Transmembrane topology and oligomeric nature of an astrocytic membrane protein, MLC1

MLC1 is a membrane protein mainly expressed in astrocytes, and genetic mutations lead to the development of a leukodystrophy, megalencephalic leukoencephalopathy with subcortical cysts disease. Currently, the biochemical properties of the MLC1 protein are largely unknown. In this study, we aimed to characterize the transmembrane (TM) topology and oligomeric nature of the MLC1 protein. Systematic immunofluorescence staining data revealed that the MLC1 protein has eight TM domains and that both the N- and C-terminus face the cytoplasm. We found that MLC1 can be purified as an oligomer and could form a trimeric complex in both detergent micelles and reconstituted proteoliposomes. Additionally, a single-molecule photobleaching experiment showed that MLC1 protein complexes could consist of three MLC1 monomers in the reconstituted proteoliposomes. These results can provide a basis for both the high-resolution structural determination and functional characterization of the MLC1 protein.

Oligomeric Structural Transition of HspB1 from Chinese Hamster

HspB1 is a mammalian sHsp that is ubiquitously expressed in almost all tissues and involved in regulating many vital functions. Although the recent crystal structure of human HspB1 showed that 24 monomers form the oligomeric complex of human HspB1 in a spherical configuration, the molecular architecture of HspB1 is still controversial. In this study, we examined the oligomeric structural change of CgHspB1 by sedimentation velocity analytical ultracentrifugation. At the low temperature of 4 °C, CgHspB1 exists as an 18-mer, probably a trimeric complex of hexamers. It is relatively unstable and partially dissociates into small oligomers, hexamers, and dodecamers. At elevated temperatures, the 24-mer was more stable than the 18-mer. The 24-mer is also in dynamic equilibrium with the dissociated oligomers in the hexameric unit. The hexamer further dissociates to dimers. The disulfide bond between conserved cysteine residues seems to be partly responsible for the stabilization of hexamers. The N-terminal domain is involved in the assembly of dimers and the interaction between hexamers. It is plausible that CgHspB1 expresses a chaperone function in the 24-mer structure.

Antibody responses to BNT162b2 mRNA vaccine: infection-naive individuals with abdominal obesity warrant attention

Objective. The excess of visceral adipose tissue might hinder and delay the immune response. How people with abdominal obesity will respond to mRNA vaccines against SARS-CoV-2 is yet to be established. We evaluated SARS-CoV-2-specific antibody responses after the first and second dose of the BNT162b2 mRNA vaccine comparing the response of individuals affected by abdominal obesity (AO) to those without, discerning between individuals with or without prior infection. Methods. IgG neutralizing antibodies against the Trimeric complex (IgG-TrimericS) were measured at four time points: at baseline, at day 21 after vaccine dose-1, at one month and three months after dose-2. Nucleocapsid antibodies were assessed to detect prior SARS-CoV-2 infection. Waist circumference was measured to determine abdominal obesity. Results. Between the first and third month after vaccine dose-2, the drop in IgG-TrimericS levels was more remarkable in individuals with AO compared to those without AO (2.44 fold [95%CI: 2.22-2.63] vs 1.82 fold [95%CI: 1.69-1.92], respectively, p<0.001). Multiple linear regression confirmed this result even when adjusting for possible confounders (p<0.001). Conclusions. Our findings highlight the need to extend the duration of serological monitoring of antibody levels in infection-naive individuals with abdominal obesity, a higher-risk population category in terms of possible weaker antibody response.

Structure of the human C9orf72-SMCR8 complex reveals a multivalent protein interaction architecture

A major cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) spectrum disorder is the hexanucleotide G4C2 repeat expansion in the first intron of the C9orf72 gene. Many underlying mechanisms lead to manifestation of disease that include toxic gain of function by repeat G4C2 RNAs, dipeptide repeat proteins, and a reduction of the C9orf72 gene product. The C9orf72 protein interacts with SMCR8 and WDR41 to form a trimeric complex and regulates multiple cellular pathways including autophagy. Here, we report the structure of the C9orf72-SMCR8 complex at 3.8 Å resolution using single-particle cryo-electron microscopy (cryo-EM). The structure reveals 2 distinct dimerization interfaces between C9orf72 and SMCR8 that involves an extensive network of interactions. Homology between C9orf72-SMCR8 and Folliculin-Folliculin Interacting Protein 2 (FLCN-FNIP2), a GTPase activating protein (GAP) complex, enabled identification of a key residue within the active site of SMCR8. Further structural analysis suggested that a coiled-coil region within the uDenn domain of SMCR8 could act as an interaction platform for other coiled-coil proteins, and its deletion reduced the interaction of the C9orf72-SMCR8 complex with FIP200 upon starvation. In summary, this study contributes toward our understanding of the biological function of the C9orf72-SMCR8 complex.

Live cell dynamics of the NF-Y transcription factor

Transcription factors (TFs) are core players in the control of gene expression, evolutionarily selected to recognise a subset of specific DNA sequences and nucleate the recruitment of the transcriptional machinery. How TFs assemble and move in the nucleus to locate and bind their DNA targets and cause a transcriptional response, remains mostly unclear. NF-Y is a highly conserved, heterotrimeric TF with important roles in both housekeeping and lineage-specific gene expression, functioning as a promoter organiser. Despite a large number of biochemical, structural and genomic studies of NF-Y, there is a lack of experiments in single living cells; therefore, basic assumptions of NF-Y biology remain unproven in vivo. Here we employ a series of dynamic fluorescence microscopy methods (FLIM-FRET, NB, RICS and FRAP) to study NF-Y dynamics and complex formation in live cells. Specifically, we provide quantitative measurement of NF-Y subunit association and diffusion kinetics in the nucleus that collectively suggest NF-Y to move and bind chromatin as a trimeric complex in vivo.

TAZ/TEAD complex regulates TGF-β1-mediated fibrosis in iPSC-derived renal organoids

Chronic kidney disease (CKD) progresses by replacement of functional tissue compartments with fibrosis, representing a maladaptive repair process. Shifting kidney repair towards a physiologically-intact architecture, rather than fibrosis, is key to blocking CKD progression. In this study, we developed a fibrosis model that uses human induced pluripotent stem cell (iPSC)-based three-dimensional renal organoids, in which exogenous TGF-β1 induces production of extracellular matrix. In these organoids, TGF- β1 increased transcription factor tafazzin (TAZ) expression. Further, in human kidney biopsies, nuclear TAZ expression was markedly increased in mild and moderate fibrosis. In cultured renal tubular cells expressing a fibrogenic program, TAZ formed a trimeric complex with phosphorylated mothers against decapentaplegic homolog 3 (p-SMAD3) and TEA domain protein (TEAD)-4. Overexpression of TEAD4 protein suppressed collagen-1α1 (COL1A1) promoter activity, and expression of TAZ attenuated this inhibition. INT-767, a dual bile acid receptor agonist binding farnesoid X receptor (FXR) and the Takeda G protein-coupled receptor 5 (TGR5), decreased the TGF-β1-induced increase in p-SMAD3 and TAZ, and preserved renal organoid architecture. These data demonstrate, in an iPSC-derived renal organoid fibrosis model, that INT767 prevents fibrosis programs early in the course of tubular injury through modulation of the TEAD4/TAZ pathway.

The dimeric Golgi protein Gorab binds to Sas6 as a monomer to mediate centriole duplication

The duplication and 9-fold symmetry of the Drosophila centriole requires that the cartwheel molecule, Sas6, physically associates with Gorab, a trans-Golgi component. How Gorab achieves these disparate associations is unclear. Here we use hydrogen-deuterium exchange mass spectrometry to define Gorab's interacting surfaces that mediate its sub-cellular localization. We identify a core stabilization sequence within Gorab's C-terminal coiled-coil domain that enables homodimerization, binding to Rab6, and thereby trans-Golgi localization. By contrast, part of the Gorab monomer's coiled-coil domain undergoes an anti-parallel interaction with a segment of the parallel coiled-coil dimer of Sas6. This stable hetero-trimeric complex can be visualized by electron microscopy. Mutation of a single leucine residue in Sas6's Gorab-binding domain generates a Sas6 variant with a 16-fold reduced binding affinity for Gorab that can not support centriole duplication. Thus Gorab dimers at the Golgi exist in equilibrium with Sas-6 associated monomers at the centriole to balance Gorab's dual role.

Homeodomain protein Six4 prevents the generation of supernumerary Drosophila type II neuroblasts and premature differentiation of intermediate neural progenitors

In order to boost the number and diversity of neurons generated from neural stem cells, intermediate neural progenitors (INPs) need to maintain their homeostasis by avoiding both dedifferentiation and premature differentiation. Elucidating how INPs maintain homeostasis is critical for understanding the generation of brain complexity and various neurological diseases resulting from defects in INP development. Here we report that Six4 expressed in Drosophila type II neuroblast (NB) lineages prevents the generation of supernumerary type II NBs and premature differentiation of INPs. We show that loss of Six4 leads to supernumerary type II NBs likely due to dedifferentiation of immature INPs (imINPs). We provide data to further demonstrate that Six4 inhibits the expression and activity of PntP1 in imINPs in part by forming a trimeric complex with Earmuff and PntP1. Furthermore, knockdown of Six4 exacerbates the loss of INPs resulting from the loss of PntP1 by enhancing ectopic Prospero expression in imINPs, suggesting that Six4 is also required for preventing premature differentiation of INPs. Taken together, our work identified a novel transcription factor that likely plays important roles in maintaining INP homeostasis.

Multimerization- and glycosylation-dependent receptor binding of SARS-CoV-2 spike proteins

Receptor binding studies on sarbecoviruses would benefit from an available toolkit of recombinant spike proteins, or domains thereof, that recapitulate receptor binding properties of native viruses. We hypothesized that trimeric Receptor Binding Domain (RBD) proteins would be suitable candidates to study receptor binding properties of SARS-CoV-1 and -2. Here we created monomeric and trimeric fluorescent RBD proteins, derived from adherent HEK293T, as well as in GnTI-/- mutant cells, to analyze the effect of complex vs high mannose glycosylation on receptor binding. The results demonstrate that trimeric, complex glycosylated proteins are superior in receptor binding compared to monomeric and immaturely glycosylated variants. Although differences in binding to commonly used cell lines were minimal between the different RBD preparations, substantial differences were observed when respiratory tissues of experimental animals were stained. The RBD trimers demonstrated distinct ACE2 expression profiles in bronchiolar ducts and confirmed the higher binding affinity of SARS-CoV-2 over SARS-CoV-1. Our results show that complex glycosylated trimeric RBD proteins are attractive to analyze sarbecovirus receptor binding and explore ACE2 expression profiles in tissues.