ABSTRACT
Clinical applications of CAR-T cells are limited by the scarcity of tumor-specific targets and are often afflicted with the same on-target/off-tumor toxicities that plague other cancer treatments. A new promising strategy to enforce tumor selectivity is the use of logic-gated, two-receptor systems. One well-described application is termed Tmod™, which originally utilized a blocking inhibitory receptor directed towards HLA-I target antigens to create a protective NOT gate. Here we show that the function of Tmod blockers targeting non-HLA-I antigens is dependent on the height of the blocker antigen and is generally compatible with small, membrane-proximal targets. We compensate for this apparent limitation by incorporating modular hinge units to artificially extend or retract the ligand-binding domains relative to the effector cell surface, thereby modulating Tmod activator and blocker function. By accounting for structural differences between activator and blocker targets, we developed a set of simple geometric parameters for Tmod receptor design that enables targeting of blocker antigens beyond HLA-I, thereby broadening the applications of logic-gated cell therapies.
Subject(s)
Neoplasms , T-Lymphocytes , Humans , Antigens/metabolismABSTRACT
Bispecific antibodies are a powerful new class of therapeutics, but their development often requires enormous amounts of time and resources. Here, we describe a high-throughput protocol for cloning, expressing, purifying, and evaluating bispecific antibodies. This protocol enables the rapid screening of large panels of bispecific molecules to identify top candidates for further development. For complete details on the use and execution of this protocol, please refer to Estes et al. (2021).
Subject(s)
Antibodies, Bispecific , Antibodies, Bispecific/therapeutic use , Cloning, MolecularABSTRACT
The worldwide pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unprecedented and the impact on public health and the global economy continues to be devastating. Although early therapies such as prophylactic antibodies and vaccines show great promise, there are concerns about the long-term efficacy and universal applicability of these therapies as the virus continues to mutate. Thus, protein-based immunogens that can quickly respond to viral changes remain of continued interest. The Spike protein, the main immunogen of this virus, displays a highly dynamic trimeric structure that presents a challenge for therapeutic development. Here, guided by the structure of the Spike trimer, we rationally design new Spike constructs that show a uniquely high stability profile while simultaneously remaining locked into the immunogen-desirable prefusion state. Furthermore, our approach emphasizes the relationship between the highly conserved S2 region and structurally dynamic Receptor Binding Domains (RBD) to enable vaccine development as well as the generation of antibodies able to resist viral mutation.
Subject(s)
Protein Interaction Domains and Motifs/genetics , Protein Interaction Domains and Motifs/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Binding Sites/genetics , Binding Sites/immunology , COVID-19/immunology , COVID-19/pathology , Cell Line , HEK293 Cells , Humans , Protein Domains/genetics , Protein Domains/immunology , Protein Stability , SARS-CoV-2/geneticsABSTRACT
Metazoan microRNAs require specific maturation steps initiated by Microprocessor, comprising Drosha and DGCR8. Lack of structural information for the assembled complex has hindered an understanding of how Microprocessor recognizes primary microRNA transcripts (pri-miRNAs). Here we present a cryoelectron microscopy structure of human Microprocessor with a pri-miRNA docked in the active site, poised for cleavage. The basal junction is recognized by a four-way intramolecular junction in Drosha, triggered by the Belt and Wedge regions that clamp over the ssRNA. The belt is important for efficiency and accuracy of pri-miRNA processing. Two dsRBDs form a molecular ruler to measure the stem length between the two dsRNA-ssRNA junctions. The specific organization of the dsRBDs near the apical junction is independent of Drosha core domains, as observed in a second structure in the partially docked state. Collectively, we derive a molecular model to explain how Microprocessor recognizes a pri-miRNA and accurately identifies the cleavage site.
Subject(s)
MicroRNAs/chemistry , RNA-Binding Proteins/chemistry , Ribonuclease III/chemistry , Cryoelectron Microscopy , Humans , MicroRNAs/metabolism , Models, Molecular , Protein Conformation , RNA, Double-Stranded/chemistry , RNA, Double-Stranded/metabolism , RNA-Binding Proteins/metabolism , Ribonuclease III/metabolismABSTRACT
DDX17, a DEAD-box ATPase, is a multifunctional helicase important for various RNA functions, including microRNA maturation. Key questions for DDX17 include how it recognizes target RNAs and influences their structures, as well as how its ATPase activity may be regulated. Through crystal structures and biochemical assays, we show the ability of the core catalytic domains of DDX17 to recognize specific sequences in target RNAs. The RNA sequence preference of the catalytic core is critical for DDX17 to directly bind and remodel a specific region of primary microRNAs 3' to the mature sequence, and consequently enhance processing by Drosha. Furthermore, we identify an intramolecular interaction between the N-terminal tail and the DEAD domain of DDX17 to have an autoregulatory role in controlling the ATPase activity. Thus, we provide the molecular basis for how cognate RNA recognition and functional outcomes are linked for DDX17.
Subject(s)
Adenosine Triphosphatases/metabolism , DEAD-box RNA Helicases/chemistry , DEAD-box RNA Helicases/metabolism , MicroRNAs/chemistry , MicroRNAs/metabolism , RNA Processing, Post-Transcriptional , Crystallography, X-Ray , DEAD-box RNA Helicases/genetics , HEK293 Cells , Homeostasis , Humans , MicroRNAs/genetics , Protein ConformationABSTRACT
The original version of this Article contained an error in Fig. 1. In panel d, the model on the right of the panel was incorrectly labeled '+Heme', and should have read '- Heme'. This has now been corrected in both the PDF and HTML versions of the Article.
ABSTRACT
MicroRNAs regulate the expression of many proteins and require specific maturation steps. Primary microRNA transcripts (pri-miRs) are cleaved by Microprocessor, a complex containing the RNase Drosha and its partner protein, DGCR8. Although DGCR8 is known to bind heme, the molecular role of heme in pri-miR processing is unknown. Here we show that heme is critical for Microprocessor to process pri-miRs with high fidelity. Furthermore, the degree of inherent heme dependence varies for different pri-miRs. Heme-dependent pri-miRs fail to properly recruit Drosha, but heme-bound DGCR8 can correct erroneous binding events. Rather than changing the oligomerization state, heme induces a conformational change in DGCR8. Finally, we demonstrate that heme activates DGCR8 to recognize pri-miRs by specifically binding the terminal loop near the 3' single-stranded segment.
Subject(s)
Heme/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , RNA-Binding Proteins/metabolism , Ribonuclease III/metabolism , Amino Acid Substitution , Base Sequence , HEK293 Cells , Heme/chemistry , Humans , MicroRNAs/chemistry , Models, Biological , Models, Molecular , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Nucleic Acid Conformation , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , RNA Processing, Post-Transcriptional , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Ribonuclease III/chemistry , Ribonuclease III/geneticsABSTRACT
The amygdala is a heterogeneous, medial temporal lobe structure that has been implicated in the formation, expression and extinction of emotional memories. This structure is composed of numerous nuclei that vary in cytoarchitectonics and neural connections. In particular the lateral nucleus of the amygdala (LA), central nucleus of the amygdala (CeA), and the basal (B) nucleus contribute an essential role to emotional learning. However, to date it is still unclear to what extent these nuclei differ at the molecular level. Therefore we have performed whole genome gene expression analysis on these nuclei to gain a better understanding of the molecular differences and similarities among these nuclei. Specifically the LA, CeA and B nuclei were laser microdissected from the rat brain, and total RNA was isolated from these nuclei and subjected to RNA amplification. Amplified RNA was analyzed by whole genome microarray analysis which revealed that 129 genes are differentially expressed among these nuclei. Notably gene expression patterns differed between the CeA nucleus and the LA and B nuclei. However gene expression differences were not considerably different between the LA and B nuclei. Secondary confirmation of numerous genes was performed by in situ hybridization to validate the microarray findings, which also revealed that for many genes, expression differences among these nuclei were consistent with the embryological origins of these nuclei. Knowing the stable gene expression differences among these nuclei will provide novel avenues of investigation into how these nuclei contribute to emotional arousal and emotional learning, and potentially offer new genetic targets to manipulate emotional learning and memory.
