The E3 ligase TRIM31 regulates hematopoietic stem cell homeostasis and MLL-AF9 leukemia.

Hematopoietic stem cells (HSC) are kept in a quiescent state to maintain their self-renewal capacity. Proper regulation of cyclin-dependent kinases (CDK) and cyclin proteins is critical for the maintenance of HSC homeostasis. Here, we found that the E3 ligase, TRIM31, regulates HSC homeostasis and leukemia through the accumulation of CDK8. TRIM31 deficiency promotes hematopoietic stem and progenitor cell proliferation and long-term HSC exhaustion. Serial competitive transplantation assays showed that TRIM31-deficient HSC exhibit impaired reconstitution ability. TRIM31 loss led to a lower rate of survival of mice under conditions of stress (5-fluorouracil administration), which was correlated with a lower number of hematopoietic stem and progenitor cells. In a murine model of acute myeloid leukemia, the initiation of leukemia was significantly accelerated upon TRIM31 deletion. Mechanistically, we found that ubiquitin-mediated degradation of CDK8 was impaired by TRIM31 deletion, which further induced transcriptional expression of PBX1 and cyclin D1. Taken together, these findings reveal the function of TRIM31 in the regulation of HSC homeostasis and leukemia initiation, and indicate the physiological importance of TRIM31 in the early stage of the development of leukemia.

Structure of TBC1D23 N-terminus reveals a novel role for rhodanese domain.

Members of the Tre2-Bub2-Cdc16 (TBC) family often function to regulate membrane trafficking and to control signaling transductions pathways. As a member of the TBC family, TBC1D23 is critical for endosome-to-Golgi cargo trafficking by serving as a bridge between Golgi-bound golgin-97/245 and the WASH/FAM21 complex on endosomal vesicles. However, the exact mechanisms by which TBC1D23 regulates cargo transport are poorly understood. Here, we present the crystal structure of the N-terminus of TBC1D23 (D23N), which consists of both the TBC and rhodanese domains. We show that the rhodanese domain is unlikely to be an active sulfurtransferase or phosphatase, despite containing a putative catalytic site. Instead, it packs against the TBC domain and forms part of the platform to interact with golgin-97/245. Using the zebrafish model, we show that impacting golgin-97/245-binding, but not the putative catalytic site, impairs neuronal growth and brain development. Altogether, our studies provide structural and functional insights into an essential protein that is required for organelle-specific trafficking and brain development.

Structural and functional studies of TBC1D23 C-terminal domain provide a link between endosomal trafficking and PCH.

Pontocerebellar hypoplasia (PCH) is a group of neurological disorders that affect the development of the brain, in particular, the pons and cerebellum. Homozygous mutations of TBC1D23 have been found recently to lead to PCH; however, the underlying molecular mechanisms remain unclear. Here, we show that the crystal structure of the TBC1D23 C-terminal domain adopts a Pleckstrin homology domain fold and selectively binds to phosphoinositides, in particular, PtdIns(4)P, through one surface while binding FAM21 via the opposite surface. Mutation of key residues of TBC1D23 or FAM21 selectively disrupts the endosomal vesicular trafficking toward the Trans-Golgi Network. Finally, using the zebrafish model, we show that PCH patient-derived mutants, impacting either phosphoinositide binding or FAM21 binding, lead to abnormal neuronal growth and brain development. Taken together, our data provide a molecular basis for the interaction between TBC1D23 and FAM21, and suggest a plausible role for PtdIns(4)P in the TBC1D23-mediating endosome-to-TGN trafficking pathway. Defects in this trafficking pathway are, at least partially, responsible for the pathogenesis of certain types of PCH.

Mechanism of inhibition of retromer transport by the bacterial effector RidL.

Retrograde vesicle trafficking pathways are responsible for returning membrane-associated components from endosomes to the Golgi apparatus and the endoplasmic reticulum (ER), and they are critical for maintaining organelle identity, lipid homeostasis, and many other cellular functions. The retrograde transport pathway has emerged as an important target for intravacuolar bacterial pathogens. The opportunistic pathogen Legionella pneumophila exploits both the secretory and recycling branches of the vesicle transport pathway for intracellular bacterial proliferation. Its Dot/Icm effector RidL inhibits the activity of the retromer by directly engaging retromer components. However, the mechanism underlying such inhibition remains unknown. Here we present the crystal structure of RidL in complex with VPS29, a subunit of the retromer. Our results demonstrate that RidL binds to a highly conserved hydrophobic pocket of VPS29. This interaction is critical for endosomal recruitment of RidL and for its inhibitory effects. RidL inhibits retromer activity by direct competition, in which it occupies the VPS29-binding site of the essential retromer regulator TBC1d5. The mechanism of retromer inhibition by RidL reveals a hotspot on VPS29 critical for recognition by its regulators that is also exploited by pathogens, and provides a structural basis for the development of small molecule inhibitors against the retromer.

Structural Biology for the Molecular Insight between Aptamers and Target Proteins.

Aptamers are promising therapeutic and diagnostic agents for various diseases due to their high affinity and specificity against target proteins. Structural determination in combination with multiple biochemical and biophysical methods could help to explore the interacting mechanism between aptamers and their targets. Regrettably, structural studies for aptamer-target interactions are still the bottleneck in this field, which are facing various difficulties. In this review, we first reviewed the methods for resolving structures of aptamer-protein complexes and for analyzing the interactions between aptamers and target proteins. We summarized the general features of the interacting nucleotides and residues involved in the interactions between aptamers and proteins. Challenges and perspectives in current methodologies were discussed. Approaches for determining the binding affinity between aptamers and target proteins as well as modification strategies for stabilizing the binding affinity of aptamers to target proteins were also reviewed. The review could help to understand how aptamers interact with their targets and how alterations such as chemical modifications in the structures affect the affinity and function of aptamers, which could facilitate the optimization and translation of aptamers-based theranostics.

A thiazole-derived oridonin analogue exhibits antitumor activity by directly and allosterically inhibiting STAT3.

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) occurs in ∼70% of human cancers, and STAT3 is regarded as one of the most promising targets for cancer therapy. However, specific direct STAT3 inhibitors remain to be developed. Oridonin is an ent-kaurane plant-derived diterpenoid with anti-cancer and anti-inflammatory activities. Here, using an array of cell-based and biochemical approaches, including cell proliferation and apoptosis assays, pulldown and reporter gene assays, site-directed mutagenesis, and molecular dynamics analyses, we report that a thiazole-derived oridonin analogue, CYD0618, potently and directly inhibits STAT3. We found that CYD0618 covalently binds to Cys-542 in STAT3 and suppresses its activity through an allosteric effect, effectively reducing STAT3 dimerization and nuclear translocation, as well as decreasing expression of STAT3-targeted oncogenes. Remarkably, CYD0618 not only strongly inhibited growth of multiple cancer cell lines that harbor constitutive STAT3 activation, but it also suppressed in vivo tumor growth via STAT3 inhibition. Taken together, our findings suggest Cys-542 as a druggable site for selectively inhibiting STAT3 and indicate that CYD0618 represents a promising lead compound for developing therapeutic agents against STAT3-driven diseases.

Manganese-Catalyzed Oxidative Azidation of C(sp3)-H Bonds under Electrophotocatalytic Conditions.

The selective installation of azide groups into C(sp3)-H bonds is a priority research topic in organic synthesis, particularly in pharmaceutical discovery and late-stage diversification. Herein, we demonstrate a generalized manganese-catalyzed oxidative azidation methodology of C(sp3)-H bonds using nucleophilic NaN3 as an azide source under electrophotocatalytic conditions. This approach allows us to perform the reaction without the necessity of adding an excess of the substrate and successfully avoiding the use of stoichiometric chemical oxidants such as iodine(III) reagent or NFSI. A series of tertiary and secondary benzylic C(sp3)-H, aliphatic C(sp3)-H, and drug-molecule-based C(sp3)-H bonds in substrates are well tolerated under our protocol. The simultaneous gram-scale synthesis and the ease of transformation of azide to amine collectively advocate for the potential application in the preparative synthesis. Good reactivity of the tertiary benzylic C(sp3)-H bond and selectivity of the tertiary aliphatic C(sp3)-H bond in substrates to incorporate nitrogen-based functionality at the tertiary alkyl group also provide opportunities to manipulate numerous potential medicinal candidates. We anticipate our synthetic protocol, consisting of metal catalysis, electrochemistry, and photochemistry, would provide a new sustainable option to execute challenging organic synthetic transformations.

Reduced thiamine binding is a novel mechanism for TPK deficiency disorder.

Thiamine pyrophosphokinase (TPK) converts thiamine (vitamin B1) into thiamine pyrophosphate (TPP), an essential cofactor for many important enzymes. TPK1 mutations lead to a rare disorder: episodic encephalopathy type thiamine metabolism dysfunction. Yet, the molecular mechanism of the disease is not entirely clear. Here we report an individual case of episodic encephalopathy, with familial history carrying a novel homozygous TPK1 mutation (p.L28S). The L28S mutation leads to reduced enzymatic activity, both in vitro and in vivo, without impairing thiamine binding and protein stability. Thiamine supplementation averted encephalopathic episodes and restored the patient's developmental progression. Biochemical characterization of reported TPK1 missense mutations suggested reduced thiamine binding as a new disease mechanism. Importantly, many disease mutants are directly or indirectly involved in thiamine binding. Thus, our study provided a novel rationale for thiamine supplementation, so far the major therapeutic intervention in TPK deficiency.

Chiral biosensing at both interband transition and plasmonic extinction regions using twisted-stacked nanowire arrays.

Chiral metal nanostructures that exhibit strong chiroptical properties and enhanced light-matter interactions have recently attracted great interest due to their potential applications including chiral sensing and asymmetric synthesis. Most studies in this field focused on chiral sensing using circular dichroism (CD) responses at the plasmonic extinction region. In comparison, little is known about their CD responses at interband transition regions and their utility in chiral biosensing. Herein, we constructed a series of twisted-stacked silver nanowire arrays (TNAs) featuring CD signals at both the interband transition and plasmonic extinction regions and that are independently controllable. These TNAs are highly sensitive towards protein secondary structures. Proteins containing more ß-sheets are more sensitive toward strong chiral plasmonic fields, whereas proteins rich in α-helices tend to generate larger CD shifts at the interband transition region of TNAs. The mutually independent optical activities at the interband transition and plasmonic extinction regions complement each other, providing more sensitivity and reliability in chiral biosensing.

Drug discovery of sclerostin inhibitors.

Sclerostin, a protein secreted from osteocytes, negatively regulates the WNT signaling pathway by binding to the LRP5/6 co-receptors and further inhibits bone formation and promotes bone resorption. Sclerostin contributes to musculoskeletal system-related diseases, making it a promising therapeutic target for the treatment of WNT-related bone diseases. Additionally, emerging evidence indicates that sclerostin contributes to the development of cancers, obesity, and diabetes, suggesting that it may be a promising therapeutic target for these diseases. Notably, cardiovascular diseases are related to the protective role of sclerostin. In this review, we summarize three distinct types of inhibitors targeting sclerostin, monoclonal antibodies, aptamers, and small-molecule inhibitors, from which monoclonal antibodies have been developed. As the first-in-class sclerostin inhibitor approved by the U.S. FDA, the monoclonal antibody romosozumab has demonstrated excellent effectiveness in the treatment of postmenopausal osteoporosis; however, it conferred high cardiovascular risk in clinical trials. Furthermore, romosozumab could only be administered by injection, which may cause compliance issues for patients who prefer oral therapy. Considering these above safety and compliance concerns, we therefore present relevant discussion and offer perspectives on the development of next-generation sclerostin inhibitors by following several ways, such as concomitant medication, artificial intelligence-based strategy, druggable modification, and bispecific inhibitors strategy.

C/EBP homologous protein deficiency enhances hematopoietic stem cell function via reducing ATF3/ROS-induced cell apoptosis.

Hematopoietic stem cells (HSCs) reside in a quiescent niche to reserve their capacity of self-renewal. Upon hematopoietic injuries, HSCs enter the cell cycle and encounter protein homeostasis problems caused by accumulation of misfolded proteins. However, the mechanism by which protein homeostasis influences HSC function and maintenance remains poorly understood. Here, we show that C/EBP homologous protein (CHOP), demonstrated previously to induces cell death upon unfolded protein response (UPR), plays an important role in HSCs regeneration. CHOP-/- mice showed normal hematopoietic stem and progenitor cell frequencies in steady state. However, when treated with 5-FU, CHOP deficiency resulted in higher survival rates, associated with an increased number of HSCs and reduced level of apoptosis. In serial competitive transplantation experiments, CHOP-/- HSCs showed a dramatic enhancement of repopulation ability and a reduction of protein aggresomes. Mechanistically, CHOP deletion causes reduced ATF3 expression and further leads to decreased protein aggregation and ROS. In addition, CHOP-/- HSCs exhibited an increased resistance to IR-induced DNA damage and improved HSCs homeostasis and function in telomere dysfunctional (G3Terc-/- ) mice. In summary, these findings disclose a new role of CHOP in the regulation of the HSCs function and homeostasis through reducing ATF3 and ROS signaling.

Effect of ionic liquids with different cations and anions on photosystem and cell structure of Scenedesmus obliquus.

The rapid increase in the production and practical application of ionic liquids (ILs) could pose potential threats to aquatic systems. In this study, we investigated the effects of four ILs with different cations and anions, including 1-hexyl-3-methylimidazolium nitrate ([HMIM]NO3), 1-hexyl-3-methylimidazolium chloride ([HMIM]Cl), N-hexyl-3-metylpyridinium chloride ([HMPy]Cl), and N-hexyl-3-metylpyridinium bromide ([HMPy]Br), on photosystem and cellular structure of Scenedesmus obliquus. The results indicated that ILs are phytotoxic to S. obliquus. The contents of chlorophyll a, chlorophyll b and total chlorophyll decreased with increasing ILs concentrations. The chlorophyll fluorescence parameters of photosynthetic system II (PSII), including minimal fluorescence yield (F0), potential efficiency of PSII (Fv/Fo), maximum quantum efficiency of PSII photochemistry (Fv/Fm), yield of photochemical quantum [Y(II)], and non-photochemical quenching coefficient without measuring F0' (NPQ), were all affected. This indicates that ILs could damage PSII, inhibit the primary reaction of photosynthesis, interdict the process of electron-transfer and lead to loss of heat-dissipating ability. ILs also increased cell membrane permeability of S. obliquus, influenced the cellular ultrastructure, changed the morphology of algae cells and destroyed the cell wall, cell membrane and organelles. The results indicated that imidazolium ILs had greater effect than pyridinium ILs, NO3--IL and Br--IL had greater effect than Cl--IL. To minimize threats to the environment, the structure of ILs should be taken into consideration.

The toxicity of ionic liquid 1-decylpyridinium bromide to the algae Scenedesmus obliquus: Growth inhibition, phototoxicity, and oxidative stress.

Although ionic liquids (ILs) are unlikely to act as air contaminants, their high solubility and slow degradation make them a potential threat to the aquatic environment. The IL 1-decylpyridinium bromide ([DPy]Br) is a common type of pyridine IL, which has varied applications such as in extraction, separation, and catalytic synthesis. Herein, the toxicity of [DPy]Br to S. obliquus is determined. Growth was inhibited by high-concentration [DPy]Br, whereas it had a hormetic effect at low concentrations. The IC50-96h was approximately 0.06mg/L. The cell membrane permeability of S. obliquus increased with [DPy]Br concentration, indicating that [DPy]Br can cause damage to the algae cell structure. Chlorophyll content decreased at high [DPy]Br concentration; chlorophyll fluorescence parameters, such as the maximum effective quantum yield of PSII (Fv/Fm), potential activity of PSII (Fv/F0), yield of the photochemical quantum [Y(II)], and the non-photochemical quenching coefficient (NPQ) were affected, suggesting that [DPy]Br can damage PSII. The ROS fluorescent images revealed that the morphology of cells changed gradually from fusiform to round. High ROS levels were observed with high concentrations of [DPy]Br, indicating that [DPy]Br induced oxidative stress on S. obliquus. The SOD and CAT activities increased when the concentration was lower than IC50, whereas they decreased when the concentration was higher than IC50. The relative ROS content was significantly correlated with growth inhibition rate, cell membrane permeability, chlorophyll content, and SOD and CAT activities. The increase of ROS content in algal cells is an important toxicological mechanism of [DPy]Br to S. obliquus.

Evaluation of interaction between imidazolium-based chloride ionic liquids and calf thymus DNA.

With ionic liquids (ILs) being widely used, the toxicity of many ILs has been studied and verified. However the mechanism underlying the interaction between ILs and DNA needs to be investigated. In this study, the interaction of three imidazolium-based ILs ([C8mim]Cl, [C12mim]Cl, and [C16mim]Cl) with calf thymus DNA (ctDNA) was investigated by UV absorption spectroscopy and fluorescence spectroscopy. An intense interaction between [Cnmim]Cl and ctDNA was observed, involving a hypochromic effect or even a hyperchromic effect, in the UV absorption spectrum of ctDNA at 260nm. The Tm of ctDNA increased over 10°C after binding with [Cnmim]Cl, and the KSV values of [Cnmim]Cl-ctDNA quenched by potassium iodide (KI) were lower than those of [Cnmim]Cl. The fluorescence intensity of ctDNA-ethidium bromide (EB) was gradually quenched as the [Cnmim]Cl concentration increased. The results indicated that ctDNA interacted with [Cnmim]Cl through an intercalation binding mode. The mechanism of fluorescence quenching of [Cnmim]Cl with ctDNA involved static quenching. The binding constant between [Cnmim]Cl and ctDNA were 1443, 11169, and 67189, and the number of binding sites were 0.89, 1.10, and 1.27 at 298K, for [C8mim]Cl, [C12mim]Cl, and [C16mim]Cl, respectively. The results indicated that the intercalation binding between the three [Cnmim]Cl and ctDNA increased with increasing IL-alkyl chain length. These results will aid in the understanding of the mechanism of toxicity and of the biologically mediated environmental processes of ILs.

Orientational behaviors of liquid crystals coupled to chitosan-disrupted phospholipid membranes at the aqueous-liquid crystal interface.

In this study, we investigated the orientational behavior of liquid crystals (LCs) which is associated with the chitosan-disrupted phospholipid membrane at the aqueous/LC interface. The optical response of LCs changed from dark to bright after the transfer of an aqueous solution of chitosan onto the LC interface decorated with self-assembled monolayers of a negatively charged phospholipid, dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG). The chitosan-lipid interactions induced a rearrangement of the membrane, and thus, resulted in an orientational transition of LCs from a homeotropic to a planar state, thereby triggering a dark-to-bright shift in the optical response. We observed that LCs exhibited a bright-to-dark shift after an aqueous solution of lysozyme was transferred onto the chitosan-disrupted membrane, which implied that an enzymatic reaction between lysozyme and chitosan took place. We found that the addition of bovine serum album (BSA) induced a bright-to-dark change in the optical response; while LCs remained to appear bright after the transfer of chymotrypsin onto the aqueous/LC interface. We then further examined the interactions between other polyelectrolytes and phospholipid membranes.