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Summary Understanding of protein complex assembly and the effect of ligand binding on their native topologies is integral to discerning how alterations in their architecture can affect function. Probing the disassembly pathway may offer insight into the mechanisms through which various subunits self-assemble into complexes. Here, a gas-phase dissociation method, surface-induced dissociation (SID) coupled with ion mobility (IM), was utilized to determine whether disassembly pathways are consistent with the assembly of three homotetramers and to probe the effects of ligand binding on conformational flexibility and tetramer stability. The results indicate that the smaller interface in the complex is initially cleaved upon dissociation, conserving the larger interface, and suggest that assembly of a D 2 homotetramer from its constituent monomers occurs via a C 2 dimer intermediate. In addition, we demonstrate that ligand-mediated changes in tetramer SID dissociation behavior are dependent on where and how the ligand binds. [ABSTRACT FROM AUTHOR]

The hypothalamus regulates innate social interactions, but how hypothalamic neurons transduce sex-related sensory signals emitted by conspecifics to trigger appropriate behaviors remains unclear. Here, we addressed this issue by identifying specific hypothalamic neurons required for sensing conspecific male cues relevant to inter-male aggression. By in vivo recording of neuronal activities in behaving mice, we showed that neurons expressing dopamine transporter (DAT+) in the ventral premammillary nucleus (PMv) of the hypothalamus responded to male urine cues in a vomeronasal organ (VNO)-dependent manner in naive males. Retrograde trans-synaptic tracing further revealed a specific group of neurons in the bed nucleus of the stria terminalis (BNST) that convey male-relevant signals from VNO to PMv. Inhibition of PMvDAT+ neurons abolished the preference for male urine cues and reduced inter-male attacks, while activation of these neurons promoted urine marking and aggression. Thus, PMvDAT+ neurons exemplify a hypothalamic node that transforms sex-related chemo-signals into recognition and behaviors. • PMvDAT+ neurons selectively tune to gonad-intact conspecific male urine cues • v-BNST relay male-relevant chemosensory information from VNO to PMvDAT+ neurons • Inhibition of PMvDAT+ neurons blocks male urine preference and decreases attack • Activation of PMvDAT+ neurons promotes urine marking and aggression How hypothalamic neurons transduce sex signals emitted by conspecifics to trigger appropriate behavioral outputs remains unclear. Chen et al. identify PMvDAT+ neurons as a key node that extract male-relevant information from the chemosensory pathway, via the v-BNST, to coordinate multiple behavioral responses, including male urine preference, inter-male aggression, and urine marking. [ABSTRACT FROM AUTHOR]

Iron (Fe) storage in plant seeds is not only necessary for seedling establishment following germination but is also a major source of dietary Fe for humans and other animals. Accumulation of Fe in seeds is known to be low during early seed development. However, the underlying mechanism and biological significance remain elusive. Here, we show that reduced expression of Arabidopsis YABBY transcription factor INNER NO OUTER (INO) increases embryonic Fe accumulation, while transgenic overexpression of INO results in the opposite effect. INO is highly expressed during early seed development, and decreased INO expression increases the expression of NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN 1 (NRAMP1), which encodes a transporter that contributes to seed Fe loading. The relatively high embryonic Fe accumulation conferred by decreased INO expression is rescued by the nramp1 loss-of-function mutation. We further demonstrated that INO represses NRAMP1 expression by binding to NRAMP1 -specific promoter region. Interestingly, we found that excessive Fe loading into developing seeds of ino mutants results in greater oxidative damage, leading to increased cell death and seed abortion, a phenotype that can be rescued by the nramp1 mutation. Taken together, these results indicate that INO plays an important role in safeguarding reproduction by reducing Fe loading into developing seeds by repressing NRAMP1 expression. Iron (Fe) accumulation in plant seeds is essential for both seedling establishment and human dietary Fe acquisition. Nevertheless, how seed Fe accumulation is controlled within plants remains largely unknown. This study reveals that the YABBY transcription factor INO plays an important role in safeguarding reproduction by reducing Fe loading into early developing seeds through the repression of NRAMP1 expression. [ABSTRACT FROM AUTHOR]

Proteolysis-targeting chimeras (PROTACs) represent a new direction in small-molecule therapeutics whereby a heterobifunctional linker to a protein of interest (POI) induces its ubiquitination-based proteolysis by recruiting an E3 ligase. Here, we show that charge reduction, native mass spectrometry, and gas-phase activation methods combine for an in-depth analysis of a PROTAC-linked ternary complex. Electron capture dissociation (ECD) of the intact POI-PROTAC-VCB complex (a trimeric subunit of an E3 ubiquitin ligase) promotes POI dissociation. Collision-induced dissociation (CID) causes elimination of the nonperipheral PROTAC, producing an intact VCB-POI complex not seen in solution but consistent with PROTAC-induced protein-protein interactions. In addition, we used ion mobility spectrometry (IMS) and collisional activation to identify the source of this unexpected dissociation. Together, the evidence shows that this integrated approach can be used to screen for ternary complex formation and PROTAC-protein contacts and may report on PROTAC-induced protein-protein interactions, a characteristic correlated with PROTAC selectivity and efficacy. • Native MS yields structural analysis of a PROTAC-induced ternary complex • Multiple activation methods probe unique subunit dissociation pathways • Conformational analysis identifies two gas-phase PROTAC complex topologies • Subsequent subunit dissociation interrogates relevant protein-protein interactions Song et al. demonstrate the utility of native mass spectrometry, ion mobility spectrometry, and multiple activation methods to characterize PROTAC-induced complex topologies and conformation-specific protein-protein interactions. [ABSTRACT FROM AUTHOR]

Generation of the first T lymphocytes in the human embryo involves the emergence, migration, and thymus seeding of lymphoid progenitors together with concomitant thymus organogenesis, which is the initial step to establish the entire adaptive immune system. However, the cellular and molecular programs regulating this process remain unclear. We constructed a single-cell transcriptional landscape of human early T lymphopoiesis by using cells from multiple hemogenic and hematopoietic sites spanning embryonic and fetal stages. Among heterogenous early thymic progenitors, one subtype shared common features with a subset of lymphoid progenitors in fetal liver that are known as thymus-seeding progenitors. Unbiased bioinformatics analysis identified a distinct type of pre-thymic lymphoid progenitors in the aorta-gonad-mesonephros (AGM) region. In parallel, we investigated thymic epithelial cell development and potential cell-cell interactions during thymus organogenesis. Together, our data provide insights into human early T lymphopoiesis that prospectively direct T lymphocyte regeneration, which might lead to development of clinical applications. • Single-cell RNA-seq resolves human early T lymphopoiesis and thymus organogenesis • The first ETPs in the thymus share transcriptional features of TSPs in human fetal liver • Pre-thymic lymphoid progenitors are transcriptomically identified in human AGM region • Cortical TECs mature earlier than medullary TECs in human thymic primordium A comprehensive and precise understanding of early T lymphopoiesis and thymus organogenesis in human embryo is lacking. By performing single-cell transcriptomic analysis on human embryonic and fetal hematopoietic organs, Zeng et al. reveal distinct subpopulations of early thymic progenitors, identify thymus-seeding progenitors in fetal liver and pre-thymic lymphoid progenitors in the aorta-gonad-mesonephros region, and characterize early development of thymic epithelial cells. [ABSTRACT FROM AUTHOR]