SRPK2 Mediates HBV Core Protein Phosphorylation and Capsid Assembly via Docking Interaction.

Members of the serine-arginine protein kinase (SRPK) family, SRPK1 and SRPK2, phosphorylate the hepatitis B core protein (Cp) and are crucial for pregenomic RNA encapsidation during viral nucleocapsid assembly. Among them, SRPK2 exhibits higher kinase activity toward Cp. In this study, we identified Cp sites that are phosphorylated by SRPK2 and demonstrated that the kinase utilizes an SRPK-specific docking groove to interact with and regulate the phosphorylation of the C-terminal arginine rich domain of Cp. We determined that direct interaction between the docking groove of SRPK2 and unphosphorylated Cp inhibited premature viral capsid assembly in vitro, whereas the phosphorylation of the viral protein reactivated the process. Pull-down assays together with the new cryo-electron microscopy structure of the HBV capsid in complex with SRPK2 revealed that the kinases decorate the surface of the viral capsid by interacting with the C-terminal domain of Cp, underscoring the importance of the docking interaction in regulating capsid assembly and pregenome packaging. Moreover, SRPK2-knockout in HepG2 cells suppressed Cp phosphorylation, indicating that SRPK2 is an important cellular kinase for HBV life cycle.

The beauty and complexity of the small heat shock proteins: a report on the proceedings of the fourth workshop on small heat shock proteins.

The Fourth Cell Stress Society International workshop on small heat shock proteins (sHSPs), a follow-up to successful workshops held in 2014, 2016 and 2018, took place as a virtual meeting on the 17-18 November 2022. The meeting was designed to provide an opportunity for those working on sHSPs to reconnect and discuss their latest work. The diversity of research in the sHSP field is reflected in the breadth of topics covered in the talks presented at this meeting. Here we summarise the presentations at this meeting and provide some perspectives on exciting future topics to be addressed in the field.

Site-Selective Lysine Acetylation of Human Immunoglobulin G for Immunoliposomes and Bispecific Antibody Complexes.

Site-selective acetylation of a single lysine residue in a protein that reaches a lysine acetyltransferase's accuracy, precision, and reliability is challenging. Here, we report a peptide-guided, proximity-driven group transfer reaction that acetylates a single lysine residue, Lys 248, of the fragment crystallizable region (Fc region) in the heavy chain of the human Immunoglobulin G (IgG). An Fc-interacting peptide bound with the Fc domain and positioned a phenolic ester close to Lys 248, which induced a nucleophilic reaction and resulted in the transfer of an acetyl group to Lys 248. The acetylation reaction proceeded to a decent yield under the physiological condition without the need for deglycosylation, unnatural amino acids, or catalysts. Along with acetylation, functional moieties such as azide, alkyne, fluorescent molecules, or biotin could also be site-selectively installed on Lys 248, allowing IgG's further derivatization. We then synthesized an antibody-lipid conjugate and constructed antibody-conjugated liposomes (immunoliposomes), targeting HER2-positive (HER2+) cancer cells. We also built a bispecific antibody complex (bsAbC) covalently linking an anti-HER2 antibody and an anti-CD3 antibody. The bsAbC showed in vitro effector-cell-mediated cytotoxicity at nanomolar concentrations. Compared with bispecific antibodies (bsAbs), bsAbCs are constructed based on native IgGs and contain two antigen-binding sites to each antigen, twice that of bsAbs. Altogether, this work reports a method of site-selective acetylation of native antibodies, highlights a facile way of site-selective IgG functionalization, and underscores the potential of bsAbCs in cancer immunotherapy.

A distinct giant coat protein complex II vesicle population in Arabidopsis thaliana.

Plants live as sessile organisms with large-scale gene duplication events and subsequent paralogue divergence during evolution. Notably, plant paralogues are expressed tissue-specifically and fine-tuned by phytohormones during various developmental processes. The coat protein complex II (COPII) is a highly conserved vesiculation machinery mediating protein transport from the endoplasmic reticulum to the Golgi apparatus in eukaryotes1. Intriguingly, Arabidopsis COPII paralogues greatly outnumber those in yeast and mammals2-6. However, the functional diversity and underlying mechanism of distinct COPII paralogues in regulating protein endoplasmic reticulum export and coping with various adverse environmental stresses are poorly understood. Here we characterize a novel population of COPII vesicles produced in response to abscisic acid, a key phytohormone regulating abiotic stress responses in plants. These hormone-induced giant COPII vesicles are regulated by an Arabidopsis-specific COPII paralogue and carry stress-related channels/transporters for alleviating stresses. This study thus provides a new mechanism underlying abscisic acid-induced stress responses via the giant COPII vesicles and answers a long-standing question on the evolutionary significance of gene duplications in Arabidopsis.

Structural basis of substrate recognition and thermal protection by a small heat shock protein.

Small heat shock proteins (sHsps) bind unfolding proteins, thereby playing a pivotal role in the maintenance of proteostasis in virtually all living organisms. Structural elucidation of sHsp-substrate complexes has been hampered by the transient and heterogeneous nature of their interactions, and the precise mechanisms underlying substrate recognition, promiscuity, and chaperone activity of sHsps remain unclear. Here we show the formation of a stable complex between Arabidopsis thaliana plastid sHsp, Hsp21, and its natural substrate 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) under heat stress, and report cryo-electron microscopy structures of Hsp21, DXPS and Hsp21-DXPS complex at near-atomic resolution. Monomeric Hsp21 binds across the dimer interface of DXPS and engages in multivalent interactions by recognizing highly dynamic structural elements in DXPS. Hsp21 partly unfolds its central α-crystallin domain to facilitate binding of DXPS, which preserves a native-like structure. This mode of interaction suggests a mechanism of sHsps anti-aggregation activity towards a broad range of substrates.

Subnanometer resolution cryo-EM structure of Arabidopsis thaliana ATG9.

Macroautophagy/autophagy is an essential process for the maintenance of cellular homeostasis by recycling macromolecules under normal and stress conditions. ATG9 (autophagy related 9) is the only integral membrane protein in the autophagy core machinery and has a central role in mediating autophagosome formation. In cells, ATG9 exists on mobile vesicles that traffic to the growing phagophore, providing an essential membrane source for the formation of autophagosomes. Here we report the three-dimensional structure of ATG9 from Arabidopsis thaliana at 7.8 Å resolution, determined by single particle cryo-electron microscopy. ATG9 organizes into a homotrimer, with each protomer contributing at least six transmembrane α-helices. At the center of the trimer, the protomers interact via their membrane-embedded and C-terminal cytoplasmic regions. Combined with prediction of protein contacts using sequence co-evolutionary information, the structure provides molecular insights into the ATG9 architecture and testable hypotheses for the molecular mechanism of autophagy progression regulated by ATG9.Abbreviations: 2D: 2-dimensional; 3D: 3-dimensional; AtATG9: Arabidopsis ATG9; Atg: autophagy-related; ATG9: autophagy-related protein 9; cryo-EM: cryo-electron microscopy; DDM: dodecyl maltoside; GraDeR: gradient-based detergent removal; LMNG: lauryl maltose-neopentyl glycol; PAS: phagophore assembly site; PtdIns3K: phosphatidylinositol 3-kinase.

Structural Biology and Electron Microscopy of the Autophagy Molecular Machinery.

Autophagy is a highly regulated bulk degradation process that plays a key role in the maintenance of cellular homeostasis. During autophagy, a double membrane-bound compartment termed the autophagosome is formed through de novo nucleation and assembly of membrane sources to engulf unwanted cytoplasmic components and targets them to the lysosome or vacuole for degradation. Central to this process are the autophagy-related (ATG) proteins, which play a critical role in plant fitness, immunity, and environmental stress response. Over the past few years, cryo-electron microscopy (cryo-EM) and single-particle analysis has matured into a powerful and versatile technique for the structural determination of protein complexes at high resolution and has contributed greatly to our current understanding of the molecular mechanisms underlying autophagosome biogenesis. Here we describe the plant-specific ATG proteins and summarize recent structural and mechanistic studies on the protein machinery involved in autophagy initiation with an emphasis on those by single-particle analysis.

Morphological Diversity, Protein Adsorption, and Cellular Uptake of Polydopamine-Coated Gold Nanoparticles.

Polydopamine (PDA)-coated nanoparticles are adhesive bionanomaterials widely utilized in intracellular applications, yet how their adhesiveness affects their colloidal stability and their interactions with serum proteins and mammalian cells remain unclear. In this work, we systematically investigate the combined effects of dopamine (DA) concentration and polymerization time (both reaction parameters spanning 2 orders of magnitude) on the morphological diversity of PDA-coated nanoparticles by coating PDA onto gold nanoparticle cores. Independent of the DA concentration, Au@PDA NPs remain largely aggregated upon several hours of limited polymerization; interestingly, extended polymerization for 2 days or longer yield randomly aggregated NPs, nearly monodisperse NPs, or worm-like NP chains in the ascending order of DA concentration. Upon exposure to serum proteins, the specific type of proteins adsorbed to the Au@PDA NPs strongly depends upon the DA concentration. As DA concentration increases, less albumin and more hemoglobin subunits adhere. Moreover, cellular uptake is a strong function of polymerization time. Serum-stabilized Au@PDA NPs prepared by limited polymerization enter Neuro-2a and HeLa cancer cells more abundantly than those prepared by extended polymerization. Our data underscore the importance of DA concentration and polymerization time for tuning the morphology and degree of intracellular delivery of PDA-coated nanostructures.

Isolation of the Plant Exocyst Complex.

The exocyst, conserved from yeast to plants to mammals, is a hetero-octameric complex that mediates tethering of secretory vesicles to designated sites on the plasma membrane during polarized exocytosis. Because structural studies of the intact exocyst complex have been greatly limited by the low yields of purified proteins, many aspects of the exocyst functions remain poorly understood. Here, we present the protocols for the isolation and purification of the recombinant and the native plant exocyst complex. Given the known diversification of the exocyst subunits in plants, our protocols will likely open the possibility of unraveling the functional significance of these subunits in the context of the fully assembled exocyst complex.

Chaperonin TRiC/CCT Modulates the Folding and Activity of Leukemogenic Fusion Oncoprotein AML1-ETO.

AML1-ETO is the most common fusion oncoprotein causing acute myeloid leukemia (AML), a disease with a 5-year survival rate of only 24%. AML1-ETO functions as a rogue transcription factor, altering the expression of genes critical for myeloid cell development and differentiation. Currently, there are no specific therapies for AML1-ETO-positive AML. While known for decades to be the translational product of a chimeric gene created by the stable chromosome translocation t(8;21)(q22;q22), it is not known how AML1-ETO achieves its native and functional conformation or whether this process can be targeted for therapeutic benefit. Here, we show that the biosynthesis and folding of the AML1-ETO protein is facilitated by interaction with the essential eukaryotic chaperonin TRiC (or CCT). We demonstrate that a folding intermediate of AML1-ETO binds to TRiC directly, mainly through its ß-strand rich, DNA-binding domain (AML-(1-175)), with the assistance of HSP70. Our results suggest that TRiC contributes to AML1-ETO proteostasis through specific interactions between the oncoprotein's DNA-binding domain, which may be targeted for therapeutic benefit.

Modulation of STAT3 folding and function by TRiC/CCT chaperonin.

Signal transducer and activator of transcription 3 (Stat3) transduces signals of many peptide hormones from the cell surface to the nucleus and functions as an oncoprotein in many types of cancers, yet little is known about how it achieves its native folded state within the cell. Here we show that Stat3 is a novel substrate of the ring-shaped hetero-oligomeric eukaryotic chaperonin, TRiC/CCT, which contributes to its biosynthesis and activity in vitro and in vivo. TRiC binding to Stat3 was mediated, at least in part, by TRiC subunit CCT3. Stat3 binding to TRiC mapped predominantly to the ß-strand rich, DNA-binding domain of Stat3. Notably, enhancing Stat3 binding to TRiC by engineering an additional TRiC-binding domain from the von Hippel-Lindau protein (vTBD), at the N-terminus of Stat3, further increased its affinity for TRiC as well as its function, as determined by Stat3's ability to bind to its phosphotyrosyl-peptide ligand, an interaction critical for Stat3 activation. Thus, Stat3 levels and function are regulated by TRiC and can be modulated by manipulating its interaction with TRiC.