Liver is a complex organ, capable of incredible regenerative capacity in the mammalian body. In addition to well-known hepatocytes, liver is composed of a complex cell microenvironment, with each cell type contributing to homeostasis, regeneration and disease. Modelling of the liver in vitro has been hampered by 2D cell lines being too simple and whole animal models too complex to underpin the mechanisms of liver biology. Liver organoids are emerging as a great tool to study liver (patho)physiology. Increasing in complexity, organoids have successfully been used to understand the cellular interplay in genetic and metabolic liver diseases, as well as liver cancer. This review will discuss the advancement in modelling cell complexity, maturation and liver architecture in a Petri dish. It will also provide a state-of-the-art perspective of what is still lacking and how the models can be further improved. Finally, we will conclude with our personal vision of where the field is going.
Heme-containing enzymes represent one of nature's most versatile catalytic platforms, capable of mediating a broad spectrum of redox and group-transfer reactions. This review provides a comprehensive overview of the structural and mechanistic principles that underpin the bioengineering of heme proteins for both natural and abiological transformations. Here, we discuss that the core of reactivity of such metalloenzymes is the iron-porphyrin cofactor, whose tunable oxidation and spin states enable the formation of diverse high-energy intermediates, including iron-oxo, carbene, and nitrene species. Using recent bioengineering applications, we also show that the catalytic behavior of these intermediates is not solely determined by intrinsic cofactor chemistry but is profoundly influenced by the surrounding protein scaffold. Key factors such as axial ligation, second coordination sphere interactions, hydrogen-bonding networks, and local electric fields collectively govern substrate binding, intermediate stabilization, and reaction selectivity. The review further discusses a few applications of de novo protein design and artificial metalloenzymes that provide unprecedented control over active-site architecture, allowing the creation of highly robust and tunable catalysts.
The p21-activated kinases (PAKs) are conserved serine/threonine kinases that couple cytoskeletal dynamics to diverse cellular processes. Despite emerging roles for PAK2 in development and disease, its regulated phosphorylation programs remain poorly defined. Here, we performed quantitative phosphoproteomic profiling of WT and Pak2-/- mouse embryos at embryonic day 9.5 and identified 151 differential phosphopeptide features, corresponding to 119 phosphosites and 116 PAK2-associated phosphoproteins. Differential events were predominantly downregulated and enriched for serine-proline motifs. Functional analyses highlighted modules involved in nuclear RNA processing, chromatin/DNA regulation, translation/RNA-binding, cytoskeletal signaling, and neurodevelopment. Finally, bulk RNA-seq and single-cell transcriptomic analyses integrated the genes encoding these phosphoproteins into cellular and developmental programs, providing transcriptomic context for the PAK2-associated post-translational events in early embryogenesis.
How do eukaryotic genomes acquire new functional parts, such as regulatory elements and long non-coding RNAs? Here, we propose that these parts arise from non-functional precursors primarily through non-adaptive processes that proliferate when selection is weak. Borrowing concepts from Markov chain theory, we represent each stage along the junk-to-function continuum as a discrete state with defined transition probabilities. This formalism makes clear why selection cannot act on future function, and why a part's current biochemical activity may be unrelated to its evolutionary past. We show how, under certain conditions, new intermediate states appear that increase the forward flux from junk DNA to new functional parts. These conditions-weak selection, abundant epistasis and quality control processes, and the accumulation of messiness-are characteristic of many eukaryotic genomes, allowing them to become more complex as new functional parts emerge.
Although chronic psychosocial stress is a well-established independent risk factor for cardiovascular disease (CVD), the underlying mechanisms linking stress to CVD remain incompletely understood. Here, we review clinical and preclinical research documenting how stress can activate innate immune cells. Based on these findings, we propose that trained innate immunity is one possible mechanism responsible for the lasting effects of stress on the innate immune system, and hence contributes to the association between stress and CVD. Trained immunity is defined as a persistent hyperinflammatory phenotype in innate immune cells, mediated by metabolic and epigenetic reprogramming of the bone marrow myeloid progenitor cells. As we describe herein, accumulating experimental evidence shows that chronic psychosocial stress can induce a maladaptive trained immunity program that accelerates atherosclerosis. By advancing our understanding of stress-induced trained immunity, we will move one step closer to designing new therapeutic, behavioral, and lifestyle strategies to counter maladaptive immune activation and reduce risk for CVD, one of the greatest causes of morbidity and mortality worldwide.
Photosystem II (PS II) is a water-plastoquinone oxidoreductase. This photosystem has a short half-life (< 1 h) which is driven by repair of oxidative damage to the D1 protein. Reactive oxygen species (ROS) produced by the photosystem under stress conditions oxidatively modify key residues, which trigger the sequential disassembly of the photosystem. We hypothesize that the principal target for ROS damage of D1 is 332His. This residue is a ligand to Mn1 of the Mn4CaO5 cluster and is the first residue exhibiting oxidative modification during photoinactivation. Recently, cryo-EM studies indicate that PS II monomers containing oxidatively modified 332His lose their manganese clusters and the extrinsic proteins associated with the photosystem, both hallmarks of the earliest stages of PS II turnover/repair.
Single-molecule biophysical techniques enable the study of biological systems by directly observing individual molecules and events, rather than measuring population averages. These approaches reveal transient states, rare events, conformational dynamics, and subpopulations that are often obscured in biochemical assays. The application of single-molecule methods has been limited by technical complexity and the need for specialized instrumentation. Recent standardization of protocols, methodologies, and platforms has lowered these barriers, enabling broader adoption and promoting interdisciplinary collaborations that facilitate integration into biochemical research. In this review, we discuss how ensemble biochemistry and single-molecule approaches are complementary, outline commonly used single-molecule techniques, and illustrate their relevance through two representative case studies: chromatin organization by SMC complexes and pathway choice during DNA double-strand break repair.
Cisplatin (CDDP), a widely used chemotherapeutic agent, enters mammalian cells through high-affinity copper transporter CTR1. We found that copper preloading reduces CDDP cytotoxicity, supporting a shared uptake mechanism for copper and cisplatin. CDDP-induced CTR1 endocytosis requires both the amino-terminal His-Met motifs and the conserved 150MXXM154 pore-motif, similar to copper-mediated transport. Following internalization, CTR1 is routed through VPS35-positive endosomes and ultimately targeted to lysosomes. However, unlike copper, CDDP suppresses local recycling of CTR1 to plasma membrane and delays its lysosomal delivery. While copper promotes rapid lysosomal clearance of CTR1, CDDP results in prolonged intracellular trafficking. Additionally, CDDP enhances lysosomal size and cathepsin activity, indicating lysosomal activation. These findings reveal distinct mechanisms underlying CTR1-mediated cisplatin uptake and its impact on lysosomal physiology.
Protein engineering relies heavily on computational characterization of constrained protein fitness landscapes, in which only a limited fraction of sequence space corresponds to stable and functional biomolecules. Advances in structural biology and machine learning are progressively shifting protein design strategies from empirical optimization toward multidimensional evaluation of sequence-structure-function relationships. This review examines current computational strategies for exploring these landscapes, including sequence-derived evolutionary descriptors, structural fitness assessment, energetic evaluation, and integrated multi-parameter scoring. Recent developments in protein language models, deep-learning-based structure prediction, generative protein design, and consensus scoring approaches support large-scale exploration of biologically accessible sequence space. Negative-design constraints, including aggregation propensity, intrinsic disorder, and developability are important in prioritizing experimentally tractable protein candidates. Finally, the integration of computational prediction with iterative experimental validation is discussed as a central framework for rational protein engineering. By framing structure prediction, sequence representation learning, and generative design as complementary strategies for navigating a single constrained fitness landscape, this review highlights integrated, multidimensional scoring and negative-design filtering as the critical link between computational candidate generation and experimentally tractable protein design.
Lineage plasticity develops as tumor cells overcome terminal differentiation barriers, adapt to environmental stressors, and acquire metastatic, therapy-resistant phenotypes. These phenotypic transitions are driven by nonmutational epigenetic mechanisms, including dynamic shifts in chromatin accessibility, histone modifications, DNA methylation, and noncoding RNA activity beyond genetic alterations. While epithelial-mesenchymal plasticity (EMP) remains a foundational model, cancer-cell plasticity extends into a broader pan-plasticity spectrum encompassing dedifferentiation, transdifferentiation into neuroendocrine or squamous lineages, and stem cell-like reprogramming. In this review, we evaluate how the antagonistic interplay between Polycomb and Trithorax complexes, chromatin modifier activity, and enhancer reprogramming affects lineage identity and enables multi-lineage plasticity in cancer. We further discuss extrinsic mechanisms reinforced by the tumor microenvironment, particularly hypoxia, chronic inflammation, and metabolic stress, that rewire the epigenetic landscape to stabilize plastic states. Collectively, these data underscore epigenetic deregulation as a primary driver of lineage plasticity and intratumoral heterogeneity, while revealing therapeutic vulnerabilities and the potential to reverse plasticity and overcome therapy resistance by targeting the epigenome.
Microbiome dysbiosis correlates with aging-associated pathological skin conditions, and our understanding of how the microbiome regulates skin aging at a molecular level is rapidly advancing. Classical hallmarks of skin aging, including genomic instability and telomere attrition, loss of proteostasis, epigenetic alterations, and altered intercellular communication, are critically regulated by the microbiome. Oxidative stress represents a key factor implicated in virtually all hallmarks of skin aging. Meanwhile, recent data underscore the role of metabolism in intercellular communication and aging. Here, we examine current evidence linking the skin microbiome to such molecular events in the aging skin.
The Golgi complex is not only a central hub for bidirectional protein trafficking, but also a key site for post-translational modifications; the enzymes responsible are trafficked to the Golgi. To determine whether Golgi-resident enzymes can be recaptured from distal sites, we employed a nanobody-based transport assay. While most enzymes studied localized predominantly to the Golgi complex, some were also observed at the plasma membrane. Using sulfation-competent and fluorescently marked nanobody, we observed that certain enzymes are capable of retrograde transport to the trans-Golgi network. In contrast, classical and abundant recycling receptors readily internalized the nanobody but did not undergo retrograde transport. Our findings provide evidence that select Golgi enzymes can be retrieved from the cell surface to the sulfation compartment.
The L-type calcium channel CaV1.2 serves as a core trigger for excitation-contraction and excitation-transcription coupling in cardiovascular cells. Its abnormalities are closely linked to multiple cardiovascular diseases, including hypertension, arrhythmias, and cardiac hypertrophy. Alternative splicing (AS) generates CaV1.2 channel isoforms with distinct electrophysiological properties and drug sensitivity, finely tuning channel functions. This review systematically summarizes AS regulation and pathological significance of CaV1.2 in the cardiovascular system. Three key splicing events, mutually exclusive exons 8/8a, cassette exon 9* and exon 33, impact channel gating, calcium influx, and drug responsiveness. These AS events are dynamically regulated by splicing factors such as Rbfox1/2, PTBP1, and RBM20. We further analyze aberrant CaV1.2 splicing in hypertension, heart failure, myocardial infarction, and diabetic cardiomyopathy. Finally, we discuss preclinical and clinical advances of antisense oligonucleotides for treating cardiovascular CaV1.2 channelopathies. This review aims to clarify the regulatory network of CaV1.2 AS, providing theoretical evidence for precision therapies based on splicing modulation.
In most Actinobacteria, the respiratory complexes CIII and CIV form an obligate supercomplex, but the exact subunit composition varies. Here, we have characterized AscF (MSMEG_4692), a subunit of the Mycobacterium smegmatis CIII-CIV supercomplex. We showed that AscF and the small, membrane-anchored AscG constitute a heteromeric TPM domain featuring a noncanonical topology. Biophysical analysis demonstrated that the isolated AscF/AscG module lacked intrinsic affinity for metals or respiratory nucleotides in vitro. Functionally, an ascF frameshift mutant exhibited abolished malate-dependent oxygen consumption and severe growth defects on nonfermentable energy sources. We conclude that AscF likely is not a sensor for metal ions or nucleotides but acts as an adapter subunit facilitating electron transfer from the tricarboxylic acid cycle to the mycobacterial respiratory supercomplex.
This Perspective explores recent methods and prospective ideas for developing hybrid AI-physics-based pipelines for protein and antibody de novo design. We argue that the highest-confidence candidates emerge where deep learning and first-principles models agree, a "sweet spot" that balances generative flexibility with thermodynamic realism. For example, although interface confidence scores such as ipTM, pDockQ2, or ipSAE are widely used to rank generated designs, we show that they are not well suited to rank similar sequences, which suggests the need to combine them with physics-based methods to improve design filtering and ranking. Furthermore, we describe a generalizable framework for implementing antibody design pipelines that combine AI with physics-based modeling and scoring methods and also showcase MadraX, a differentiable and AI-compatible implementation of the FoldX force field. In addition, we classify three tiers of AI-physics integration, from post hoc filtering to full embedding of differentiable physics inside deep learning models. Finally, we discuss the future of the protein design community and underline the need to support current initiatives for community wide blind assessments of the growing number of de novo design pipelines.
Lysosomes are dynamic organelles regulating metabolic signaling by recruiting cytosolic molecules to protein platforms on their limiting membrane. We used proximity labeling to define interactors and vicinal proteins of LAMTOR3, a component of the Ragulator scaffold that controls mTORC1 signaling and lysosome positioning. The screen has yielded several previously unappreciated interactors, including an actin remodeling network. Here, we characterize the RhoGEF PLEKHG3 as a LAMTOR3 vicinal protein colocalizing with peripheral lysosomes and cortical F-actin at focal adhesion sites. Forced peripheral dispersion of lysosomes drives PLEKHG3 accumulation at focal adhesions and decreases protrusive activity in both wild-type and PLEKHG3-deficient cells. Thus, lysosome positioning governs both PLEKHG3 localization and protrusive activity, yet the protrusion changes can occur independently of PLEKHG3.
Transmembrane-spanning cell surface proteins undergo maturation from the site of synthesis in the endoplasmic reticulum to the plasma membrane, and may be endocytosed back into the cell. Microscopy allows the visual separation of such populations at the cell surface and in intracellular compartments and to monitor trafficking and dissect the relative abundance of such pools. Protein tags, like SNAP-tag, HTP, and FAST, combined with their respective (im)permeable ligands fused to fluorescent dyes, open up new possibilities to distinguish between intra- and extracellular protein pools. Introducing cleavable groups in dye-ligand molecules allows interrogation of endocytosed pools by stripping the remaining surface pools. Latest developments, for instance, fluorescence lifetime imaging microscopy and brightness demixing, give further opportunities in microscopy.
Age-related decline in neurovascular integrity is an increasingly recognized contributor to cognitive impairment and neurodegenerative vulnerability. A central feature is blood-brain barrier (BBB) dysfunction arising from endothelial senescence, altered barrier regulation, and chronic low-grade inflammation. In parallel, aging remodels the gut microbiota, with reduced diversity, loss of short-chain fatty acid-producing commensals, and expansion of pro-inflammatory taxa. Converging evidence indicates that age-related shifts in the gut microbiota alter microbiome function and can modulate BBB physiology through microbial metabolites, immune-endothelial signaling, and systemic metabolic pathways. These data position the gut-brain axis as an important, but not sole, modulator of neurovascular aging. Preclinical and emerging human data suggest that dysbiosis lowers the threshold for BBB dysfunction, and microbiome-targeted interventions in experimental models can improve barrier-relevant features. Notably, no human trial has yet demonstrated that microbiome modulation prevents or reverses BBB impairment using validated neuroimaging or fluid biomarkers. This review synthesizes mechanisms of microbiota-BBB crosstalk in aging, distinguishes correlation from causation, and outlines translational opportunities and limitations of dietary, probiotic, and fecal microbiota-based strategies for preserving neurovascular health in older adults.
Claudins regulate molecular transport between cells via the paracellular route at tight junctions in epithelial and endothelial tissues and are prime targets for biologic therapies. Claudin-claudin interactions through their extracellular segments facilitate form and function of tight junction barriers, making for defined targetable surfaces. However, developing biologic or small molecule binders against claudin extracellular segments is challenging because they are small, dynamic, and no structures of assemblies exist to guide efforts to rationally design binders. Here, I review claudin targeting strategies, recent advances using natural and synthetic molecules, and the impact these molecules have had on claudin or tight junction biology. I surmise that the therapeutic promise of any molecule is dependent on deeply considering how claudins assemble and what structures they take within and outside of tight junctions. This review thus focuses on structure-forward themes, with the hope of providing a framework for researchers to apply this knowledge in the design and development of new claudin-binding molecules with diverse properties, mechanisms, and functions.
G protein-coupled receptor 35 (GPR35) has been implicated in cancer, but the functional roles of its isoforms remain unresolved. Here, we characterize GPR35-long, an N-terminally extended epithelial isoform selectively enriched in colorectal cancer and cholangiocarcinoma. Mass spectrometry and immunohistochemistry confirmed GPR35-long protein expression in tumor cells. Functional analyses revealed that GPR35-long supports tumor-associated transcriptional programs, enhances cellular ATP production, and exhibits increased constitutive and ligand-induced beta-arrestin signaling. These phenotypes were selectively suppressed by the inverse agonist CID-2745687, identifying GPR35-long as a functionally distinct isoform with selective pharmacological sensitivity in tumor cells.
