Journal of Cellular Biochemistry

Despite advances in treatment, HIV-1 infection continues to remain a major global health challenge, prompting ongoing efforts to understand the mechanisms that enable natural viral suppression and immune control. Elite controllers (ECs), a rare subset of PLWH individuals, naturally suppress HIV-1 replication without antiretroviral therapy, highlighting the importance of host-related factors in viral control. Understanding the mechanisms underlying this unique phenotype is crucial for developing novel therapeutic strategies. Previous studies from our group identified certain EC-specific metabolites, called dipeptides (DPs), and investigated their antiviral properties. We hypothesize that these dipeptides may potentially affect epithelial barrier integrity by modulating the expression of tight junction proteins, which in turn influences the mucosal barrier function, a key factor in HIV-1 pathogenesis. Therefore, in this study we investigated the impact of ten EC-specific DPs on tight junction (TJ) gene and protein expression in epithelial models derived from the female reproductive and gastrointestinal tracts, where we observed enhanced expression of different TJ genes (CLDN1, CLDN3, CLDN4, CLDN7, CLDN14, TJP1, TJP2, OCLN) and proteins (CLDN1, CLDN7, and CLDN14), suggesting the potential influence of these dipeptides on epithelial barrier function. Furthermore, we also examined different proteomic profiles between dipeptide (WG)-treated HeLa CD4+ CCR5+ cells compared with the untreated ones, and observed significantly reduced abundance of pro-inflammatory proteins, such as RELB Proto-Oncogene (RELB), TNF--induced protein 1 (TNFAIP1), TNF receptor superfamily member 1A (TNFRSF1A), and IL-32, in dipeptide-treated cells; and increased expression of proteins associated with tissue homeostasis (SMAD family member 5 [SMAD5]), cellular proliferation (transforming growth factor {beta} receptor 3 [TGFBR3]), and epithelial integrity, like CD81. Interestingly, KEGG analysis revealed possible attenuation of NF-{kappa}B, MAPK, TNF, and JAK-STAT signaling pathways, along with the enrichment of mTOR and PI3K-AKT pathways in treated HeLa CD4+ CCR5+ cells. Overall, this study investigated the potential interplay between tight junction proteins and key signaling pathways involved in maintaining epithelial barrier integrity and modulating immune activation, potentially contributing to both HIV-1 control and to the chronic inflammation associated with infection.

Leucine-rich repeat kinase 2 (LRRK2) is a complex multidomain protein whose catalytic and protein-protein interaction domains regulate a wide range of cellular processes. To investigate whether evolutionary divergence of these domains contributes to species-specific differences in LRRK2 biology, we combined phylogenetic, sequence, interactome and structural analyses of human and mouse LRRK2. Phylogenetic analysis revealed that the catalytic core predates the acquisition of the N-terminal and C-terminal protein-protein interaction domains during LRRK2 evolution. Accordingly, despite the high overall sequence similarity between human and mouse LRRK2, sequence divergence was not uniformly distributed across the protein but was concentrated within protein-protein interaction domains, whereas the catalytic ROC-COR- kinase core displayed markedly higher conservation. Consistent with this pattern, comparison of curated human and mouse interactomes revealed substantial differences in protein interaction networks and associated biological pathways. Structural modelling of a subset of interactors further showed that predicted interaction interfaces are enriched for residues that differ between the two species, providing a structural rationale for altered interaction specificity. Together, these findings support the view that evolutionary divergence of LRRK2 protein-protein interaction domains contributes to species-specific interactome organization. These results provide an evolutionary framework for interpreting differences between human and mouse LRRK2 and highlight the importance of considering species-specific interaction networks when translating findings from experimental models.

Background and PurposePARP inhibitors have been evaluated in clinical trials for several cancers and Olaparib is FDA approved for treating BRCA deficient ovarian cancer. Numerous reports have suggested Poly(ADP-ribose) polymerase1(PARP1) overexpression in a variety of cancers including breast carcinomas and proposed the role of PARP1 in metastasis. However, the mechanism of PARP1 in regulating metastatic process in BRCA proficient and deficient TNBC is not studied thoroughly. In this study, we propose that PARP1 mediated breast carcinoma progression is gene transcription mediated, where it regulates several steps of pro-metastasis. Experimental ApproachPARP inhibitors effect on metastasis was monitored by migration and invasion assay, modulation in protein expression was assessed by proteomic analysis and further confirmed by immunoblotting. Chromatin immunoprecipitation was performed to study the transcriptional role of PARP1. Ectopic expression and siRhoGDI, and immunofluorescence assessed the cytoskeleton changes. PARP inhibitor was administered in xenograft mice to study metastasis. Immunohistochemical analysis was done on patient and mice tissues. Key resultsBreast cancer cells exhibited reduced migration and invasion due to PARP1 inhibition. PARP1 regulates expression of vimentin and RhoGDI and hence cytoskeletal rearrangement causing a change in migrating potential of a cell. Metastasis in mice was reduced upon PARP inhibition. PARP1 was identified to be a novel transcriptional regulator of RhoGDI. Furthermore, RhoGDI ectopic expression substantiated the PARP inhibitor effects, suggesting the PARP inhibitor downstream signaling to be mediated through RhoGDI. Conclusions and ImplicationsWe identified a novel aspect of PARP1 as promoter of metastasis via transcriptional regulation of RhoGDI. Assessing RhoGDI levels in TNBC patients might be useful to predict sensitivity to PARP inhibitors.

Significance Missense mutations can lead to pathological effects in human cells. Predictive methods that account for structural context, such as AlphaMissense, can provide pathogenicity scores. The accumulation of pathogenicity hotspots can reveal important structural features within individual proteins of protein families, such as GLUT transporters. Mapping pathogenicity scores onto the structure can thus provide a mechanistic explanation of the protein function necessary for its role in the cell. Abstract Non-synonymous amino acid substitutions (missense mutations) are common in the general population; some are causative of serious disease. Depending on their structural context, they can disrupt protein function, folding, or dynamics. Computational predictive methods developed in recent years, such as AlphaMissense, provide new insights into how missense mutations affect protein structure by predicting and mapping their pathogenicity across each amino acid in the human proteome. In this study, we identify recurring patterns of pathogenicity prediction across the GLUT family membrane transporters encoded by genes slc2a1-14. Within the GLUT transporter family, we observe higher pathogenicity profiles in the transmembrane domains, particularly in pore-lining and binding-site residues. Predicted missense pathogenicity is elevated throughout residues assigned to the central cavity, suggesting sensitivity of the transport pathway. Another finding shows higher pathogenicity in specific transmembrane helices of the protein, with the same pattern across all proteins. On the other hand, we observed lower pathogenicity values in some representatives of the GLUT family. These findings show that the pathogenicity of glucose transport within the GLUT family may be shaped by functional redundancy and physiological essentiality across GLUT groups.

The methyltransferase EZH2 (Enhancer of Zest Homolog 2) and the demethylase KDM6B (Lysine Demethylase 6B) have been associated with epithelial to mesenchymal transition (EMT) and poor prognosis in various cancers. These enzymes methylate and demethylate H3K27me3 and regulate distinct sets of genes controlling EMT induction, despite having opposite catalytic activities. This could be due to their recruitment or the modulation of their activity by partner proteins on specific loci. This work sought to identify proteins associated with chromatin and interacting with EZH2 or with KDM6B during EMT. To do so, co-immunoprecipitation and mass spectroscopy was used under TGF{beta} (Tumor growth factor {beta}) and TNF (Tumor necrosis factor ) treatment to induce EMT in A549 lung cancer cells. Surprisingly, numerous proteins related to focal adhesions were identified to interact with EZH2 or KDM6B. These proteins are part of a nuclear protein interaction network previously described as nucleo-adhesome. Among these proteins, TGFB1I1 (transforming growth factor induced peptide 1) and CSRP2 (cysteine and glycine rich protein 2) were further confirmed to interact with KDM6B in the nucleus and even more so during EMT. The target genes of these complexes were then sought by knocking down KDM6B, TGFB1I1 or CSRP2. Three genes (coding Integrin alpha 5, Laminin y2 and Matrix Metalloproteinase 9) were confirmed to be regulated by KDM6B, TGFB1I1 and CSRP2. These findings may have clinical relevance, as immunohistochemistry analyses performed on a cohort of lung cancer patients revealed increased nuclear localization of TGFB1I1 and CSRP2 in cells undergoing EMT.

The phenotypic plasticity, slow replication, and complex, hydrophobic cell envelope of Mycobacterium tuberculosis contribute to its successful survival as a pathogen and its drug tolerance. Consequently, the global threat of multidrug-resistant Tuberculosis (MDR-TB), coupled with lengthy and highly toxic treatment regimens, necessitates the development of innovative treatment solutions. Mycobacteriophages are natural viruses of mycobacteria that typically encode two endolysins, which cooperatively facilitate host cell lysis at the end of the lytic life cycle: LysA, a peptidoglycan hydrolase, and LysB, a lipolytic enzyme, targeting the mycolylarabinogalactan-peptidoglycan complex. Their precise and efficient lytic activity, along with their low propensity to induce resistance, make them, particularly LysBs, promising candidates for new treatment solutions. In this study, we report MTB-LysB1, a novel LysB enzyme from an F1 sub-cluster mycobacteriophage isolated from our laboratory collection. While studying its structural features by comparing the modelled structure with representative mycobacteriophage LysB homologues, we found that the /{beta}-hydrolase fold and key motifs are conserved. Also, we identified putative membrane-interaction motifs that may play a role in LysB1s cell permeation. Significantly, we found MTB-LysB1 to be active against both drug-susceptible and multidrug-resistant (MDR) M. tuberculosis strains at nanomolar concentrations, comparable to the well-characterised D29 LysB reference enzyme. Beyond its standalone activity, MTB-LysB1 exhibits an additive effect when combined with the TB drugs rifampicin and moxifloxacin, and co-administration reduces the drugs minimum inhibitory concentrations (MICs), which holds clinical significance. By structurally damaging the mycobacterial cell wall, the enzyme appears to act as a permeability enhancer for the chemotherapeutic drugs, thereby improving antibiotic efficacy. Collectively, our findings position the enzyme not only as a novel antimycobacterial agent but also provide a structural framework for its rational engineering as a promising next-generation adjunct to TB drug regimens. HighlightsO_LIA novel F1 sub-cluster phage-derived LysB is discovered and characterised using integrated computational, biochemical and microbiological methods. C_LIO_LIAlphaFold2 modelling, molecular dynamics simulations and comparative structural analyses revealed an /{beta}-hydrolase fold with conserved catalytic and membrane-interaction features. C_LIO_LIThe enzyme exhibited high esterase activity, thermal stability and potent lytic activity against Mycobacterium tuberculosis. C_LIO_LIAn additive effect with TB drugs rifampicin and moxifloxacin highlights MTB-LysB1s potential as an adjunct therapeutic. C_LI

Long non-coding RNAs (lncRNAs) have attracted increasing interest because of their roles as modulators of tumor progression, acting either as oncogenic drivers or tumor suppressors, depending on the cellular context. LINC01133 has been implicated in regulation of multiple tumor-related mechanisms; however, its role in breast cancer, particularly in the triple-negative subtype, remains poorly characterized. In this study, we investigated the impact of LINC01133 depletion on malignant phenotypes and on the expression of migration- and invasion-associated genes using the Hs578T triple-negative breast cancer (TNBC) cell line, through comparative analyses of parental, control, and LINC01133-knockout cell lines, namely Hs578T_wt, Hs578T_ctr, and Hs578T_ko. Functional characterization included morphological analysis, growth assays, anchorage-independent colony formation, migration, invasion, and quantitative biomolecular experiments. Depletion of LINC01133 led to reduction of cell diameter, a significant increase in colony-forming capacity, and marked enhancement of migratory and invasive potential. At the molecular level, LINC01133 loss induced the expression of genes associated with extracellular matrix remodeling and cellular plasticity, including fibronectin, vimentin, integrins, FOXC1, and TWIST1, concomitant with reduced expression of ZEB1, TWIST2, and N-cadherin. Collectively, these data indicate that LINC01133 acts as a potential fine regulator of in vitro migration and invasion processes in TNBC, with its expression favoring a more asymptomatic mode of tumor progression, whereas its loss markedly enhances tumor malignancy.

In terms of cancer-related death, non-small cell lung cancer (NSCLC), the worlds leading cause, highlights the need for continued research into the genetic factors that influence tumor growth. Long non-coding RNAs (lncRNAs) are now well recognized as essential regulators of oncogenic signaling cascades; nevertheless, the specific role and molecular basis of the SOX2 overlapping transcript (SOX2OT) in NSCLC are not entirely understood. This study examined the functional importance of SOX2OT and its regulatory interactions with tumor-suppressive microRNAs in NSCLC cells. In A549 and Calu-3 cells, RNA interference-mediated SOX2OT silencing dramatically reduced cellular proliferation, migration, and invasiveness. Moreover, SOX2OT knockdown was associated with inhibition of epithelial-mesenchymal transition (EMT), alongside induction of cell cycle arrest and activation of apoptotic pathways. Integrated transcriptomic profiling and bioinformatic prediction analyses identified miR-143 as a putative downstream effector of SOX2OT activity. Consistently, depletion of SOX2OT resulted in marked elevation of miR-143 expression, which corresponded with downregulation of oncogenic mediators, including STAT3, EZH2, and CXCL13. As a result of SOX2OT suppression, both the transcript and the protein levels of PTEN were restored. Further functional characterization demonstrated that SOX2OT knockdown inhibits EMT progression by decreasing mesenchymal markers and EMT-related transcription factors (TFs) while concomitantly enhancing epithelial marker expression. Collectively, these findings suggest that SOX2OT contributes to NSCLC pathogenesis through regulation of a miR-143-centered signaling network that influences oncogenic signaling, cellular survival, and metastatic potential. Targeting the SOX2OT/miR-143 regulatory axis may therefore represent a promising therapeutic approach for NSCLC, while also underscoring the broader importance of lncRNA-mediated post-transcriptional regulation in lung cancer biology.

The lack of effective anti-hepatitis B virus (HBV) therapies highlights the need for a new type of treatment that targets different stages of the viral life cycle. The HBV core protein (HBc) is a critical component of this cycle. Various capsid assembly modulators (CAMs) have been developed to target the HBc and inhibit HBV replication. We recently described a subset of capsid assembly modulators (CAMs) that induce the formation of aberrant structures from the HBc in the nucleus, leading to cell death via annexin A1 (ANXA1)-driven apoptosis. Thus, we further elucidated the mechanism of HBc aggregation in the nucleus, with a particular focus on the interplay between nuclear HBc aggregates and PML nuclear bodies. We found that long-term treatment with CAM-A induced the formation of enlarged PML bodies, approximately 1-2 m in diameter, that accumulated aggregated HBc. PML silencing in HBc-overexpressing HepG2-NTCP cells led to a dramatic increase in apoptosis following CAM-A-induced HBc aggregation, which was associated with elevated ANXA1. Next, we showed that PML nuclear bodies orchestrate proteasomal degradation of nuclear HBc aggregates via sumoylation-dependent recruitment of RNF4. Collectively, our results suggest that PML nuclear bodies act as storage compartments for aggregated HBc proteins in the nucleus, thereby counteracting the apoptotic elimination of cells. Further study of PML function and the targeting of PML nuclear bodies in HBV-infected hepatocytes could reveal new ways to enhance the effectiveness of CAMs.

In TMPRSS2 fusion-positive prostate cancer, ERR is involved in regulation of ERG and promotes the androgen receptor independent signaling in the cancer progression. The ERR binds to the ERREs (estrogen-related receptor response elements) present at -5042 bp of the TMPRSS2- promoter and enhances the ERG overexpression that causes prostate cancer progression. To dissect the structural basis of the ERR recognition to the TMPRSS2 promoter DNA, we have purified the full-length ERR (ERRFL), NTD deleted construct (ERR{Delta}NTD), and the DNA-binding domain (ERRDBD) proteins and performed the binding analysis with 30 bp TMPRSS2-promoter DNA (5' -AGTCCAAGGTCGGTGGATC ACAAGGTCAGG-3'). Circular dichroism analysis showed that all three ERR proteins adopt native secondary structures. DNA binding induced subtle changes in the secondary structures, while enhancing the thermal stability (Tm) of all ERRa proteins. Binding analysis showed that ERRDBD bound weakly to the DNA, whereas ERRFL and ERR{Delta}NTD exhibited substantially higher affinities ~120-fold and ~131-fold than ERRaDBD, respectively. Small-angle X-ray scattering (SAXS) analyses revealed a dimeric ERRFL structure and an ERRFL-DNA complex (2:1) structure in solution and fitted well with Alpha Fold model of apo and DNA bound complex of ERRFL. Furthermore, 100 ns dynamics simulations on apo and DNA-bound ERRa proteins showed that all proteins remained structurally stable, with flexibility largely confined to loop regions of ERRa proteins. Our biophysical, DNA binding and structural analyses have revealed the mechanism involved in ERR recognition of the TMPRSS2- promoter DNA, which provides insight into ERR-mediated transcriptional regulation and development of anticancer drugs against ERR-driven prostate cancer.

It has been previously shown that chronic social defeat stress caused by paired agonistic interactions between male mice is accompanied by the development of depression-like state and immune deficiency. The aim of this study was to investigate changes in the expression of C1qtnf superfamily genes (encoding the complement component related with tumor necrosis factor) in the hypothalamus, thymus and lungs against the background of the Lewis lung adenocarcinoma growth. In the experiments, on the 5th day of social stress, male mice were injected with tumor cells into the tail vein. Chronic social stress continued for the next two weeks. The transcriptomes of the hypothalamus, thymus and lungs of mice were sequenced at the Genoanalytica Collective Center (http://genoanalytica.ru/, Moscow). Changes in the expression of the C1qtnf genes in the tissues of stressed mice were studied compared with the control and mice that were additionally injected with tumor cells. Overall, significant correlations were found between expression of most genes in each tissue of the experimental groups. In the hypothalamus of stressed animals, when tumor cells were introduced, an increase in the expression of the genes C1qtnf1, C1qtnf2, C1qtnf3, C1qtnf6 and C1qtnf7 was observed compared to controls. In the thymus of these animals, tumor cell injection increased expression of the C1qtnf1, C1qtnf5, and C1qtnf6 genes. In the lung of tumor-injected stressed mice, expression of the C1qtnf1, C1qtnf2, C1qtnf7, and C1qtnf9 genes was decreased relative to controls and non-tumor-injected depressed mice, reaching near-zero levels in some mice. Analysis of C1qtnf superfamily gene expression in the all tissues revealed negative correlations between the expression of the C1qtnf1, C1qtnf2, and C1qtnf7 genes in the hypothalamus and lungs indicating synchronization of processes against the background of social stress and Levis lung adenocarcinoma.

Analyses of human breast carcinomas (BCs) and premalignant breast lesions show that the loss of RASSF1A is an early event in the development of ER+ BCs, which correlates linearly with malignant progression. This observation suggests that RASSF1A inhibition is important for the development and progression of ER+ BCs. In addition to RASSF1A, concurrent caveolin-1 (Cav-1) inhibition may further promote ER+ breast carcinogenesis. In the present study, transgenic Rassf1a-/- and Cav-1(-/-) single as well as Rassf1a-/-, Cav-1(-/-) double knockout mice were used to investigate the impact of single or combined Rassf1a and Cav-1 inactivation on BC initiation. Loss of either one or both proteins led to different, pre-malignant histopathological alterations within the mammary glands of the mice, but not to fully developed BC, confirming that Rassf1a and Cav-1 are both important for maintaining the integrity of mammary gland epithelial structure, but suggesting that further intracellular changes or extracellular factors are required for the development of luminal BC when both genes are lost.

Cowpox virus CPXV012 inhibits MHC class I antigen presentation by interfering with TAP-dependent peptide transport, but its membrane-dependent structural organization and dynamic behavior remain incompletely defined. Here, we investigated the conformational properties of CPXV012 in membrane-mimicking environments and in a model of the CPXV012-TAP complex. CPXV012 was divided into three peptide constructs corresponding to the N-terminal cytosolic region, transmembrane segment, and C-terminal ER-luminal domain. The peptides were analyzed by circular dichroism spectroscopy, multidimensional NMR spectroscopy, and molecular dynamics simulations, and the resulting structural information was integrated into a full-length CPXV012 model. CD spectra showed that CPX-E1 and CPX-C2 are predominantly disordered in aqueous solution but acquire ordered, mainly -helical features in DPC micelles. NMR analysis in DPC-d38 micelles provided residue-level assignments and structural restraints supporting restrained structure calculations for both peptides. In three independent 1 {micro}s molecular dynamics simulations of the CPXV012-TAP complex, CPXV012 preserved a reproducible two-helical organization. The N-terminal/transmembrane region behaved as a relatively stable structural element, whereas the ER-luminal segment showed greater local flexibility. Interface analysis indicated that CPXV012 contacts both TAP1 and TAP2, with recurrent interactions concentrated in the luminal Y47-I69 region and involving polar and charge-complementary contacts. These results support a model in which membrane-associated structuring positions CPXV012 for TAP recognition, while the flexible ER-luminal region forms the main TAP-interacting surface. This structural framework complements existing functional models of CPXV012-mediated TAP inhibition.

Lysophosphatidic acid (LPA) is widely recognized as an extracellular lipid mediator; however, the functional significance of intracellularly produced LPA remains poorly understood. Here, we investigated the regulatory mechanism and functional role of a LPA-producing lysophospholipase D GDE4, also known as GDPD1, in prostate cancer cells. GDE4 expression is induced under ER stress conditions in a PERK-dependent manner and requires the transcription factor ATF3. Disruption of GDE4 expression resulted in altered intracellular levels of LPA and LPA precursor lysophosphatidylethanolamine, accompanied by reduced cell proliferation. RNA sequencing and subsequent validation identified a set of genes downregulated in GDE4-depleted cells. Pharmacological inhibition experiments indicated that peroxisome proliferator-activated receptor and {gamma} (PPAR and PPAR{gamma}) signaling pathways contribute to the regulation of these GDE4-dependent genes. Collectively, our findings suggest that GDE4-dependent lipid remodeling is associated with PPAR/{gamma}-mediated transcriptional regulation under ER stress conditions. These results provide a potential framework for understanding the link between intracellular lipid metabolism and stress-responsive gene regulation.

Mutations in p53 and its 12 isoforms can alter its functions. As N-terminally truncated isoforms of p53 (delta40p53, delta133p53, and delta160p53) participate in tetramer formation, they are important regulators of cancer fate. Although delta40p53- and delta133p53-mediated regulation of cancer is well reported, the mechanism underlying delta160p53 production and its functional role remains unclear. We investigated the internal ribosomal entry site (IRES)-mediated translation of {Delta}160p53 and its role in cancer regulation. As differential synthesis of delta160p53 was observed under different stress conditions, IRES-mediated translation of this isoform was demonstrated using bicistronic luciferase constructs. No cryptic promoters or splicing sites were detected in the IRES sequence. Cell death and late apoptosis were significantly decreased, while proliferation, the number of cells in the S phase, and drug resistance were induced by delta160p53. Furthermore, delta160p53 did not induce p53-responsive promoters. RNA sequencing analysis of delta160p53 overexpression showed similar results, along with the inhibition of other tumor suppressor genes. Overall, our results provide insights into IRES-mediated translation of delta160p53, which can be considered a novel target for cancer treatment.

The Epstein-Barr virus (EBV) is a highly prevalent virus worldwide that is associated with several lymphoid and epithelial malignancies. However, extensive research on EBV integral membrane proteins BILF1, LMP1 and LMP2, has been scarce due to their hydrophobic transmembrane domains. Our study applies the QTY code (glutamine, threonine, tyrosine) to design water-soluble analogs of BILF1, LMP1 and LMP2 with reduced hydrophobicity, where we systematically replaced hydrophobic amino acid residues leucine (L), isoleucine (I), valine (V), and phenylalanine (F) with structurally similar polar residues glutamine (Q), threonine (T), and tyrosine (Y). We retrieved their native sequences from UniProt, identified transmembrane domains using Protter, then performed QTY design through the Protein Solubilizing Server (PSS). We then predicted native and QTY structures using in silico prediction tools AlphaFold3, ColabFold, and Boltz-2. Our analyses demonstrate that despite significant protein sequence replacements in their transmembrane domains (54.15%-61.59%) and increased intrinsic solubility, the QTY analogs exhibited minimal changes in isoelectric point (0.00-0.15 decrease) and molecular weight (0.7-1.2 kDa increase). Additionally, structural superpositions between QTY analogs and native structures using PyMOL yield low RMSD values (0.217[A] -1.202[A]). Our results demonstrate the QTY codes ability to design detergent-free analogs of BILF1, LMP1 and LMP2 with substantially reduced hydrophobicity and aggregation propensity whilst preserving native-like structures. Our results may facilitate protein characterization studies, therapeutic research on EBV, and other protocols that typically require protein solubilization.

Neuroblastoma is a pediatric, sympathoadrenal tumour accounting for 7-10% of childhood malignancies. Some neuroblastomas are driven by activating mutations in ALK kinase and inhibitors show promise in clinical trials. Nevertheless, with resistance an ever-present concern, it remains important to understand better the effectors and modulators of ALK signaling. Wild type ALK promotes ERK activation, raising expression of negative regulators such as the dual-specificity phosphatase (DUSP) DUSP6. DUSP6 though can be pro- or anti-oncogenic in different cancers and its role in neuroblastoma cells remains unclear. We sought to understand its role in cells with either wild type or mutated ALK. Mutated ALK strongly promotes DUSP6 transcription, but apparently not through the ERK pathway. DUSP6 also appears to promote neuroblastoma cell proliferation without affecting ERK. Additionally, when DUSP6 is lost, the cells become more sensitive to ALK inhibitors lorlatinib and crizotinib. Phosphoproteomic analysis of such cells demonstrates that mutated ALK cooperates with DUSP6 to maximise signaling through several potential pathways, but again not through ERK or AKT. Their cooperation may also maintain optimal levels of N-Myc in MYCN-amplified neuroblastoma cells. While key substrates of DUSP6 remain to be determined in neuroblastoma cells, our study defines a novel role for this phosphatase in supporting the action of oncogenic ALK.

Urease is a nickel-dependent enzyme that plays an important role in urea hydrolysis and in a process named as microbial-induced calcium carbonate precipitation (MICP), which is widely used in sustainable environmental biotechnology. Despite its ecological importance, urease powers Biogrout (biocementation), a promising green technology for soil stabilization and infrastructure repair. Yet, the relationship between nickel availability, enzyme activation, and bacterial fitness remains poorly understood. In this study, we reveal a striking dual effect of nickel on Sporosarcina pasteurii: while high Ni{superscript 2} concentrations strongly inhibit growth (IC {approx} 637.7 {micro}M), they simultaneously boost specific urease activity up to six-fold. This uncoupling between biomass and enzymatic efficiency highlights a previously overlooked adaptive strategy under metal stress. Using structural bioinformatics and molecular docking, we show that Ure1--the catalytic subunit--exhibits the strongest nickel affinity (-4.3 kcal{middle dot}mol-{superscript 1}), supported by highly conserved active-site residues, whereas accessory proteins UreE and UreG display moderate and weak binding, consistent with their roles in metal delivery and GTP-dependent maturation. In addition, microscopic observations confirmed that calcium carbonate precipitation was most pronounced at intermediate nickel concentrations (approximately 400-1000 {micro}M), whereas higher concentrations ([≥]1000-1300 {micro}M) led to reduced mineral formation due to loss viable cells. Taken together, these results indicates that nickel availability controls both urease activation and bacterial fitness, and that an optimal balance is required to maximize biomenerilization efficiency in environmental applications, particularly in biocementation technology. ImportanceUrease-driven biomineralization is widely used in sustainable technologies such as soil stabilization and self-healing concrete. However, optimizing these systems requires a clear understanding of how environmental factors influence enzyme performance. This study shows that nickel, an essential cofactor for urease, plays a dual role by enhancing enzymatic activity while inhibiting bacterial growth at high concentrations. By integrating experimental data with computational analysis, we demonstrate that efficient biomineralization depends on maintaining nickel within an optimal range that balances enzyme activation and microbial viability. These findings provide practical guidance for improving biocementation processes and highlight nickel as a key regulator of urease-based environmental biotechnology applications.

The epidermal growth factor receptor (EGFR) expression is often dysregulated in head and neck squamous cell carcinoma (HNSCC), driving cancer cell proliferation, invasion, and metastasis through diverse pathways, thereby contributing to aggressive chemo- and radio-therapy resistance. A GPI-anchored protein, CD109 is upregulated in multiple cancers, including HNSCC. While membrane-anchored CD109 (mCD109) is pro-tumorigenic in SCC via EGFR/STAT3 activation, the role of protease-cleaved soluble CD109 (sCD109) is poorly understood. Our groundbreaking findings demonstrate that sCD109 antagonizes EGFR signaling by directly binding to the EGFR extracellular domain, preventing mCD109-EGFR stabilizing interactions on the cell surface, followed by inhibition of EGFR phosphorylation at Y1068 and downstream signaling cascades (AKT, MAPK, and STAT3) consequently suppressing cancer cell migration, invasion, 3D tumor spheroid formation and angiogenic tube formation. In addition, we found that sCD109 regulates EGFR fates by inhibiting nuclear localization of phosphorylated EGFR and promoting EGFR degradation. Additionally, sCD109 significantly reduces EGF-induced expression of cancer stem cell markers (CD44 and CD133) and embryonic stem cell markers (Nanog and Sox2), suggesting a suppressive role in cancer stemness. Taken together, these results underscore the opposing roles of mCD109 and sCD109: with sCD109 acting as an antagonist by inhibiting mCD109/EGFR-driven oncogenic signaling and phenotypes. Our current findings reveal a complex interplay among mCD109, sCD109, and EGFR, identifying a mechanism for targeting EGFRs degradation in HNSCC, and lay the groundwork for future research on investigating sCD109s modulatory role in preclinical models of HNSCC.

The UL49.5 protein of bovine alphaherpesvirus 1 (BoHV-1) is known to inhibit the transporter associated with antigen processing (TAP) and interfere with antigen presentation, in part by promoting TAP degradation. However, the role of electrostatic interactions within the N-terminal luminal domain in controlling these processes remains unclear. Here, we combined circular dichroism (CD), solution nuclear magnetic resonance spectroscopy (NMR), all-atom molecular dynamics simulations, and cell-based assays to define the structural and functional contribution of charged residues within the N-terminal luminal domain of UL49.5. Two N-terminal variants of UL49.5, UL49.522-56RR(30-31)DD and UL49.522-56D36K, with substitutions of charged-reversal amino acid residues, were designed. These two mutants formed membrane-induced -helical structures but showed altered helix stability and interaction patterns. Molecular dynamics simulations of the UL49.5-TAP complexes revealed that wild-type UL49.5 forms a stable electrostatic interface with TAP, particularly in the unkinked conformation, while charge-reversal mutations remodel salt-bridge networks, destabilize the luminal helix, and alter the positioning and dynamics of the transmembrane and cytoplasmic C-terminal regions. The structural changes within the N-terminus alter the exposure of the C-terminal degron required for KLHDC3-dependent degradation. Consistent with these findings, the mutants did not induce proteasomal degradation of TAP, despite maintaining near wild-type levels of downregulation of MHC class I. Together, these results identify N-terminal electrostatic interactions as allosteric determinants of UL49.5-driven TAP degradation and demonstrate that TAP degradation can be mechanically uncoupled from downregulation of MHC class I. This study improves our understanding of viral immune evasion strategies and potential therapeutic targets.