Journal of Biological Chemistry最新文献

Approximately 80% of eukaryotic and 65% of prokaryotic proteins are composed of multiple folding units (i.e., domains) connected by flexible linkers. These dynamic protein architectures, facilitated by linker regions, support essential functions such as electron transfer, respiration, and biosynthesis. This review critically assesses recent advancements in methods for studying protein dynamics, with a particular focus on modular, multidomain nitric oxide synthase (NOS) enzymes. Moving beyond traditional static "snapshots" of protein structures, current research emphasizes the dynamic nature of proteins, viewing them as flexible architectures modulated by conformational changes and interactions. In this context, the review discusses key developments in the integration of quantitative crosslinking mass spectrometry (qXL MS) with AlphaFold 2 predictions, which provides a powerful approach to disentangling NOS structural dynamics and understanding their modulation by external regulatory cues. Additionally, advances in site-specific infrared (IR) spectroscopy offer exciting potential in providing rich details about the conformational dynamics of NOSs in docked states. Moreover, optimization of genetic code expansion machinery enables the generation of genuine phosphorylated NOS enzymes, allowing detailed biophysical and functional analysis of phosphorylation's role in shaping NOS activity and structural flexibility; notably, this approach also empowers site-specific IR probe labeling with cyano groups. By embracing and leveraging artificial intelligence-driven tools like AlphaFold 2 for structural and conformational modeling, alongside solution-based biophysical methods such as site-specific IR spectroscopy and qXL MS, researchers will gain integrative insights into functional protein dynamics. Collectively, these breakthroughs highlight the transformative potential of modern approaches in driving fundamental biological chemistry research.

Hereditary folate malabsorption (HFM) is a rare, autosomal recessive disorder characterized by impaired intestinal absorption and impaired transport of folates across the choroid plexus into cerebral spinal fluid due to inactivating mutations in the hPCFT gene, which encodes the proton-coupled folate transporter (PCFT-SLC46A1). Understanding the structural impact of these mutations is crucial for elucidating the mechanistic basis for PCFT function and the pathophysiology of HFM. Recently, the cryo-electron microscopic structural characterization of the Gallus gallus PCFT (gPCFT) was obtained, which shares significant sequence identity with hPCFT. We conducted molecular dynamics (MD) simulations of human PCFT (hPCFT) based on this structure, to explore structural changes induced by functionally defective disease-causing and other mutant proteins and mutations that restore function. Simulations revealed that the mutually mechanistic basis for the loss of function is partial loss of structural integrity of hPCFT primarily manifested in an enlarged and distorted pore accompanied by loss of long-range contacts, less stable, fluctuating inner helices with reduced solvent accessibility and a marked loss of ordered secondary structures. These changes are reversed by the introduction of compensatory mutations. These findings provide novel insights into the structural and functional consequences of PCFT mutations associated with HFM and provide correlations with kinetic and biochemical properties of the mutant proteins.

Inositol phosphates (IPs) and inositol pyrophosphate play critical roles in many biological processes as signaling molecules in pathways responsible for cellular functions involved in growth and maintenance. The biosynthesis of IPs is carried out by a family of inositol phosphate kinases. In mammals, Inositol tetrakisphosphate kinase-1 (ITPK1) phosphorylates inositol-1,3,4-trisphosphate (Ins(1,3,4)P₃) and inositol-3,4,5,6-tetrakisphosphate (IP₄), generating inositol-1,3,4,5,6-pentakisphosphate (IP₅), which can be further phosphorylated to become inositol hexakisphosphate (IP₆). ITPK1 also possesses phosphatase activity that can convert IP₅ back to IP₄; therefore, ITPK1 may serve as a regulatory step in IP₆ production. IP₆ utilization has been implicated in processes fundamental to cellular sustainability that are severely perturbed in many disease states including RNA editing, DNA repair, chromatin structure organization, and ubiquitin ligation. Therefore, ITPK1, with no known inhibitors in the literature, is a potential molecular target for modulating important processes in several human diseases. By independently coupling ITPK1 phosphatase and kinase activities to luciferase activity, we have developed and used biochemical high throughput assays to discover eight ITPK1 inhibitors. Further analysis revealed that three of these leads inhibit ITPK1 in an ATP-competitive manner, with low micromolar to nanomolar affinities. We further demonstrate that the most potent ITPK1 inhibitor can regulate cellular ITPK1 activity. We determined the crystal structure of ITPK1 in complex with this inhibitor at a resolution of 2.25 Å. This work provides insight into the design of potential next-generation inhibitors.

Mirtrons are the predominant class of non-canonical miRNAs derived from introns through a Drosha-independent, splicing-dependent pathway. Unregulated splicing of introns containing hairpins may adversely impact Dicer/Ago-mediated canonical miRNA biogenesis. However, the mechanism regulating mirtron biogenesis remains poorly understood. We found that the 5' arm of plant mirtrons and invertebrate mirtrons are enriched for uracils (Us); in contrast, the 5' arm vertebrate mirtrons are enriched for guanines (Gs). Further analysis revealed that most of the mammalian mirtrons contain an RNA G-quadruplex (rG4); this was not observed among plant/invertebrate mirtrons. Interestingly, almost all the rG4s in mammalian mirtrons were present in the 5' arm. Predicted rG4s in human mirtrons form a G-quadruplex structure in-vitro and rG4 formation in the 5' arm of mirtrons facilitates splicing-mediated biogenesis of mirtrons. Notably, the disruption of rG4s in the 5' arm of mirtrons inhibits splicing and maturation; while mutations outside the rG4-motif do not impact mirtron biogenesis. Our findings support the notion that rG4s at the 5' arm are key regulatory elements in the evolutionary landscape of mammalian mirtrons. This work advances our current understanding of mirtron biogenesis and highlights additional roles for rG4s in small RNA biology.

The complement system plays an important role in antibacterial infection and immune regulation. Ba, an important complement component, is produced and released by the cleavage of complement factor B (CFB) during complement activation. However, the immune functions of Ba are unclear. In this study, we reported that recombinant Ba exerted direct bactericidal and immune regulatory effects. Recombinant Paralichthys olivaceus Ba (rPoBa) bound bacteria via interaction with the bacterial wall component lipopolysaccharide (LPS), resulting in bacterial membrane permeabilization and bacterial death. Furthermore, rPoBa exhibited bactericidal activity against Gram-negative bacteria in a manner that depended on concentration, time, temperature, pH, and metal ions. Structure prediction analysis showed that PoBa contained three distinct CCP domains. CCP1 was mainly responsible for binding to LPS, and both CCP1 and CCP3 might be required for bacterial membranous permeabilization. The bactericidal effects of Ba were observed only in lower vertebrates, with no such effects observed in mammals. In addition, rPoBa could protect P. olivaceus against Vibrio harveyi infection both in vitro and in vivo by significantly improving the immune activity of peripheral blood leukocytes and reducing tissue bacterial loads. Consistently, when PoCFB expression in P. olivaceus was knocked down, the PoBa production and complement activity were decreased, and bacterial replication was significantly enhanced. In conclusion, this study revealed that the complement-activated recombinant Ba fragment improved the immune defense against bacterial infection and provided a potential strategy to control disease in lower vertebrates.

Dentin sialophosphoprotein (DSPP) is highly expressed by odontoblasts, the cell type responsible for dentin formation. DSPP therefore has been extensively studied as a regulator of dentinogenesis. Besides defective dentinogenesis in teeth, Dspp deficient mice also display reduced blood vessels in the transition zone of femurs. However, the exact role and underlying mechanisms of DSPP in the process of blood vessel formation remain enigmatic. Here, we show that dentin sialoprotein (DSP), the NH₂-terminal cleavage product of DSPP, promotes the migration and capillary-like structure formation of human umbilical vein endothelial cells (HUVECs) as well as the migration and endothelial differentiation of human dental pulp stem cells (DPSCs). Further experiments demonstrate that endoglin (ENG), a membrane receptor associated with angiogenesis, can be co-immunoprecipitated by DSP. Flow cytometry assays show that HUVECs and DPSCs, two cell types with endogenous ENG expression, display obvious binding signals of supplemented DSP protein, but human embryonic kidney 293T (HEK293T) cells, a cell type without endogenous ENG expression do not. Pretreatment with an anti-ENG antibody or knockdown of ENG inhibits the binding of DSP to DPSCs, while ENG overexpression enhances binding signals of DSP to HEK293T cells. Meanwhile, multiple experiments demonstrate that knockdown of ENG impairs DSP-induced migration and endothelial differentiation of DPSCs. Therefore, ENG is essential for the angiogenic effects of DSP. Moreover, Dspp deficient mice exhibit defective capillary formation in molars, supporting the positive role of DSP in blood vessel development. Collectively, these findings identify that DSP acts as an angiogenic factor through association with ENG.

The taste system extends beyond the oral cavity, with various taste receptors found in extraoral organs. Mice deficient in the taste receptor type 1 (TAS1R) family member, TAS1R3, and fed a high-fat, high-sugar diet showed high bone mass without altering food consumption. However, the underlying mechanisms, including the cell types responsible for TAS1R3 expression, remain unclear. Here, we demonstrate the expression and function of TAS1R3 in osteoclasts, which are responsible for bone resorption. The expression of Tas1r3 but not Tas1r1 or Tas1r2, is evoked during osteoclast differentiation. Osteoclastogenesis-related genes were downregulated in TAS1R3-deficient mice, whereas the opposite phenotypes were elicited by TAS1R3 overexpression. Contrary to the common heterodimerization with TAS1R1 or TAS1R2, TAS1R3 formed a homodimer that functioned to detect glucose, enhance p38 phosphorylation, and induce osteoclastogenesis. These results provide novel insights into the role of TAS1R3 in bone metabolism and suggest that TAS1R3 may be a viable target for therapeutic agents in bone metabolic diseases.

Hyperglycemia is a hallmark of metabolic disorders, yet the precise mechanisms linking epigenetic regulation to glucose metabolism remain underexplored. Coactivator-associated arginine methyltransferase 1 (CARM1), a type I histone methyltransferase, promotes transcriptional activation through the methylation of histone H3 at arginine residues H3R17 and H3R26. Here, we identify a novel mechanism by which metformin, a widely prescribed antidiabetic drug, inhibits CARM1 activity. Using biochemical and biophysical assays, we show that metformin binds to the substrate-binding site of CARM1, reducing histone H3 methylation levels in CARM1-overexpressing hepatic cells and liver tissues from metformin-fed mice. This epigenetic modulation suppresses the expression of gluconeogenic enzymes (G6Pase, FBPase, and PCK1), thereby reversing CARM1-induced glycolytic suppression and regulating gluconeogenesis. Importantly, metformin does not alter CARM1 protein levels and its recruitment to gluconeogenic gene promoters but diminishes H3R17me2a marks at these loci. Our findings reveal a previously unrecognized epigenetic mechanism of metformin action, offering new therapeutic insights for hyperglycemia management.

The intrinsically disordered reflectin proteins drive tunable reflectivity for dynamic camouflage and communication in the recently evolved Loliginidae family of squid. Previous work revealed that reflectin A1 forms discrete assemblies whose size is precisely predicted by protein net charge density (NCD) and charge screening by the local anion concentration. Using dynamic light scattering (DLS), Forster resonant energy transfer (FRET) and confocal microscopy, we show that these assemblies, of which 95-99% of bulk protein in solution is partitioned into, are dynamic intermediates to liquid protein-dense condensates formed by liquid-liquid phase separation (LLPS). Increasing salt concentration drives this progression by anionic screening of the cationic protein's Coulombic repulsion, and by increasing the contribution of the hydrophobic effect which tips the balance between short-range attraction and long-range repulsion (SALR) to drive protein assembly and ultimately LLPS. Measuring fluorescence recovery after photobleaching (FRAP) and droplet fusion dynamics, we demonstrate that reflectin diffusivity in condensates is tuned by protein NCD. These results illuminate the physical processes governing reflectin A1 assembly and LLPS, and demonstrate the potential for reflectin A1 condensate-based tunable biomaterials. They also compliment previous observations of liquid phase separation in the Bragg lamellae of activated iridocytes and suggest that LLPS behavior may serve a critical role in governing the tunable and reversible dehydration of the membrane-bounded Bragg lamellae and vesicles containing reflectin in biophotonically active cells.

The p38 MAPKs' family includes four isoforms, of which only p38α has been considered essential for numerous important processes including mice embryogenesis. It is also considered essential for myoblast to myotube differentiation, as exposure of myoblasts to p38α/β inhibitors or to siRNA that targets p38α suppresses the process. The functions of p38β and p38γ in myoblast differentiation are not clear. We knocked out p38α in C2C12 myoblasts, assuming that the resulting C2p38α^-/- cells would not differentiate. They did, however, form mature fibers. We found elevated levels and activation of the p38 activator MKK6 in the C2p38α^-/- cells, leading to activation of p38β and p38γ, which are not active in differentiating parental C2C12 cells. Thus, p38α is an inhibitor of p38β+p38γ, that perhaps replace it in promoting differentiation. To test this notion, we generated C2p38α/γ^-/- and C2p38α/β^-/- cells and found that in both clones, the myogenic program was induced. C2p38β/γ^-/- cells also formed myotubes. These observations could be interpreted in two ways: either each p38 isoform can promote, by itself, the myogenic program, or p38 activity is not required at all for the process. Generating C2p38α/β/γ^-/- cells in which the myogenic program was shut-off altogether, showed that p38 activity is critical for differentiation. Notably, C2p38α/β/γ^-/- cells proliferate uncontrollably and give rise to foci, reminiscence of oncogenically-transformed cells. In summary, our study shows that a crosstalk between p38 isoforms functions in C2C12 cells as a safeguard mechanism that ensures resilience of the p38 activity in promoting the myogenic program and enforcing cell cycle arrest.