Adaptor Protein Research Articles

Characterization of a novel immune deficiency gene of Macrobrachium rosenbergii reveals antibacterial and antiviral defenses.

We sought to identify and characterize an immune deficiency (IMD) homolog from the giant freshwater prawn (also known as the giant river prawn) Macrobrachium rosenbergii. The IMD is a death-domain-containing protein that plays a crucial role as an adaptor protein in the IMD pathway-one of the most important response mechanisms to viral and bacterial invasion of invertebrates. An IMD homolog gene from M. rosenbergii (MrIMD) was isolated using rapid amplification of complementary DNA ends. The tissue distribution and response to immune challenge of MrIMD were analyzed by real-time reverse transcription polymerase chain reaction to understand the regulatory mechanism of MrIMD messenger RNA (mRNA) expression in M. rosenbergii. The open reading frame of MrIMD comprised 555 nucleotides encoding a protein consisting of 184 amino acids, with a conserved death domain at the C-terminus. The MrIMD protein demonstrated 53-74% similarity with IMDs from other crustaceans; the highest similarity was with the IMD from the oriental river prawn M. nipponense. Gene expression analysis revealed that MrIMD mRNA levels were highest in gill tissues. After Aeromonas hydrophila stimulation, MrIMD was significantly upregulated in the muscle, gills, and intestine, whereas there was no significant difference in the hemocytes and hepatopancreas. In the case of Macrobrachium rosenbergii nodavirus stimulation, MrIMD was dramatically upregulated in the muscle and hepatopancreas, whereas downregulation was observed in the gills. These results suggest that the MrIMD gene may play different roles in response to gram-negative bacteria and viral infection and plays a crucial role in innate immunity as an important key molecule in the defense against bacterial and viral infections.

RUVBL1 ubiquitination by DTL promotes RUVBL1/2-β-catenin-mediated transcriptional regulation of NHEJ pathway and enhances radiation resistance in breast cancer

Radiotherapy effectiveness in breast cancer is limited by radioresistance. Nevertheless, the mechanisms behind radioresistance are not yet fully understood. RUVBL1 and RUVBL2, referred to as RUVBL1/2, are crucial AAA+ ATPases that act as co-chaperones and are connected to cancer. Our research revealed that RUVBL1, also known as pontin/TIP49, is excessively expressed in MMTV-PyMT mouse models undergoing radiotherapy, which is considered a murine spontaneous breast-tumor model. Our findings suggest that RUVBL1 enhances DNA damage repair and radioresistance in breast cancer cells both in vitro and in vivo. Mechanistically, we discovered that DTL, also known as CDT2 or DCAF2, which is a substrate adapter protein of CRL4, promotes the ubiquitination of RUVBL1 and facilitates its binding to RUVBL2 and transcription cofactor β-catenin. This interaction, in turn, attenuates its binding to acetyltransferase Tat-interacting protein 60 (TIP60), a comodulator of nuclear receptors. Subsequently, ubiquitinated RUVBL1 promotes the transcriptional regulation of RUVBL1/2-β-catenin on genes associated with the non-homologous end-joining (NHEJ) repair pathway. This process also attenuates TIP60-mediated H4K16 acetylation and the homologous recombination (HR) repair process. Expanding upon the prior study’s discoveries, we exhibited that the ubiquitination of RUVBL1 by DTL advances the interosculation of RUVBL1/2-β-catenin. And, it then regulates the transcription of NHEJ repair pathway protein. Resulting in an elevated resistance of breast cancer cells to radiation therapy. From the aforementioned, it is evident that targeting DTL-RUVBL1/2-β-catenin provides a potential radiosensitization approach when treating breast cancer.

NOD2 Responds to Dengue Virus Type 2 Infection in Macrophage-like Cells Interacting with MAVS Adaptor and Affecting IFN-α Production and Virus Titers

In pathogen recognition, the nucleotide-binding domain (NBD) and leucine rich repeat receptors (NLRs) have noteworthy functions in the activation of the innate immune response. These receptors respond to several viral infections, among them NOD2, a very dynamic NLR, whose role in dengue virus (DENV) infection remains unclear. This research aimed to determine the role of human NOD2 in THP-1 macrophage-like cells during DENV-2 infection. NOD2 levels in DENV-2 infected THP-1 macrophage-like cells was evaluated by RT-PCR and Western blot, and an increase was observed at both mRNA and protein levels. We observed using confocal microscopy and co-immunoprecipitation assays that NOD2 interacts with the effector protein MAVS (mitochondrial antiviral signaling protein), an adaptor protein promoting antiviral activity, this occurring mainly at 12 h into the infection. After silencing NOD2, we detected increased viral loads of DENV-2 and lower levels of IFN-α in supernatants from THP-1 macrophage-like cells with NOD2 knock-down and further infected with DENV-2, compared with mock-control or cells transfected with Scramble-siRNA. Thus, NOD2 is activated in response to DENV-2 in THP-1 macrophage-like cells and participates in IFN-α production, in addition to limiting virus replication at the examined time points.

FADD promotes type I interferon production to suppress porcine reproductive and respiratory syndrome virus infection

Porcine reproductive and respiratory syndrome (PRRS) is an epidemic animal infectious disease worldwide, causing huge economic losses to the global swine industry. Fas-associated death domain (FADD) was previously reported to be an adaptor protein that functions in transferring the apoptotic signals regulated by the death receptors. In the current study, we unravel its unidentified role in promoting type I interferon (IFN) production during PRRS virus (PRRSV) infection. We identified that FADD inhibited PRRSV infection via promotion of type I IFN transcription. Overexpression of FADD suppressed the replication of PRRSV, while knockout of FADD increased viral titer and nucleocapsid protein expression. Mechanistically, FADD promoted mitochondrial antiviral signaling protein (MAVS)-mediated production of IFN-β and some IFN-stimulated genes (ISGs). Furthermore, FADD exerted anti-PRRSV effects in a MAVS-dependent manner and increased the type I IFN signaling during PRRSV infection. This study highlights the importance of FADD in PRRSV replication, which may have implications for the future control of PRRS.

Abstract LB433: Targeting of ABI1 via DNA-based PROTACs

Abstract Breast cancer is the most commonly diagnosed non-cutaneous cancer in American women. Although current therapies reduce initial tumor burden, patients often relapse with aggressive tumors which can metastasize. Abelson Interactor 1 (ABI1) is an adaptor protein which is traditionally seen as a WAVE complex component- regulating actin polymerization, cell migration and adhesion. In this role, ABI1 serves as a driver of breast cancer metastasis. Recent studies have discovered that ABI1 contains a homeobox homology region (HHR) which is able to bind DNA and regulate transcription. In order to specifically target and degrade ABI1, we designed a novel PROteolysis-TArgeting Chimera (PROTAC) system using DNA as the bait for ABI1. We hypothesized that by linking ABI1 DNA targets to the ubiquitin system, we would be able to effectively target ABI1 for degradation. Lenalidomide, an E3 ubiquitin ligase ligand, was attached to an oligonucleotide via click chemistry. Cells were then treated with PROTAC systems via direct addition to media or via lipofection reagents, and levels of ABI1 were detected using western blotting. Using this DNA-based PROTAC system in cell-culture models of both breast and ABI1-elevated prostate cancer suggested that the system is able to target and degrade ABI1. Usage of lipofection reagents were able to improve drug delivery and effectiveness. Further research is needed to identify off-target effects and effectiveness in vivo models of disease to reduce metastasis. In conclusion, PROTACs targeting ABI1 may prove to be an effective and novel method to reduce metastasis and improve outcomes for cancer treatment. Citation Format: Kevin M. Lin, Xiang Li, Leszek Kotula. Targeting of ABI1 via DNA-based PROTACs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB433.

Abstract SY05-01: Targeting chromatin adaptor proteins in cancer

Abstract Menin interacts with oncogenic fusion proteins produced by MLL1 rearrangements, and small molecules capable of disrupting these interactions are currently under investigation in clinical trials for the treatment of leukemia. Our research, which combines chromatin-focused and genome-wide CRISPR screenings with a range of genetic, pharmacological, and biochemical strategies in both mouse and human models, has led to the discovery of a molecular mechanism that acts as a switch between the MLL1-Menin and MLL3/4-UTX chromatin modifying complexes, influencing the efficacy of Menin-MLL inhibitors. We demonstrate that the MLL1-Menin complex plays a critical role in maintaining leukemia cell survival by preventing the MLL3/4-UTX complex from binding to certain gene promoters. Interruption of the Menin-MLL1 interaction results in the UTX-dependent transcriptional activation of a tumor suppressor gene program, which significantly influences therapeutic outcomes in both murine and human leukemia models. Notably, the therapeutic activation of this gene program through CDK4/6 inhibitors can overcome resistance in leukemia cells that do not respond to Menin inhibitors. The identification of this molecular switch between MLL1-Menin and MLL3/4-UTX complexes on chromatin highlights the complex roles of these evolutionarily conserved epigenetic regulators and underscores their importance in understanding and addressing the molecular mechanisms underlying response and resistance in leukemia treatment trials. Citation Format: Yadira Soto-Feliciano. Targeting chromatin adaptor proteins in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY05-01.

Abstract LE21: Mechanisms of gene regulation by chromatin adaptors in development and disease

Abstract Complex biological processes, such as organismal development, are directly influenced by the interplay between genes and the cellular structures and environmental factors that regulate them. Key players in this regulation are chromatin adaptor proteins, which can impact the genetic and epigenetic programs of cells. However, our understanding of how these proteins interact with chromatin - the physiological form of our genomes - and regulate gene activity is limited, especially in the context of diseases like cancer. To test our central hypothesis and to understand how chromatin adaptors contribute to cell function, we are developing innovative high-throughput and biochemical approaches. These will help us systematically identify specific histone modifications that affect the binding of Menin and other adaptors to chromatin. Our experimental approach includes profiling, validation, and functional characterization using advanced genomic and epigenomic technologies, transcriptional profiling, and CRISPR gene editing. The application of these techniques will reveal, for the first time, the molecular mechanisms by which these proteins bind to specific parts of the genome and regulate gene activity. Our findings will enhance our understanding of how chromatin adaptors, like Menin, interact with chromatin and regulate gene activity in different tissues, and how these functions are disrupted by cancer-associated mutations. This will also contribute to the development of potential new therapies that target these altered mechanisms. Furthermore, our findings can be extended to other proteins involved in chromatin and epigenetic regulation, enabling a broader application in understanding and treating cancer and other human diseases. Citation Format: Yadira Marie Soto-Feliciano. Mechanisms of gene regulation by chromatin adaptors in development and disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LE21.

Abstract LB188: PEA-15 phosphorylation mediates pro-tumor effects by promoting β-catenin activity in TNBC

Abstract Triple negative breast cancer (TNBC) is a subtype of breast cancer that is defined by a lack of estrogen receptor, progesterone receptor, and HER2-neu gene product expression. Previously, we found that TNBC cells with phosphorylated PEA-15 adaptor protein exhibited decreased clonogenic and anchorage-independent growth in vitro, and decreased growth and lung metastasis in vivo. Further, we found lower levels of phosphorylated β-catenin at S675 in PEA-15-AA (non-phosphorylatable) mutants than in PEA-15-DD (phosphomimetic) mutants. We therefore hypothesized that phosphorylated PEA-15 enhances the aggressiveness of TNBC by regulating the phosphorylation of β-catenin at S675. We first screened a panel of TNBC cells for β-catenin levels via western blot. Next to assess the biological role of β-catenin we knocked down its expression in TNBC cells (SUM149, BT549). In the β-catenin knockouts, proliferation decreased by 20%(P<0.0001) mammosphere formation by 69% (P<0.001), and clonogenic growth by 65% (P<0.0001), when compared to the vector control. To determine if PEA-15 mediates pro-tumor effects by phosphorylating β-catenin at S675, we created a nonphosphorylatable (S675A) and a phosphomimetic (S675D) form of β-catenin. We transfected BT549 cells with S675A and observed a decrease of 26% (P=0.011) in proliferation, 65% (P<0.001) in mammospheres, and 54% (P=0.0001) in clonogenic formation when compared with WT β-catenin transfected cells. Next, we transfected SUM149 cells with S675D and found an increase of 58% (P<0.001) in proliferation, 79% (P=0.001) in mammospheres, and 23% (P=0.0303) in clonogenic formation when compared with WT β-catenin transfected cells. To test if PEA-15 phosphorylation mediates β-catenin activity, we transfected the S675A and S675D form of β-catenin into MDA-MB-468 cell lines which stably express PEA-15-AA and PEA-15-DD. When we performed a rescue experiment with PEA-15-AA cells transfected with S675D we found a 78% (P=0.0008) increase in mammospheres when compared to vector transfected cells. Conversely, when PEA-15-DD cells were transfected with S675A we found a 53% (P=0.0004) decrease in mammospheres when compared to vector transfected cells. Next, we did a multiplex staining of a tissuemicroarray (n= 102 human TNBC tumors). We observed that in patients with grade 3 tumors, the percentages of cells expressing total PEA-15(P=0.005), P-PEA-15-S116(P=0.038), and p-β-catenin (P=0.0005) were significantly higher than those in patients with grade 2 tumors. Together, these results show that PEA-15 phosphorylation mediates tumor aggressiveness in TNBC by promoting β-catenin activity. Future studies will evaluate the clinical significance of PEA-15 and β-catenin and their phosphorylated forms in TNBC patients. These results will establish β-catenin and PEA-15 phosphorylation as potential markers of, and targets against TNBC progression. Citation Format: Alex W. Tan, Mohd Mughees, Wendy A. Woodward, Debu Tripathy, Natalie Fowlkes, Chandra Bartholomeusz. PEA-15 phosphorylation mediates pro-tumor effects by promoting β-catenin activity in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB188.

Abstract LB114: STING licensing of type I dendritic cells potentiates antitumor immunity

Abstract Stimulator of interferon genes (STING) is an immune adaptor protein that senses cyclic GMP-AMP (cGAMP) in response to self or microbial cytosolic DNA as a danger signal. STING is ubiquitously expressed in diverse cell populations including cancer cells with distinct cellular functions such as activation of type I interferons, autophagy induction, or triggering apoptosis. It is not well understood whether and which subsets of immune cells, stromal cells, or cancer cells are particularly important for STING-mediated antitumor immunity. Here using a polymeric STING-activating nanoparticle (PolySTING) with a “shock-and-lock” dual activation mechanism, we show type 1 conventional dendritic cell (cDC1) is essential for STING-mediated rejection of multiple established and metastatic murine tumors. STING status in the host but not in the cancer cells (Tmem173− / − ) is important for antitumor efficacy. Specific depletion of cDC1 (Batf3− / − ) or STING deficiency in cDC1 (XCR1creSTINGfl/fl ) abolished PolySTING efficacy, whereas depletion of other myeloid cells had little effect. Adoptive transfer of wildtype cDC1 in Batf3− / − mice restored antitumor efficacy while transfer of cDC1 with STING or IRF3 deficiency failed to rescue. PolySTING induced a specific chemokine signature in wildtype but not Batf3− / − mice. Multiplexed immunohistochemistry analysis of STING-activating cDC1s in resected tumors correlates with patient survival while also showing increased expressions after neoadjuvant pembrolizumab therapy in non-small cell lung cancer patients. Therefore, we have defined that a subset of myeloid cells is essential for STING-mediated antitumor immunity with associated biomarkers for prognosis. Citation Format: Jian Wang, Suxin Li, Maggie Wang, Xu Wang, Shuqing Chen, Zhichen Sun, Xiubao Ren, Gang Huang, Baran D. Sumer, Nan Yan, Yang-Xin Fu, Jinming Gao. STING licensing of type I dendritic cells potentiates antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB114.

Molecular dynamics simulations to explore the binding mode between the amyloid-β protein precursor (APP) and adaptor protein Mint2

Alzheimer's disease (AD) presents a significant challenge in neurodegenerative disease management, with limited therapeutic options available for its prevention and treatment. At the heart of AD pathogenesis is the amyloid-β (Aβ) protein precursor (APP), with the interaction between APP and the adaptor protein Mint2 being crucial. Despite previous explorations into the APP-Mint2 interaction, the dynamic regulatory mechanisms by which Mint2 modulates APP binding remain poorly understood. This study undertakes molecular dynamics simulations across four distinct systems—free Mint2, Mint2 bound to APP, a mutant form of Mint2, and the mutant form bound to APP—over an extensive 400 ns timeframe. Our findings reveal that the mutant Mint2 experiences significant secondary structural transformations, notably the formation of an α-helix in residues S55-K65 upon APP binding, within the 400 ns simulation period. Additionally, we observed a reduction in the active pocket size of the mutant Mint2 compared to its wild-type counterpart, enhancing its APP binding affinity. These insights hold promise for guiding the development of novel inhibitors targeting the Mints family, potentially paving the way for new therapeutic strategies in AD prevention and treatment.

Recombinant probiotic Lactococcus lactis delivering P62 mitigates moderate colitis in mice

Introduction and objectivep62 is a human multifunctional adaptor protein involved in key cellular processes such as tissue homeostasis, inflammation, and cancer. It acts as a negative regulator of inflammasome complexes. It may thus be considered a good candidate for therapeutic use in inflammatory bowel diseases (IBD), such as colitis. Probiotics, including recombinant probiotic strains producing or delivering therapeutic biomolecules to the host mucosal surfaces, could help prevent and mitigate chronic intestinal inflammation. The objective of the present study was to combine the intrinsic immunomodulatory properties of the probiotic Lactococcus lactis NCDO2118 with its ability to deliver health-promoting molecules to enhance its protective and preventive effects in the context of ulcerative colitis (UC).Material and methodsThis study was realized in vivo in which mice were supplemented with the recombinant strain. The intestinal barrier function was analyzed by monitoring permeability, secretory IgA total levels, mucin expression, and tight junction genes. Its integrity was evaluated by histological analyses. Regarding inflammation, colonic cytokine levels, myeloperoxidase (MPO), and expression of key genes were monitored. The intestinal microbiota composition was investigated using 16S rRNA Gene Sequencing.Results and discussionNo protective effect of L. lactis NCDO2118 pExu:p62 was observed regarding mice clinical parameters compared to the L. lactis NCDO2118 pExu: empty. However, the recombinant strain, expressing p62, increased the goblet cell counts, upregulated Muc2 gene expression in the colon, and downregulated pro-inflammatory cytokines Tnf and Ifng when compared to L. lactis NCDO2118 pExu: empty and inflamed groups. This recombinant strain also decreased colonic MPO activity. No difference in the intestinal microbiota was observed between all treatments. Altogether, our results show that recombinant L. lactis NCDO2118 delivering p62 protein protected the intestinal mucosa and mitigated inflammatory damages caused by dextran sodium sulfate (DSS). We thus suggest that p62 may constitute part of a therapeutic approach targeting inflammation.

Enzymatic activity of cGAS in the presence of three types of DNAs: limited cGAS stimulation by single-stranded HIV-1 SL2 DNA.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is activated by binding to DNA. Activated cGAS produces 2'3'-cGAMP, which subsequently binds to the adaptor protein STING (stimulator of interferon genes). This interaction triggers the cGAS/STING signaling pathway, leading to the production of type I interferons. Three types of DNA, namely double-stranded DNA longer than 40 base pairs, a 70-nucleotide single-stranded HIV-1 DNA known as SL2, and Y-form DNA with unpaired guanosine trimers (G3 Y-form DNA), induce interferon production by activating cGAS/STING signaling. However, the extent of cGAS activation by each specific DNA type remains unclear. The comparison of cGAS stimulation by various DNAs is crucial for understanding the mechanisms underlying cGAS-mediated type I interferon production in the innate immune response. Here, we revealed that cGAS produces 2'3'-cGAMP at a significantly lower rate in the presence of single-stranded SL2 DNA than in the presence of double-stranded DNA or G3 Y-form DNA. Furthermore, the guanine-to-cytosine mutations and the deletion of unpaired guanosine trimers significantly reduced the 2'3'-cGAMP production rate and the binding of cGAS to Y-form DNA. These studies will provide new insights into the cGAS-mediated DNA-sensing in immune response.

SLAM Family Receptors in B Cell Chronic Lymphoproliferative Disorders.

The signaling lymphocytic activation molecule (SLAM) receptor family (SLAMF) consists of nine glycoproteins that belong to the CD2 superfamily of immunoglobulin (Ig) domain-containing molecules. SLAMF receptors modulate the differentiation and activation of a wide range of immune cells. Individual SLAMF receptors are expressed on the surface of hematopoietic stem cells, hematopoietic progenitor cells, B cells, T cells, NK cells, NKT cells, monocytes, macrophages, dendritic cells, neutrophils, and platelets. The expression of SLAMF receptors was studied during normal B cell maturation. Several SLAMF receptors were also detected in cancer cell lines of B-lymphoid origin and in pathological B cells from patients with B cell chronic lymphoproliferative disorders (B-CLPD), the most frequent hematological malignancies in adults. This review summarizes current knowledge on the expression of SLAMF receptors and their adaptor proteins SAP and EAT-2 in B-CLPD. Several SLAMF receptors could be regarded as potential diagnostic and differential diagnostic markers, prognostic factors, and targets for the development of novel drugs for patients with B-CLPD.

Proteomic Analyses Reveal the Role of Alpha-2-Macroglobulin in Canine Osteosarcoma Cell Migration.

Canine osteosarcoma (OSA) is an aggressive bone neoplasia with high metastatic potential. Metastasis is the main cause of death associated with OSA, and there is no current treatment available for metastatic disease. Proteomic analyses, including matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI TOF/TOF MS), are widely used to select molecular targets and identify proteins that may play a key role in primary tumours and at various steps of the metastatic cascade. The main aim of this study was to identify proteins differently expressed in canine OSA cell lines with different malignancy phenotypes (OSCA-8 and OSCA-32) compared to canine osteoblasts (CnOb). The intermediate aim of the study was to compare canine OSA cell migration capacity and assess its correlation with the malignancy phenotypes of each cell line. Using MALDI-TOF/TOF MS analyses, we identified eight proteins that were significantly differentially expressed (p ≤ 0.05) in canine OSA cell lines compared to CnOb: cilia- and flagella-associated protein 298 (CFAP298), general transcription factor II-I (GTF2I), mirror-image polydactyly gene 1 protein (MIPOL1), alpha-2 macroglobulin (A2M), phosphoglycerate mutase 1 (PGAM1), ubiquitin (UB2L6), ectodysplasin-A receptor-associated adapter protein (EDARADD), and leucine-rich-repeat-containing protein 72 (LRRC72). Using the Simple Western technique, we confirmed high A2M expression in CnOb compared to OSCA-8 and OSCA-32 cell lines (with intermediate and low A2M expression, respectively). Then, we confirmed the role of A2M in cancer cell migration by demonstrating significantly inhibited OSA cell migration by treatment with A2M (both at 10 and 30 mM concentrations after 12 and 24 h) in a wound-healing assay. This study may be the first report indicating A2M's role in OSA cell metastasis; however, further in vitro and in vivo studies are needed to confirm its possible role as an anti-metastatic agent in this malignancy.

Iron Oxide Nanoparticles Inhibit Tumor Progression and Suppress Lung Metastases in Mouse Models of Breast Cancer.

Systemic exposure to starch-coated iron oxide nanoparticles (IONPs) can stimulate antitumor T cell responses, even when little IONP is retained within the tumor. Here, we demonstrate in mouse models of metastatic breast cancer that IONPs can alter the host immune landscape, leading to systemic immune-mediated disease suppression. We report that a single intravenous injection of IONPs can inhibit primary tumor growth, suppress metastases, and extend survival. Gene expression analysis revealed the activation of Toll-like receptor (TLR) pathways involving signaling via Toll/Interleukin-1 receptor domain-containing adaptor-inducing IFN-β (TRIF), a TLR pathway adaptor protein. Requisite participation of TRIF in suppressing tumor progression was demonstrated with histopathologic evidence of upregulated IFN-regulatory factor 3 (IRF3), a downstream protein, and confirmed in a TRIF knockout syngeneic mouse model of metastatic breast cancer. Neither starch-coated polystyrene nanoparticles lacking iron, nor iron-containing dextran-coated parenteral iron replacement agent, induced significant antitumor effects, suggesting a dependence on the type of IONP formulation. Analysis of multiple independent clinical databases supports a hypothesis that upregulation of TLR3 and IRF3 correlates with increased overall survival among breast cancer patients. Taken together, these data support a compelling rationale to re-examine IONP formulations as harboring anticancer immune (nano)adjuvant properties to generate a therapeutic benefit without requiring uptake by cancer cells.

Cargo specificity, regulation, and therapeutic potential of cytoplasmic dynein.

Intracellular retrograde transport in eukaryotic cells relies exclusively on the molecular motor cytoplasmic dynein 1. Unlike its counterpart, kinesin, dynein has a single isoform, which raises questions about its cargo specificity and regulatory mechanisms. The precision of dynein-mediated cargo transport is governed by a multitude of factors, including temperature, phosphorylation, the microtubule track, and interactions with a family of activating adaptor proteins. Activating adaptors are of particular importance because they not only activate the unidirectional motility of the motor but also connect a diverse array of cargoes with the dynein motor. Therefore, it is unsurprising that dysregulation of the dynein-activating adaptor transport machinery can lead to diseases such as spinal muscular atrophy, lower extremity, and dominant. Here, we discuss dynein motor motility within cells and in in vitro, and we present several methodologies employed to track the motion of the motor. We highlight several newly identified activating adaptors and their roles in regulating dynein. Finally, we explore the potential therapeutic applications of manipulating dynein transport to address diseases linked to dynein malfunction.

An Ultra-Rare Mixed Phenotype with Combined AP-4 and ERF Mutations: The First Report in a Pediatric Patient and a Literature Review

The adaptor protein 4 (AP-4) constitutes a conserved hetero-tetrameric complex within the family of adaptor protein (AP) complex, crucial for the signal-mediated trafficking of integral membrane proteins. Mutations affecting all subunits of the AP-4 complex have been linked to autosomal-recessive cerebral palsy and a complex hereditary spastic paraparesis (HSP) phenotype. Our report details the case of a 14-year-old boy born to consanguineous parents, presenting psychomotor delay, severe intellectual disability, microcephaly, and trigonocephaly. Despite a history of febrile seizures, subsequent years were devoid of seizures, with normal EEG. Exome sequencing revealed pathogenic variants in both the AP4B1 and ERF genes. Significantly, the patient exhibited features associated with AP4B1 mutations, including distinctive traits such as cranial malformations. The ERF gene variant, linked to craniosynostosis, likely contributes to the observed trigonocephaly. This case represents the initial documentation of a concurrent mutation in the AP4B1 and ERF genes, underscoring the critical role of exome analysis in unraveling complex phenotypes. Understanding these complex genotypes offers valuable insights into broader syndromic conditions, facilitating comprehensive patient management.

Structural analysis of resistance-nodulation cell division transporters.

SUMMARYInfectious bacteria have both intrinsic and acquired mechanisms to combat harmful biocides that enter the cell. Through adaptive pressures, many of these pathogens have become resistant to many, if not all, of the current antibiotics used today to treat these often deadly infections. One prominent mechanism is the upregulation of efflux systems, especially the resistance-nodulation-cell division class of exporters. These tripartite systems consist of an inner membrane transporter coupled with a periplasmic adaptor protein and an outer membrane channel to efficiently transport a diverse array of substrates from inside the cell to the extracellular space. Detailed mechanistic insight into how these inner membrane transporters recognize and shuttle their substrates can ultimately inform both new antibiotic and efflux pump inhibitor design. This review examines the structural basis of substrate recognition of these pumps and the molecular mechanisms underlying multidrug extrusion, which in turn mediate antimicrobial resistance in bacterial pathogens.

Gulp1 regulates chondrocyte growth arrest and differentiation via the TGF-β/SMAD2/3 pathway.

Chondrocyte differentiation is crucial for cartilage formation. However, the complex processes and mechanisms coordinating chondrocyte proliferation and differentiation remain incompletely understood. Here, we report a novel function of the adaptor protein Gulp1 in chondrocyte differentiation. Gulp1 expression is upregulated during chondrogenic differentiation. Gulp1 knockdown in chondrogenic ATDC5 cells reduces the expression of chondrogenic and hypertrophic marker genes during differentiation. Furthermore, Gulp1 knockdown impairs cell growth arrest during chondrocyte differentiation and reduces the expression of the cyclin-dependent kinase inhibitor p21. The activation of the TGF-β/SMAD2/3 pathway, which is associated with p21 expression in chondrocytes, is impaired in Gulp1 knockdown cells. Collectively, these results demonstrate that Gulp1 contributes to cell growth arrest and chondrocyte differentiation by modulating the TGF-β/SMAD2/3 pathway.

Conserved Functions of Orthohepadnavirus X Proteins to Inhibit Type-I Interferon Signaling.

Orthohepadnavirus causes chronic hepatitis in a broad range of mammals, including primates, cats, woodchucks, and bats. Hepatitis B virus (HBV) X protein inhibits type-I interferon (IFN) signaling, thereby promoting HBV escape from the human innate immune system and establishing persistent infection. However, whether X proteins of Orthohepadnavirus viruses in other species display a similar inhibitory activity remains unknown. Here, we investigated the anti-IFN activity of 17 Orthohepadnavirus X proteins derived from various hosts. We observed conserved activity of Orthohepadnavirus X proteins in inhibiting TIR-domain-containing adaptor protein inducing IFN-β (TRIF)-mediated IFN-β signaling pathway through TRIF degradation. X proteins from domestic cat hepadnavirus (DCH), a novel member of Orthohepadnavirus, inhibited mitochondrial antiviral signaling protein (MAVS)-mediated IFNβ signaling pathway comparable with HBV X. These results indicate that inhibition of IFN signaling is conserved in Orthohepadnavirus X proteins.