Study of individual domains contributing to MALT1 dimerization in BCL10-independent and dependent assembly.

The CARMA-BCL10-MALT1 (CBM) signalosome functions as a pivotal supramolecular module, integrating diverse receptor-induced signaling pathways to regulate BCL10-dependent NF-kB activation in innate and adaptive immunity. Conversely, the API2-MALT1 fusion protein in t(11; 18)(q21; q21) MALT lymphoma constitutively induces BCL10-independent NF-kB activation. MALT1 dimer formation is indispensable for the requisite proteolytic activity and is critical for NF-kB activation regulation in both scenarios. However, the molecular assembly of MALT1 individual domains in CBM activation remains elusive. Here we report the crystal structure of the MALT1 death domain (DD) at a resolution of 2.1 Å, incorporating reconstructed residues in previously disordered loops 1 and 2. Additionally, we observe a conformational regulation element (CRE) regulating stem-helix formation in NLRPs pyrin (PYD) within the MALT1 DD structure. The structure reveals a stem-helix-mediated dimer further corroborated in solution. To elucidate how the BCL10 filament facilitates MALT1 dimerization, we reconstitute a BCL10-CARD-MALT1-DD-IG1-IG2 complex model. We propose a N+7 rule for BCL10-dependent MALT1 dimerization via the IG1-IG2 domain and for MALT1-dependent cleavage in trans. Biochemical data further indicates concentration-dependent dimerization of the MALT1 IG1-IG2 domain, facilitating MALT1 dimerization in BCL10-independent manner. Our findings provide a structural and biochemical foundation for understanding MALT1 dimeric mechanisms, shedding light on potential BCL10-independent MALT1 dimer formation and high-order BCL10-MALT1 assembly.

Lambda-free light chain: A serum marker of dengue disease via NS3 protease-mediated antibody cleavage.

Dengue poses a significant global public health threat, with diverse clinical manifestations due to complex interactions between the host and the pathogen. Recent reports have highlighted elevated serum-free light chain (FLC) levels in viral infectious diseases. Hence, our study aimed to investigate serum FLC levels in dengue patients. The findings revealed elevated serum λ FLCs, which were associated with the severity of dengue. Receiver operating characteristic curve (ROC) analysis demonstrated that λ FLCs may serve as a serum marker for identifying dengue disease (AUC: 0.7825, sensitivity: 80, specificity: 71.43) and classifying severe dengue (AUC: 0.8102, sensitivity: 75, specificity: 79.52). The viral protease, Dengue virus (DENV) nonstructural protein 3 (NS3), acts as a protease that cleaves viral polyproteins as well as host substrates. Therefore, we proposed that antibodies might be potential targets of NS3 protease, leading to an increase in FLCs. LC/MS-MS analysis confirmed that λ FLCs were the predominant products after antibody degradation by NS3 protease. Additionally, purified NS3 protease cleaved both human IgG and DENV2-neutralizing antibodies, resulting in the presence of λ FLCs. Moreover, NS3 protease administration in vitro led to a reduction in the neutralizing efficacy of DENV2-neutralizing antibodies. In summary, the elevated serum λ FLC levels effectively differentiate dengue patients from healthy individuals and identify severe dengue. Furthermore, the elevation of serum λ FLCs is, at least in part, mediated through NS3 protease-mediated antibody cleavage. These findings provide new insights for developing diagnostic tools and understanding the pathogenesis of DENV infection.

Disruption of the pentraxin 3/CD44 interaction as an efficient therapy for triple-negative breast cancers.

Due to the heterogeneity and high frequency of genome mutations in cancer cells, targeting vital protumour factors found in stromal cells in the tumour microenvironment may represent an ideal strategy in cancer therapy. However, the regulation and mechanisms of potential targetable therapeutic candidates need to be investigated. An in vivo study demonstrated that loss of pentraxin 3 (PTX3) in stromal cells significantly decreased the metastasis and growth of cancer cells. Clinically, our results indicate that stromal PTX3 expression correlates with adverse prognostic features and is associated with worse survival outcomes in triple-negative breast cancer (TNBC). We also found that transforming growth factor beta 1 (TGF-ß1) induces PTX3 expression by activating the transcription factor CCAAT/enhancer binding protein delta (CEBPD) in stromal fibroblasts. Following PTX3 stimulation, CD44, a PTX3 receptor, activates the downstream ERK1/2, AKT and NF-κB pathways to specifically contribute to the metastasis/invasion and stemness of TNBC MDA-MB-231 cells. Two types of PTX3 inhibitors were developed to disrupt the PTX3/CD44 interaction and they showed a significant effect on attenuating growth and restricting the metastasis/invasion of MDA-MB-231 cells, suggesting that targeting the PTX3/CD44 interaction could be a new strategy for future TNBC therapies.

Protein-based soft actuator with high photo-response and easy modulation for anisotropic cell alignment and proliferation in a liquid environment.

Cell alignment and elongation, which are critical factors correlated with differentiation and maturation in cell biology and tissue engineering, have been widely studied in organisms. Several strategies such as external mechanical strain, geometric topography, micropatterning approaches, and microfabricated substrates have been developed to guide cell alignment, but these methodologies cannot be used for easily denatured natural proteins to modulate the cell behaviour. Herein, for the first time, a novel biocompatible light-controlled protein-based bilayer soft actuator composed of elastin-like polypeptides (ELPs), silk fibroin (SF), graphene oxide (GO), and reduced graphene oxide (rGO), named ESGRG, is developed for efficiently driving cellular orientation and elongation with anisotropic features on soft actuator via remote NIR laser exposure. The actuation of ESGRG could be manipulated by modulating the intensity of NIR and the relative ratio of GO to rGO for promoting myoblasts alignment and nucleus elongation to generate different motions. The results indicate that the YAP and MHC protein expression of C2C12 skeletal muscle cells on ESGRG can be rapidly induced and enhanced by controlling the relative ratio of rGO/GO = 1/4 at a multiple-cycle stimulation with a very low power intensity of 1.2 W cm-2 in friendly liquid environments. This study demonstrates that the ESGRG hydrogel actuator system can modulate the cell-level behaviors via light-driven cyclic bending-motions and can be utilized in applications of soft robotic and tissue engineering such as artificial muscle and maturation of cardiomyocytes.

Development of a computational promoter with highly efficient expression in tumors.

BACKGROUND: Gene therapy is a potent method to increase the therapeutic efficacy against cancer. However, a gene that is specifically expressed in the tumor area has not been identified. In addition, nonspecific expression of therapeutic genes in normal tissues may cause side effects that can harm the patients' health. Certain promoters have been reported to drive therapeutic gene expression specifically in cancer cells; however, low expression levels of the target gene are a problem for providing good therapeutic efficacy. Therefore, a specific and highly expressive promoter is needed for cancer gene therapy. METHODS: Bioinformatics approaches were utilized to analyze transcription factors (TFs) from high-throughput data. Reverse transcription polymerase chain reaction, western blotting and cell transfection were applied for the measurement of mRNA, protein expression and activity. C57BL/6JNarl mice were injected with pD5-hrGFP to evaluate the expression of TFs. RESULTS: We analyzed bioinformatics data and identified three TFs, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), cyclic AMP response element binding protein (CREB), and hypoxia-inducible factor-1α (HIF-1α), that are highly active in tumor cells. Here, we constructed a novel mini-promoter, D5, that is composed of the binding sites of the three TFs. The results show that the D5 promoter specifically drives therapeutic gene expression in tumor tissues and that the strength of the D5 promoter is directly proportional to tumor size. CONCLUSIONS: Our results show that bioinformatics may be a good tool for the selection of appropriate TFs and for the design of specific mini-promoters to improve cancer gene therapy.

CCAAT/enhancer-binding protein delta promotes intracellular lipid accumulation in M1 macrophages of vascular lesions.

AIMS: Lipid homeostasis is reprogrammed in the presence of inflammation, which results in excessive lipid accumulation in macrophages, and leads to the formation of lipid-laden foam cells. We aimed to link an inflammation-responsive transcription factor CCAAT/enhancer-binding protein delta (CEBPD) with polarized macrophages and dissect its contribution to lipid accumulation. METHODS AND RESULTS: We found that CEBPD protein colocalized with macrophages in human and mouse (C57BL/6, Apoe-/-) atherosclerotic plaques and that Cebpd deficiency in bone marrow cells suppressed atherosclerotic lesions in hyperlipidemic Apoe-/- mice. CEBPD was responsive to modified low-density lipoprotein (LDL) via the p38MAPK/CREB pathway, and it promoted lipid accumulation in M1 macrophages but not in M2 macrophages. CEBPD up-regulated pentraxin 3 (PTX3), which promoted the macropinocytosis of LDL, and down-regulated ATP-binding cassette subfamily A member 1 (ABCA1), which impaired the intracellular cholesterol efflux in M1 macrophages. We further found that simvastatin (a HMG-CoA reductase inhibitor) could target CEBPD to block lipid accumulation in a manner not directly related to its cholesterol-lowering effect in M1 macrophages. CONCLUSION: This study underscores how CEBPD functions at the junction of inflammation and lipid accumulation in M1 macrophages. Therefore, CEBPD-mediated lipid accumulation in M1 macrophages could represent a new therapeutic target for the treatment of cardiovascular diseases.

Conductive nanogel-interfaced neural microelectrode arrays with electrically controlled in-situ delivery of manganese ions enabling high-resolution MEMRI for synchronous neural tracing with deep brain stimulation.

Chronic brain stimulation has become a promising physical therapy with increased efficacy and efficiency in the treatment of neurodegenerative diseases. The application of deep brain electrical stimulation (DBS) combined with manganese-enhanced magnetic resonance imaging (MEMRI) provides an unbiased representation of the functional anatomy, which shows the communication between areas of the brain responding to the therapy. However, it is challenging for the current system to provide a real-time high-resolution image because the incorporated MnCl2 solution through microinjection usually results in image blurring or toxicity due to the uncontrollable diffusion of Mn2+. In this study, we developed a new type of conductive nanogel-based neural interface composed of amphiphilic chitosan-modified poly(3,4 -ethylenedioxythiophene) (PMSDT) that can exhibit biomimic structural/mechanical properties and ionic/electrical conductivity comparable to that of Au. More importantly, the PMSDT enables metal-ligand bonding with Mn2+ ions, so that the system can release Mn2+ ions rather than MnCl2 solution directly and precisely controlled by electrical stimulation (ES) to achieve real-time high-resolution MEMRI. With the integration of PMSDT nanogel-based coating in polyimide-based microelectrode arrays, the post-implantation DBS enables frequency-dependent MR imaging in vivo, as well as small focal imaging in response to channel site-specific stimulation on the implant. The MR imaging of the implanted brain treated with 5-min electrical stimulation showed a thalamocortical neuronal pathway after 36 h, confirming the effective activation of a downstream neuronal circuit following DBS. By eliminating the susceptibility to artifact and toxicity, this system, in combination with a MR-compatible implant and a bio-compliant neural interface, provides a harmless and synchronic functional anatomy for DBS. The study demonstrates a model of MEMRI-functionalized DBS based on functional neural interface engineering and controllable delivery technology, which can be utilized in more detailed exploration of the functional anatomy in the treatment of neurodegenerative diseases.

Structural Insights into Linear Tri-ubiquitin Recognition by A20-Binding Inhibitor of NF-κB, ABIN-2.

Recognition of linear polyubiquitin by specific ubiquitin-binding proteins plays an important role in mediating nuclear factor-κB (NF-κB) signaling. A20 binding proteins, ABINs, recognize linear polyubiquitin and A20 through UBAN and AHD1, respectively, for the inhibition of NF-κB activation. Here we report the crystal structure of the AHD1-UBAN fragment of ABIN2 in complex with linear tri-ubiquitin, which reveals a 2:1 stoichiometry of the complex. Structural analyses together with mutagenesis, pull-down, and isothermal titration calorimetry assays show that the hABIN2:tri-ubiquitin interaction is mainly through the primary ubiquitin-binding site, and also through the secondary ubiquitin-binding site under a high local protein concentration. Surprisingly, three ubiquitin units could form a right-handed helical trimer to bridge two ABIN2 dimers. The residues around the M1-linkage are crucial for ABIN2 to recognize tri-ubiquitin. The tri-ubiquitin bridging two ABIN2 dimers model suggests a possible higher-order signaling complex assembled between M1-linked polyubiquitinated proteins, ubiquitin-binding proteins, and effector signaling proteins in signal transduction.

Infectious dengue vesicles derived from CD61+ cells in acute patient plasma exhibited a diaphanous appearance.

The levels of neutralizing antibody to a pathogen are an effective indicator to predict efficacy of a vaccine in trial. And yet not all the trial vaccines are in line with the theory. Using dengue virus (DENV) to investigate the viral morphology affecting the predictive value, we evaluated the viral morphology in acute dengue plasma compared to that of Vero cells derived DENV. The virions in plasma were infectious and heterogeneous in shape with a "sunny-side up egg" appearance, viral RNA was enclosed with CD61+ cell-derived membrane interspersed by the viral envelope protein, defined as dengue vesicles. The unique viral features were also observed from ex vivo infected human bone marrow. Dengue vesicles were less efficiently neutralized by convalescent patient serum, compared to virions produced from Vero cells. Our results exhibit a reason why potencies of protective immunity fail in vivo and significantly impact dengue vaccine and drug development.

Targeting chemotherapy-induced PTX3 in tumor stroma to prevent the progression of drug-resistant cancers.

The tumor microenvironment has been suggested to participate in tumorigenesis, but the nature of the communication between cancer cells and the microenvironment, especially in response to anticancer drugs, remains obscure. We determined that activation of the CCAAT/enhancer binding protein delta (CEBPD) response to Cisplatin and 5-Fluorouracil in cancer-associated macrophages and fibroblasts contributed to the metastasis, invasion, acquired chemoresistance and stemness of cancer cells by in vitro and in vivo assays. Specifically, reporter and in vivo DNA binding assays were used to determine that Pentraxin 3 (PTX3) is a CEBPD responsive gene and serves a protumor role upon anticancer drug treatment. Finally, a PTX3 peptide inhibitor RI37 was developed and assessed the antitumor effects by in vivo assays. RI37 could function as a promising inhibitor for preventing cancer progression and the metastasis, invasion and progression of drug-resistant cancers. The identification of PTX3 provided a new insight in the interaction between host and tumor and the RI37 peptide showed a great opportunity to largely reduce the risk of invasion and metastasis of cancer and drug-resistant cancers.

Tandem DEDs and CARDs suggest novel mechanisms of signaling complex assembly.

Apoptosis is an important process to maintain cellular homeostasis. Deregulated apoptosis has linked to a number of diseases, such as inflammatory diseases, neurodegenerative disorder, and cancers. A major signaling complex in the death receptor signaling pathway leading to apoptosis is death-induced signaling complex (DISC), which is regulated mainly by death effector domain (DED)-containing proteins. There are seven DED-containing proteins in human, including FADD, c-FLIP, caspase-8, caspase-10, DEDD, DEDD2, and PEA-15. The main players in DISC formation employ tandem DEDs for regulating signaling complex formation. The regulatory mechanism of signaling complex formation is important and yet remains unclear. Interestingly, three caspase recruitment domain (CARD)-containing members, which belong to the same DD superfamily as DED-containing proteins, also contains similar tandem CARDs. Recent structural studies have shown that tandem CARDs are essential for the formation of a helical signaling complex. This review summarizes recent structural studies on DED-containing proteins and especially discusses the studies on tandem DEDs and tandem CARDs, which suggest new mechanisms of signaling complex assembly.

E2 interaction and dimerization in the crystal structure of TRAF6.

Tumor necrosis factor (TNF) receptor-associated factor (TRAF)-6 mediates Lys63-linked polyubiquitination for NF-kappaB activation via its N-terminal RING and zinc finger domains. Here we report the crystal structures of TRAF6 and its complex with the ubiquitin-conjugating enzyme (E2) Ubc13. The RING and zinc fingers of TRAF6 assume a rigid, elongated structure. Interaction of TRAF6 with Ubc13 involves direct contacts of the RING and the preceding residues, and the first zinc finger has a structural role. Unexpectedly, this region of TRAF6 is dimeric both in the crystal and in solution, different from the trimeric C-terminal TRAF domain. Structure-based mutagenesis reveals that TRAF6 dimerization is crucial for polyubiquitin synthesis and autoubiquitination. Fluorescence resonance energy transfer analysis shows that TRAF6 dimerization induces higher-order oligomerization of full-length TRAF6. The mismatch of dimeric and trimeric symmetry may provide a mode of infinite oligomerization that facilitates ligand-dependent signal transduction of many immune receptors.

Molecular basis for the unique deubiquitinating activity of the NF-kappaB inhibitor A20.

Nuclear factor kappaB (NF-kappaB) activation in tumor necrosis factor, interleukin-1, and Toll-like receptor pathways requires Lys63-linked nondegradative polyubiquitination. A20 is a specific feedback inhibitor of NF-kappaB activation in these pathways that possesses dual ubiquitin-editing functions. While the N-terminal domain of A20 is a deubiquitinating enzyme (DUB) for Lys63-linked polyubiquitinated signaling mediators such as TRAF6 and RIP, its C-terminal domain is a ubiquitin ligase (E3) for Lys48-linked degradative polyubiquitination of the same substrates. To elucidate the molecular basis for the DUB activity of A20, we determined its crystal structure and performed a series of biochemical and cell biological studies. The structure reveals the potential catalytic mechanism of A20, which may be significantly different from papain-like cysteine proteases. Ubiquitin can be docked onto a conserved A20 surface; this interaction exhibits charge complementarity and no steric clash. Surprisingly, A20 does not have specificity for Lys63-linked polyubiquitin chains. Instead, it effectively removes Lys63-linked polyubiquitin chains from TRAF6 without dissembling the chains themselves. Our studies suggest that A20 does not act as a general DUB but has the specificity for particular polyubiquitinated substrates to assure its fidelity in regulating NF-kappaB activation in the tumor necrosis factor, interleukin-1, and Toll-like receptor pathways.

Crystal structure of the BIR1 domain of XIAP in two crystal forms.

X-linked inhibitor of apoptosis (XIAP) is a potent negative regulator of apoptosis. It also plays a role in BMP signaling, TGF-beta signaling, and copper homeostasis. Previous structural studies have shown that the baculoviral IAP repeat (BIR2 and BIR3) domains of XIAP interact with the IAP-binding-motifs (IBM) in several apoptosis proteins such as Smac and caspase-9 via the conserved IBM-binding groove. Here, we report the crystal structure in two crystal forms of the BIR1 domain of XIAP, which does not possess this IBM-binding groove and cannot interact with Smac or caspase-9. Instead, the BIR1 domain forms a conserved dimer through the region corresponding to the IBM-binding groove. Structural and sequence analyses suggest that this dimerization of BIR1 in XIAP may be conserved in other IAP family members such as cIAP1 and cIAP2 and may be important for the action of XIAP in TGF-beta and BMP signaling and the action of cIAP1 and cIAP2 in TNF receptor signaling.

Crystal structures and electron micrographs of fungal volvatoxin A2.

Membrane adhesion and insertion of protein are essential to all organisms, but the underlying mechanisms remain largely unknown. Membrane pore-forming toxins (PFTs) are potential model systems for studying these mechanisms. We have determined the crystal structures of volvatoxin A2 (VVA2), a fungal PFT from Volvariella volvacea, using Br-multiple-wavelength anomalous diffraction (MAD). The VVA2 structures obtained at pH 4.6, pH 5.5 and pH 6.5 were refined to resolutions of 1.42 A, 2.6 A and 3.2 A, respectively. The structures reveal that the VVA2 monomer contains a single alpha/beta domain. Most of the VVA2 surface is occupied by its oligomerization motif and two putative heparin-binding motifs. Residues Ala91 to Ala101 display several conformations at different pH values, which might be under the control of His87. We also found that the shape of one putative heparin-binding motif in VVA2 appears similar to those found in fibroblast growth factors, and the other one displays a linear polypeptide. Our results suggest several possible intermediates of protein assembly in solution and protein adhering to cell membranes before conformational changes. The electron micrographs of VVA2 molecules in solution, at a protein concentration of 1 microg ml(-1), show that they can assemble into filament-like or braid-like oligomers in a pH-dependent way. In addition, the arc-shaped VVA2 structure obtained at pH 6.5 suggests that VVA2 could form a two-layered helical oligomer with 18 subunits per turn. The structures presented here could be used to elucidate the pore-formation mechanisms of VVA2 and its structural neighbors, Cyt toxins from Bacillus thuringiensis.