chlamydial infections along with negative effects of current broad-spectrum treatment strategies, new antichlamydial agents with novel targets are desperately needed. In this context, bacterial Clp proteases have emerged as promising new antibiotic targets, since they often play central roles in bacterial physiology and, for some bacterial species, are even essential for survival. Here, we report on the chlamydial AAA+ unfoldase ClpC, its functional reconstitution and characterization, individually and as part of the ClpCP2P1 protease, and establish an essential role for ClpC in chlamydial growth and intracellular development, thereby identifying ClpC as a potential target for antichlamydial compounds.

Optimization of Campesterol-Producing Yeast Strains as a Feasible Platform for the Functional Reconstitution of Plant Membrane-Bound Enzymes.

Campesterol is a major phytosterol that plays important roles in regulating membrane properties and serves as the precursor to multiple specialized metabolites, such as the phytohormone brassinosteroids. Recently, we established a campesterol-producing yeast strain and extended the bioproduction to 22-hydroxycampesterol and 22-hydroxycampest-4-en-3-one, the precursors to brassinolide. However, there is a trade-off in growth due to the disrupted sterol metabolism. In this study, we enhanced the growth of the campesterol-producing yeast by partially restoring the activity of the sterol acyltransferase and engineering upstream FPP supply. Furthermore, genome sequencing analysis also revealed a pool of genes possibly associated with the altered sterol metabolism. Retro engineering implies an essential role of ASG1, especially the C-terminal asparagine-rich domain of ASG1, in the sterol metabolism of yeast especially under stress. The performance of the campesterol-producing yeast strain was enhanced with the titer of campesterol to 18.4 mg/L, and the stationary OD600 was improved by ∼33% compared to the unoptimized strain. In addition, we examined the activity of a plant cytochrome P450 in the engineered strain, which exhibits more than 9-fold higher activity than when expressed in the wild-type yeast strain. Therefore, the engineered campesterol-producing yeast strain also serves as a robust host for the functional expression of plant membrane proteins.

Retained functional normal and preleukemic HSCs at diagnosis are associated with good prognosis in DNMT3AmutNPM1mut AMLs.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by high rate of relapse and mortality. Current chemotherapies whilst successful in eradicating blasts, are less effective in eliminating relapse-causing leukemic stem cells (LSCs). Although LSCs are usually identified as CD34+CD38- cells, there is significant heterogeneity in surface marker expression, and CD34- LSCs exist particularly in NPM1mut AMLs. By analyzing diagnostic primary DNMT3AmutNPM1mut AML samples, we suggest a novel flow cytometry sorting strategy particularly useful for CD34neg AML subtypes. To enrich for LSCs independently of CD34 status, positive selection for GPR56 and negative selection for NKG2D ligands are used. We show that the functional reconstitution capacity of CD34- and CD34+ LSCs as well as their transcriptomes are very similar which support phenotypic plasticity. Furthermore, we show that although CD34+ subpopulations can contain next to LSCs also normal and/or preleukemic hematopoietic stem cells (HSCs), this is not the case in CD34-GPR56+NKG2DL- enriched LSCs which thus can be isolated with high purity. Finally, we show that patients with AML, who retain at the time of diagnosis a reserve of normal and/or preleukemic HSCs in their bone marrow able to reconstitute immunocompromised mice, have significantly longer relapse-free and overall survival than patients with AML in whom functional HSCs are no longer detectable.

Functional reconstitution of a steroidal hydroxylase from the fungus Thanatephorus cucumeris in Mycolicibacterium neoaurum for 15α-hydroxylation of progesterone

Hydroxylated steroids are important intermediates in the manufacture of clinically important medicines. Filamentous fungi play a vital role in steroid hydroxylation in industry, but they usually exhibit additional undesired hydroxylation activities. The lack of fungal molecular manipulation techniques hinders the development and application of efficient industrial hydroxylases. Therefore, for industrial production of hydroxylated steroids, it is increasingly necessary to obtain a more efficient biotransformation system with higher regio- and stereo-specific activities. In this study, we successfully developed an expression system for the Thanatephorus cucumeris CYP5150AP2 and its cognate cytochrome P450 reductase TcCPR in the engineered prokaryotic host, Mycolicibacterium neoaurum YS10 for 15α-hydroxylation of progesterone. Combined strategies were used to improve protein expression and product yield. Finally, the titer of 15α-OH progesterone reached 205.6 mg/L (with 500 mg/L progesterone as the substrate) using N-terminal sequence of MnCYP2 and CP6 promoter at 25 ℃. This is the first time a fungal P450 system was successfully functionally reconstituted in Mycolicibacterium neoaurum and significantly optimized to produce hydroxylated progesterone utilizing combinatorial optimization strategies. This work further confirmed the superiority of utilizing Mycolicibacteria as cell factories for hydroxylated steroids production via expression of eukaryotic cytochrome P450s in comparison to E. coli.

A synthetic biology tool for contact-dependent synthetic cell communication.

Cell-cell communication and signal transduction through direct cell-cell contact are found ubiquitously in biology, with examples including T-cell activation upon contact formation with antigen-presenting cell in immunological synapses and electrical signal transduction in neuronal synapses. Despite the prevalence of intercellular communication at cell-cell interfaces, it is difficult to engineer and reconstitute functional proteins at membrane-membrane interfaces. A synthetic biology tool that can recapitulate signal transduction exclusively at membrane-membrane interfaces will be useful for designing synthetic intercellular communication pathways or providing a potential replacement for damaged and malfunctional natural cell-cell communication systems. Here, we present programmable activation of a split bioluminescent protein, NanoBiT, at the membrane-membrane interface between synthetic cells. We utilize a peptide-protein pair called SpyTag and SpyCatcher as adapter proteins to mediate functional reconstitution of NanoBiT. We illustrate an example of downstream protein activation through a designed light-based communication system by using a pair of photoactivatable proteins, iLID and SspB inside synthetic cells and demonstrate iLID-SspB dimerization triggered by reconstituted NanoBiT bioluminescence.

Functional reconstitution and structural characterization of the plant hormone receptor ETR1 in lipid nanodiscs.

The plant hormone receptor ETR1 regulates many highly relevant agronomic processes. Today, significant functional and structural questions remain unanswered regarding its multi-pass transmembrane sensor domain able to bind and respond to the gaseous plant hormone ethylene at femtomolar concentrations. A significant reason for this is the lack of structural data on full-length ETR1 in a lipid environment. Herein, we present the functional reconstitution of recombinant full-length ETR1 purified and solubilized from a bacterial host into lipid nanodiscs, allowing the study of the purified plant receptor for the first time in a detergent-free membrane-like environment.

Abstract 2555: Functional reconstitution of the bacterial CO2 concentrating mechanism

Abstract 2555: Functional reconstitution of the bacterial CO2 concentrating mechanism

Final Clinical Outcomes from a Phase 2 Trial of Posoleucel, an Off-the-Shelf, Multivirus-Specific T-Cell Therapy, for the Prevention of Clinically Significant Viral Infections Post-HCT

Background: Allogeneic hematopoietic cell transplant (allo-HCT) recipients are highly susceptible to reactivation of double-stranded DNA viruses. Approximately 70% of high-risk allo-HCT recipients develop clinically significant infections (CSI, defined as viremia + treatment or end organ disease). We present results from a phase 2 trial evaluating the safety and efficacy of posoleucel, a partially HLA-matched, off-the-shelf, multivirus-specific T cell investigational product, for preventing CSIs due to adenovirus (AdV), BK virus (BKV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), or JC virus (JCV). Methods: This open-label trial enrolled high-risk allo-HCT recipients, including those with grafts from haploidentical, umbilical cord blood, mismatched unrelated donors, and matched unrelated donors with lymphocytes <180/mm3 as well as recipients who underwent T cell depletion. To be eligible, patients had to be engrafted and within 15-49 days of allo-HCT. Those with grade ≥3 GVHD and those requiring steroids (>0.5 mg/kg/day prednisone equivalent) at enrollment were ineligible. Patients received up to 7 infusions of posoleucel once every 14 days and were tested weekly for viremia using quantitative PCR assays. The primary endpoint was the number of CSIs due to AdV, BKV, CMV, EBV, HHV-6, or JCV by week 14. IFN-γ ELISpot analysis was performed for each targeted virus on serial peripheral blood samples to assess functional T cell immune reconstitution and determine the frequency of circulating virus-specific T cells detected over time. To evaluate the persistence and potential contribution of posoleucel to antiviral immune reconstitution, T-cell receptor (TCR) β immunosequencing was performed at selected timepoints. TCR sequences unique to posoleucel were identified computationally and used to track in vivo persistence in serial samples. Results: Twenty-six patients were dosed. Posoleucel dosing began a median of 42 days after allo-HCT. Twenty (77%) patients had viral DNA detected in blood (reactivation) from one or more viruses, 13 (50%) had 2 or more viral reactivations, and 7 (27%) had 3 or more viral reactivations in the 14 weeks after the initiation of posoleucel dosing (Figure). However, by week 14, only 3 patients (12%) had CSIs (2 CMV viremia requiring treatment and 1 EBV-post-transplant lymphoproliferative disorder). No patient had a CSI with more than 1 virus. Nine patients (35%) experienced adverse events that were possibly or probably related to treatment. Eight patients (31%) had serious adverse events (SAEs), 2 of which were judged to be possibly related to treatment (skin and pulmonary GVHD) and 1 probably related to treatment (allergic reaction). Overall, 8 patients (31%) had acute GVHD Grade II-IV. One patient (4%) discontinued posoleucel due to a treatment-emergent SAE of chronic pulmonary GVHD. No patient experienced cytokine release syndrome. TCR sequencing data (N=12) indicated that posoleucel was detectable in vivo during the dosing phase of the study and for up to 12 weeks after the last infusion, with polyclonal VSTs detected in all 12 patients. Six of the 7 patients with available ELISpot data demonstrated IFN-γ-producing virus-specific T cell responses coincident with viral replication. The confirmed persistence of posoleucel and expansion of IFN-γ-producing virus-specific T cells directed against reactivating viruses, suggests a contribution of posoleucel to functional immune reconstitution. Conclusion: Results from this open-label phase 2 trial show that high-risk allo-HCT patients who received posoleucel had lower than expected rates of clinically significant viral infections from the 6 targeted viruses despite the observed and expected low-level viral reactivations throughout the study. Repeat dosing of posoleucel was generally safe and well tolerated. Posoleucel VSTs were detected in vivo for up to 26 weeks, and reconstituting T cells demonstrated functional specificity for multiple viruses. A placebo-controlled global phase 3 trial evaluating the safety and tolerability of posoleucel for the prevention of clinically significant AdV, BKV, CMV, EBV, HHV-6, and JCV infections is ongoing (NCT05305040). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Early stimulated immune responses predict clinical disease severity in hospitalized COVID-19 patients

BackgroundThe immune pathogenesis underlying the diverse clinical course of COVID-19 is poorly understood. Currently, there is an unmet need in daily clinical practice for early biomarkers and improved risk stratification tools to help identify and monitor COVID-19 patients at risk of severe disease.MethodsWe performed longitudinal assessment of stimulated immune responses in 30 patients hospitalized with COVID-19. We used the TruCulture whole-blood ligand-stimulation assay applying standardized stimuli to activate distinct immune pathways, allowing quantification of cytokine responses. We further characterized immune cell subsets by flow cytometry and used this deep immunophenotyping data to map the course of clinical disease within and between patients.ResultsHere we demonstrate impairments in innate immune response pathways at time of COVID-19 hospitalization that are associated with the development of severe disease. We show that these impairments are transient in those discharged from hospital, as illustrated by functional and cellular immune reconstitution. Specifically, we identify lower levels of LPS-stimulated IL-1β, and R848-stimulated IL-12 and IL-17A, at hospital admission to be significantly associated with increasing COVID-19 disease severity during hospitalization. Furthermore, we propose a stimulated immune response signature for predicting risk of developing severe or critical COVID-19 disease at time of hospitalization, to validate in larger cohorts.ConclusionsWe identify early impairments in innate immune responses that are associated with subsequent COVID-19 disease severity. Our findings provide basis for early identification of patients at risk of severe disease which may have significant implications for the early management of patients hospitalized with COVID-19.

Non-Ionic Inulin-Based Polymer Nanodiscs Enable Functional Reconstitution of a Redox Complex Composed of Oppositely Charged CYP450 and CPR in a Lipid Bilayer Membrane.

Although polymer-based lipid nanodiscs are increasingly used in the structural studies of membrane proteins, the charge of the belt-forming polymer is a major limitation for functional reconstitution of membrane proteins possessing an opposite net charge to that of the polymer. This limitation also rules out the reconstitution of a protein-protein complex composed of oppositely charged membrane proteins. In this study, we report the first successful functional reconstitution of a membrane-bound redox complex constituting a cationic cytochrome P450 (CYP450) and an anionic cytochrome P450 reductase (CPR) in non-ionic inulin-based lipid nanodiscs. The gel-to-liquid-crystalline phase-transition temperature (Tm) of DMPC:DMPG (7:3 w/w) lipids in polymer nanodiscs was determined by differential scanning calorimetry (DSC) and 31P NMR experiments. The CYP450-CPR redox complex reconstitution in polymer nanodiscs was characterized by size-exclusion chromatography (SEC), and the electron transfer kinetics was carried out using the stopped-flow technique under anaerobic conditions. The Tm of DMPC:DMPG (7:3 w/w) in polymer nanodiscs measured from 31P NMR agrees with that obtained from DSC and was found to be higher than that for liposomes due to the decreased cooperativity of lipids present in the nanodiscs. The stopped-flow measurements revealed the CYP450-CPR redox complex reconstituted in nanodiscs to be functional, and the electron transfer kinetics was found to be temperature-dependent. Based on the successful demonstration of the use of non-ionic inulin-based polymer nanodiscs reported in this study, we expect them to be useful in studying the function and structures of a variety of membrane proteins/complexes irrespective of the charge of the molecular components. Since the polymer nanodiscs were shown to align in an externally applied magnetic field, they can also be used to measure residual dipolar couplings (RDCs) and residual quadrupolar couplings (RQCs) for various molecules ranging from small molecules to soluble proteins and nucleic acids.

The physiological cargo adaptor of kinesin-2 functions as an evolutionary conserved lockpick

Specific recognition of cellular cargo and efficient transport to its correct intracellular destination is an infrastructural challenge faced by most eukaryotic cells. This remarkable deed is accomplished by processive motor proteins that are subject to robust regulatory mechanisms. The first level of regulation entails the ability of the motor to suppress its own activity. This autoinhibition is eventually relieved by specific cargo binding. To better understand the role of the cargo during motor activation, we dissected the activation mechanism of the ciliary homodimeric kinesin-2 from Caenorhabditis elegans by its physiological cargo. In functional reconstitution assays, we identified two cargo adaptor proteins that together are necessary and sufficient to allosterically activate the autoinhibited motor. Surprisingly, the orthologous adaptor proteins from the unicellular green algae Chlamydomonasreinhardtii also fully activated the kinesin-2 from worm, even though C. reinhardtii itself lacks a homodimeric kinesin-2 motor. The latter suggested that a motor activation mechanism similar to the C. elegans model existed already well before metazoans evolved, and prompted us to scrutinize predicted homodimeric kinesin-2 orthologs in other evolutionarily distant eukaryotes. We show that the ciliate Tetrahymena thermophila not only possesses a homodimeric kinesin-2 but that it also shares the same allosteric activation mechanism that we delineated in the C. elegans model. Our results point to a much more fundamental role of homodimeric kinesin-2 in intraflagellar transport (IFT) than previously thought and warrant further scrutiny of distantly related organisms toward a comprehensive picture of the IFT process and its evolution.