Deciphering the Molecular Landscape of the FAM72 Gene Family: Implications for Stem Cell Biology and Cancer.

Family with sequence similarity 72 (FAM72) is a protein-coding gene family located on chromosome 1 in humans, uniquely featuring four paralogs: FAM72A, FAM72B, FAM72C, and FAM72D. While FAM72's presence as a gene pair with the SLIT-ROBO Rho GTPase-activating protein 2 (SRGAP2) is intriguing, its functional roles, particularly in neural stem cells, remain incompletely understood. This review explores the distinct characteristics of FAM72, shedding light on its expression patterns, potential roles in cell cycle regulation, stem cell renewal and implications in neurogenesis and tumorigenesis.

Advancing Bioactivity Prediction through Molecular Docking and Self-Attention.

Bioactivity refers to the ability of a substance to induce biological effects within living systems, often describing the influence of molecules, drugs, or chemicals on organisms. In drug discovery, predicting bioactivity streamlines early-stage candidate screening by swiftly identifying potential active molecules. The popular deep learning methods in bioactivity prediction primarily model the ligand structure-bioactivity relationship under the premise of Quantitative Structure-Activity Relationship (QSAR). However, bioactivity is determined by multiple factors, including not only the ligand structure but also drug-target interactions, signaling pathways, reaction environments, pharmacokinetic properties, and species differences. Our study first integrates drug-target interactions into bioactivity prediction using protein-ligand complex data from molecular docking. We devise a Drug-Target Interaction Graph Neural Network (DTIGN), infusing interatomic forces into intermolecular graphs. DTIGN employs multi-head self-attention to identify native-like binding pockets and poses within molecular docking results. To validate the fidelity of the self-attention mechanism, we gather ground truth data from crystal structure databases. Subsequently, we employ these limited native structures to refine bioactivity prediction via semi-supervised learning. For this study, we establish a unique benchmark dataset for evaluating bioactivity prediction models in the context of protein-ligand complexes, showcasing the superior performance of our method (with an average improvement of 27.03%) through comparison with 9 leading deep learning-based bioactivity prediction methods.

A linker protein from a red-type pyrenoid phase separates with Rubisco via oligomerizing sticker motifs.

The slow kinetics and poor substrate specificity of the key photosynthetic CO2-fixing enzyme Rubisco have prompted the repeated evolution of Rubisco-containing biomolecular condensates known as pyrenoids in the majority of eukaryotic microalgae. Diatoms dominate marine photosynthesis, but the interactions underlying their pyrenoids are unknown. Here, we identify and characterize the Rubisco linker protein PYCO1 from Phaeodactylum tricornutum. PYCO1 is a tandem repeat protein containing prion-like domains that localizes to the pyrenoid. It undergoes homotypic liquid-liquid phase separation (LLPS) to form condensates that specifically partition diatom Rubisco. Saturation of PYCO1 condensates with Rubisco greatly reduces the mobility of droplet components. Cryo-electron microscopy and mutagenesis data revealed the sticker motifs required for homotypic and heterotypic phase separation. Our data indicate that the PYCO1-Rubisco network is cross-linked by PYCO1 stickers that oligomerize to bind to the small subunits lining the central solvent channel of the Rubisco holoenzyme. A second sticker motif binds to the large subunit. Pyrenoidal Rubisco condensates are highly diverse and tractable models of functional LLPS.

Zyxin regulates embryonic stem cell fate by modulating mechanical and biochemical signaling interface.

Biochemical signaling and mechano-transduction are both critical in regulating stem cell fate. How crosstalk between mechanical and biochemical cues influences embryonic development, however, is not extensively investigated. Using a comparative study of focal adhesion constituents between mouse embryonic stem cell (mESC) and their differentiated counterparts, we find while zyxin is lowly expressed in mESCs, its levels increase dramatically during early differentiation. Interestingly, overexpression of zyxin in mESCs suppresses Oct4 and Nanog. Using an integrative biochemical and biophysical approach, we demonstrate involvement of zyxin in regulating pluripotency through actin stress fibres and focal adhesions which are known to modulate cellular traction stress and facilitate substrate rigidity-sensing. YAP signaling is identified as an important biochemical effector of zyxin-induced mechanotransduction. These results provide insights into the role of zyxin in the integration of mechanical and biochemical cues for the regulation of embryonic stem cell fate.

SRRM2 organizes splicing condensates to regulate alternative splicing.

SRRM2 is a nuclear-speckle marker containing multiple disordered domains, whose dysfunction is associated with several human diseases. Using mainly EGFP-SRRM2 knock-in HEK293T cells, we show that SRRM2 forms biomolecular condensates satisfying most hallmarks of liquid-liquid phase separation, including spherical shape, dynamic rearrangement, coalescence and concentration dependence supported by in vitro experiments. Live-cell imaging shows that SRRM2 organizes nuclear speckles along the cell cycle. As bona-fide splicing factor present in spliceosome structures, SRRM2 deficiency induces skipping of cassette exons with short introns and weak splice sites, tending to change large protein domains. In THP-1 myeloid-like cells, SRRM2 depletion compromises cell viability, upregulates differentiation markers, and sensitizes cells to anti-leukemia drugs. SRRM2 induces a FES splice isoform that attenuates innate inflammatory responses, and MUC1 isoforms that undergo shedding with oncogenic properties. We conclude that SRRM2 acts as a scaffold to organize nuclear speckles, regulating alternative splicing in innate immunity and cell homeostasis.

Dynamic swimming pattern of Pseudomonas aeruginosa near a vertical wall during initial attachment stages of biofilm formation.

Studying the swimming behaviour of bacteria in 3 dimensions (3D) allows us to understand critical biological processes, such as biofilm formation. It is still unclear how near wall swimming behaviour may regulate the initial attachment and biofilm formation. It is challenging to address this as visualizing the movement of bacteria with reasonable spatial and temporal resolution in a high-throughput manner is technically difficult. Here, we compared the near wall (vertical) swimming behaviour of P. aeruginosa (PAO1) and its mutants ΔdipA (reduced in swarming motility and increased in biofilm formation) and ΔfimX (deficient in twitching motility and reduced in biofilm formation) using our new imaging technique based on light sheet microscopy. We found that P. aeruginosa (PAO1) increases its speed and changes its swimming angle drastically when it gets closer to a wall. In contrast, ΔdipA mutant moves toward the wall with steady speed without changing of swimming angle. The near wall behavior of ΔdipA allows it to be more effective to interact with the wall or wall-attached cells, thus leading to more adhesion events and a larger biofilm volume during initial attachment when compared with PAO1. Furthermore, we found that ΔfimX has a similar near wall swimming behavior as PAO1. However, it has a higher dispersal frequency and smaller biofilm formation when compared with PAO1 which can be explained by its poor twitching motility. Together, we propose that near wall swimming behavior of P. aeruginosa plays an important role in the regulation of initial attachment and biofilm formation.

A processing product of the Plasmodium falciparum reticulocyte binding protein RH1 shows a close association with AMA1 during junction formation.

Plasmodium falciparum responsible for the most virulent form of malaria invades human erythrocytes through multiple ligand-receptor interactions. The P. falciparum reticulocyte binding protein homologues (PfRHs) are expressed at the apical end of merozoites and form interactions with distinct erythrocyte surface receptors that are important for invasion. Here using a range of monoclonal antibodies (mAbs) against different regions of PfRH1 we have investigated the role of PfRH processing during merozoite invasion. We show that PfRH1 gets differentially processed during merozoite maturation and invasion and provide evidence that the different PfRH1 processing products have distinct functions during invasion. Using in-situ Proximity Ligation and FRET assays that allow probing of interactions at the nanometre level we show that a subset of PfRH1 products form close association with micronemal proteins Apical Membrane Antigen 1 (AMA1) in the moving junction suggesting a critical role in facilitating junction formation and active invasion. Our data provides evidence that time dependent processing of PfRH proteins is a mechanism by which the parasite is able to regulate distinct functional activities of these large processes. The identification of a specific close association with AMA1 in the junction now may also provide new avenues to target these interactions to prevent merozoite invasion.

A novel function of AAA-ATPase p97/VCP in the regulation of cell motility.

High level of the multifunctional AAA-ATPase p97/VCP is often correlated to the development of cancer; however, the underlying mechanism is not understood completely. Here, we report a novel function of p97/VCP in actin regulation and cell motility. We found that loss of p97/VCP promotes stabilization of F-actin, which cannot be reversed by actin-destabilizing agent, Cytochalasin D. Live-cell imaging demonstrated reduced actin dynamics in p97/VCP-knockdown cells, leading to compromised cell motility. We further examined the underlying mechanism and found elevated RhoA protein levels along with increased phosphorylation of its downstream effectors, ROCK, LIMK, and MLC upon the knockdown of p97/VCP. Since p97/VCP is indispensable in the ubiquitination-dependent protein degradation pathway, we investigated if the loss of p97/VCP hinders the protein degradation of RhoA. Knockdown of p97/VCP resulted in a higher amount of ubiquitinated RhoA, suggesting p97/VCP involvement in the proteasome-dependent protein degradation pathway. Finally, we found that p97/VCP interacts with FBXL19, a molecular chaperone known to guide ubiquitinated RhoA for proteasomal degradation. Reduction of p97/VCP may result in the accumulation of RhoA which, in turn, enhances cytoplasmic F-actin formation. In summary, our study uncovered a novel function of p97/VCP in actin regulation and cell motility via the Rho-ROCK dependent pathway which provides fundamental insights into how p97/VCP is involved in cancer development.

Regulation of flagellar motor switching by c-di-GMP phosphodiesterases in Pseudomonas aeruginosa.

The second messenger cyclic diguanylate (c-di-GMP) plays a prominent role in regulating flagellum-dependent motility in the single-flagellated pathogenic bacterium Pseudomonas aeruginosa The c-di-GMP-mediated signaling pathways and mechanisms that control flagellar output remain to be fully unveiled. Studying surface-tethered and free-swimming P. aeruginosa PAO1 cells, we found that the overexpression of an exogenous diguanylate cyclase (DGC) raises the global cellular c-di-GMP concentration and thereby inhibits flagellar motor switching and decreases motor speed, reducing swimming speed and reversal frequency, respectively. We noted that the inhibiting effect of c-di-GMP on flagellar motor switching, but not motor speed, is exerted through the c-di-GMP-binding adaptor protein MapZ and associated chemotactic pathways. Among the 22 putative c-di-GMP phosphodiesterases, we found that three of them (DipA, NbdA, and RbdA) can significantly inhibit flagellar motor switching and swimming directional reversal in a MapZ-dependent manner. These results disclose a network of c-di-GMP-signaling proteins that regulate chemotactic responses and flagellar motor switching in P. aeruginosa and establish MapZ as a key signaling hub that integrates inputs from different c-di-GMP-signaling pathways to control flagellar output and bacterial motility. We rationalized these experimental findings by invoking a model that postulates the regulation of flagellar motor switching by subcellular c-di-GMP pools.

The MapZ-Mediated Methylation of Chemoreceptors Contributes to Pathogenicity of Pseudomonas aeruginosa.

The pathogenic bacterium Pseudomonas aeruginosa is notorious for causing acute and chronic infections in humans. The ability to infect host by P. aeruginosa is dependent on a complex cellular signaling network, which includes a large number of chemosensory signaling pathways that rely on the methyl-accepting chemotaxis proteins (MCPs). We previously found that the second messenger c-di-GMP-binding adaptor MapZ modulates the methylation of an amino acid-detecting MCP by directly interacting with a chemotaxis methyltransferase CheR1. The current study further expands our understanding of the role of MapZ in regulating chemosensory pathways by demonstrating that MapZ suppresses the methylation of multiple MCPs in P. aeruginosa PAO1. The MCPs under the control of MapZ include five MCPs (Aer, CtpH, CptM, PctA, and PctB) for detecting oxygen/energy, inorganic phosphate, malate and amino acids, and three MCPs (PA1251, PA1608, and PA2867) for detecting unknown chemoattractant or chemorepellent. Chemotaxis assays showed that overexpression of MapZ hampered the taxis of P. aeruginosa toward chemoattractants and scratch-wounded human cells. Mouse infection experiments demonstrated that a dysfunction in MapZ regulation had a profound negative impact on the dissemination of P. aeruginosa and resulted in attenuated bacterial virulence. Together, the results imply that by controlling the methylation of various MCPs via the adaptor protein MapZ, c-di-GMP exerts a profound influence on chemotactic responses and bacterial pathogenesis.

The phase separation underlying the pyrenoid-based microalgal Rubisco supercharger.

The slow and promiscuous properties of the CO2-fixing enzyme Rubisco constrain photosynthetic efficiency and have prompted the evolution of powerful CO2 concentrating mechanisms (CCMs). In eukaryotic microalgae a key strategy involves sequestration of the enzyme in the pyrenoid, a liquid non-membranous compartment of the chloroplast stroma. Here we show using pure components that two proteins, Rubisco and the linker protein Essential Pyrenoid Component 1 (EPYC1), are both necessary and sufficient to phase separate and form liquid droplets. The phase-separated Rubisco is functional. Droplet composition is dynamic and components rapidly exchange with the bulk solution. Heterologous and chimeric Rubiscos exhibit variability in their tendency to demix with EPYC1. The ability to dissect aspects of pyrenoid biochemistry in vitro will permit us to inform and guide synthetic biology ambitions aiming to engineer microalgal CCMs into crop plants.

Live-Cell Imaging and Functional Dissection of Xist RNA Reveal Mechanisms of X Chromosome Inactivation and Reactivation.

We double-tagged Xist (inactivated X chromosome-specific transcript), a prototype long non-coding RNA pivotal for X chromosome inactivation (XCI), using the programmable RNA sequence binding domain of Pumilio protein, one tag for live-cell imaging and the other replacing A-repeat (a critical domain of Xist) to generate "ΔA mutant" and to tether effector proteins for dissecting Xist functionality. Based on the observation in live cells that the induced XCI in undifferentiated embryonic stem (ES) cells is counteracted by the intrinsic X chromosome reactivation (XCR), we identified Kat8 and Msl2, homologs of Drosophila dosage compensation proteins, as players involved in mammalian XCR. Furthermore, live-cell imaging revealed the obviously undersized ΔA Xist cloud signals, clarifying an issue regarding the previous RNA fluorescence in situ hybridization results. Tethering candidate proteins onto the ΔA mutant reveals the significant roles of Ythdc1, Ezh2, and SPOC (Spen) in Xist-mediated gene silencing and the significant role of Ezh2 in Xist RNA spreading.

A bilayer photoreceptor-retinal tissue model with gradient cell density design: A study of microvalve-based bioprinting.

ARPE-19 and Y79 cells were precisely and effectively delivered to form an in vitro retinal tissue model via 3D cell bioprinting technology. The samples were characterized by cell viability assay, haematoxylin and eosin and immunofluorescent staining, scanning electrical microscopy and confocal microscopy, and so forth. The bioprinted ARPE-19 cells formed a high-quality cell monolayer in 14 days. Manually seeded ARPE-19 cells were poorly controlled during and after cell seeding, and they aggregated to form uneven cell layer. The Y79 cells were subsequently bioprinted on the ARPE-19 cell monolayer to form 2 distinctive patterns. The microvalve-based bioprinting is efficient and accurate to build the in vitro tissue models with the potential to provide similar pathological responses and mechanism to human diseases, to mimic the phenotypic endpoints that are comparable with clinical studies, and to provide a realistic prediction of clinical efficacy.

P. falciparum RH5-Basigin interaction induces changes in the cytoskeleton of the host RBC.

The successful invasion of Plasmodium is an essential step in their life cycle. The parasite reticulocyte-binding protein homologues (RHs) and erythrocyte-binding like proteins are two families involved in the invasion leading to merozoite-red blood cell (RBC) junction formation. Ca2+ signaling has been shown to play a critical role in the invasion. RHs have been linked to Ca2+ signaling, which triggers the erythrocyte-binding like proteins release ahead of junction formation, consistent with RHs performing an initial sensing function in identifying suitable RBCs. RH5, the only essential RHs, is a highly promising vaccine candidate. RH5-basigin interaction is essential for merozoite invasion and also important in determining host tropism. Here, we show that RH5 has a distinct function from the other RHs. We show that RH5-Basigin interaction on its own triggers a Ca2+ signal in the RBC resulting in changes in RBC cytoskeletal proteins phosphorylation and overall alterations in RBC cytoskeleton architecture. Antibodies targeting RH5 that block the signal prevent invasion before junction formation consistent with the Ca2+ signal in the RBC leading to rearrangement of the cytoskeleton required for invasion. This work provides the first time a functional context for the essential role of RH5 and will now open up new avenues to target merozoite invasion.

POPX2 phosphatase regulates cell polarity and centrosome placement.

Proper centrosome positioning is critical for many cellular functions, such as cell migration and maintenance of polarity. During wound healing, fibroblasts orient their centrosomes such that they face the wound edge. The centrosome orientation determines the direction of cells' migration so that they can close the wound effectively. In this study, we investigated the regulation of centrosome polarization and have identified the phosphatase POPX2 as an important regulator of centrosome orientation. We found that POPX2 inhibits centrosome centration, but not rearward nuclear movement, by regulating multiple proteins that function in centrosome positioning. High POPX2 levels result in reduced motility of the kinesin-2 motor, which, in turn, inhibits the transport of N-cadherin to the cell periphery and cell junctions. Loss of N-cadherin localization to the cell membrane affects the localization of focal adhesions and perturbs CDC42-Par6/PKCζ signaling. In addition, overexpression of POPX2 also results in a loss of Par3 localization to the cell periphery and reduced levels of LIC2 (dynein light intermediate chain 2), leading to defects in microtubule tethering and dynamics at cell-cell contacts. Therefore, POPX2 functions as a regulator of signaling pathways to modulate the positioning of centrosome in fibroblast during wound healing.

POPX2 phosphatase regulates the KIF3 kinesin motor complex.

The kinesin motors are important in the regulation of cellular functions such as protein trafficking, spindle organization and centrosome separation. In this study, we have identified POPX2, a serine-threonine phosphatase, as an interacting partner of the KAP3 subunit of the kinesin-2 motor. The kinesin-2 motor is a heterotrimeric complex composed of KIF3A, KIF3B motor subunits and KAP3, the non-motor subunit, which binds the cargo. Here we report that the phosphatase POPX2 is a negative regulator of the trafficking of N-cadherin and other cargoes; consequently, it markedly influences cell-cell adhesion. POPX2 affects trafficking by determining the phosphorylation status of KIF3A at serine 690. This is consistent with the observation that the KIF3A-S690A mutant is defective in cargo trafficking. Our studies also implicate CaMKII as the kinase that phosphorylates KIF3A at serine 690. These results strongly suggest that POPX2 and CaMKII are a phosphatase-kinase pair that regulates kinesin-mediated transport and cell-cell adhesion.

In vivo FRET imaging revealed a regulatory role of RanGTP in kinetochore-microtubule attachments via Aurora B kinase.

Under the fluctuating circumstances provided by the innate dynamics of microtubules and opposing tensions resulted from microtubule-associated motors, it is vital to ensure stable kinetochore-microtubule attachments for accurate segregation. However, a comprehensive understanding of how this regulation is mechanistically achieved remains elusive. Using our newly designed live cell FRET time-lapse imaging, we found that post-metaphase RanGTP is crucial in the maintenance of stable kinetochore-microtubule attachments by regulating Aurora B kinase via the NES-bearing Mst1. More importantly, our study demonstrates that by ensuring stable alignment of metaphase chromosomes prior to segregation, RanGTP is indispensible in governing the genomic integrity and the fidelity of cell cycle progression. Our findings suggest an additional role of RanGTP beyond its known function in mitotic spindle assembly during the prometaphase-metaphase transition.

RUNX3 interactome reveals novel centrosomal targeting of RUNX family of transcription factors.

RUNX family proteins are critical regulators of lineage differentiation during development. The high prevalence of RUNX mutation/epigenetic inactivation in human cancer indicates a causative role for dysfunctional RUNX in carcinogenesis. This is supported by well-documented evidence of functional interaction of RUNX with components of major oncogenic or tumor suppressive signaling pathways such as TGFß and Wnt. Here, we explore the binding partners of RUNX3 proteins to further define the scope of RUNX3 function. Using a mass spectrometry-based approach, we found that RUNX3 binds to centrosomal protein rootletin. This led us to uncover the presence of RUNX proteins at the centrosome. Our findings suggest a potential function for RUNX3 during mitosis.

ANGPTL4 modulates vascular junction integrity by integrin signaling and disruption of intercellular VE-cadherin and claudin-5 clusters.

Vascular disruption induced by interactions between tumor-secreted permeability factors and adhesive proteins on endothelial cells facilitates metastasis. The role of tumor-secreted C-terminal fibrinogen-like domain of angiopoietin-like 4 (cANGPTL4) in vascular leakiness and metastasis is controversial because of the lack of understanding of how cANGPTL4 modulates vascular integrity. Here, we show that cANGPTL4 instigated the disruption of endothelial continuity by directly interacting with 3 novel binding partners, integrin α5ß1, VE-cadherin, and claudin-5, in a temporally sequential manner, thus facilitating metastasis. We showed that cANGPTL4 binds and activates integrin α5ß1-mediated Rac1/PAK signaling to weaken cell-cell contacts. cANGPTL4 subsequently associated with and declustered VE-cadherin and claudin-5, leading to endothelial disruption. Interfering with the formation of these cANGPTL4 complexes delayed vascular disruption. In vivo vascular permeability and metastatic assays performed using ANGPTL4-knockout and wild-type mice injected with either control or ANGPTL4-knockdown tumors confirmed that cANGPTL4 induced vascular leakiness and facilitated lung metastasis in mice. Thus, our findings elucidate how cANGPTL4 induces endothelial disruption. Our findings have direct implications for targeting cANGPTL4 to treat cancer and other vascular pathologies.