Koshi Imami

Nearly a century ago, restricting diet during early-life periods was suggested to extend lifespan in rats and in Daphnia ^1,2 . The effect of juvenile diet on adult physiology and lifespan has subsequently been described in other model organisms, including fruit flies ^3–5 and mice ^6–8 ; however, its mechanism remains poorly understood. Here, using Drosophila as a model, we show that restricting protein intake during the larval stage (early-life protein restriction; ePR) promotes adult lifespan by reducing the levels of storage proteins. Using stable-isotope tracing, we show that dietary amino acids obtained in the larval stage are retained into early adulthood, and are incorporated into ribosomal proteins in particular. This is mediated by larval serum protein 2 (Lsp2), a major storage protein, the expression of which is durably downregulated by ePR in the early adult stage. Genetic silencing of Lsp2 phenocopies ePR, attenuating ribosomal-protein abundance and translational activity in early adulthood, and extending lifespan. Restricting specific amino acids that are especially enriched in these storage proteins, such as phenylalanine and tyrosine, is sufficient to decrease the levels of early-life Lsp2 and promote longevity. These findings identify Lsp2 as a molecular carrier of nutritional history across developmental transitions, linking juvenile nutritional status to adult translational capacity and lifespan. Our study uncovers a previously unrecognized mechanism of nutritional memory that links early-life diet to lifelong organismal health.

In bottom-up proteomics, protease specificity determines peptide length and LC/MS/MS detectability; in N-terminomics, this is critical because each protein N-terminal peptide is unique and cannot be substituted by other peptides. CHromatographic AMplification of Protein N-terminal peptides (CHAMP-N) combines LysargiNase digestion with StageTip-based strong cation exchange chromatography (SCX), removing internal peptides by exploiting their newly generated N-terminal Lys/Arg residues and thereby selectively isolating protein N-terminal peptides. However, LysargiNase cleavage near the protein N-terminus can generate excessively short peptides. Here, we developed two complementary CHAMP-N approaches that modurate N-terminal peptide length while retaining the charge-based SCX separation principle. LysN digestion avoided cleavage at N-terminal-proximal Arg residues, whereas D3-acetylation blocked Lys cleavage by LysargiNase to produce ArgN-like specificity. Both approaches extended otherwise short N-terminal peptides while preserving N-terminal basic residues on internal peptides. Conventional LysargiNase, LysN, and ArgN-like CHAMP-N generated complementary sets of N-terminal peptides. Integration of the three workflows identified 2,552 protein N-termini, approximately twice the number identified by conventional CHAMP-N alone. The median relative standard deviations of N-terminal peptide intensities were approximately 15% for all three workflows, indicating comparable technical reproducibility. These results demonstrate that coordinating protease cleavage specificity with SCX selectivity reduces sequence-dependent losses and expands N-terminome coverage while preserving the simplicity and reproducibility of CHAMP-N.

Although extracellular vesicles (EVs) facilitate selective molecular exchange between cells, their low yields and inherent heterogeneity limit translational applications. Cell‐derived nanovesicles (CDNs), produced by mechanical extrusion of donor cells, offer a scalable alternative while retaining key membrane features of EVs. Yet how fabrication reshapes vesicle–cell communication remains poorly understood. Here, vesicle‐side proteomics is integrated with TurboID proximity labeling of recipient‐cell proteins to construct quantitative, dual‐sided maps of CDN and small extracellular vesicle (sEV) interactions. Despite similar size and surface charge, CDNs displayed a substantially more diverse proteome and a broader repertoire of predicted uptake‐associated signatures. Recipient‐cell proximity proteomics further resolved distinct molecular cohorts. The CDN‐associated cohort contained recipient‐derived CALR and NCL, which are literature‐linked to LRP1/CD91‐associated efferocytic recognition and NCL‐associated macropinocytic processes, respectively, whereas the sEV‐associated cohort contained signatures consistent with HSPG‐assisted docking and clathrin‐mediated or CLIC/GEEC‐related uptake. Together, these complementary datasets reveal molecular interfaces for extrusion‐generated CDNs that are distinct from those of naturally secreted sEVs. Receptor dependence and the contributions of surface association and internalization require direct testing; however, the identified associations define specific mechanistic targets for further investigation. This dual‐sided proteomic strategy establishes a quantitative framework for dissecting vesicle–cell communication and engineering membrane‐based nanocarriers.

Mammalian genomes encode multiple transposable element (TE) silencing pathways that distinguish their targets through different molecular features, with KRAB zinc finger proteins recognizing DNA sequence, the HUSH complex sensing intronless transcripts, and the PIWI-piRNA pathway using small RNA guides. How chromatin state itself contributes to TE recognition remains less defined. Here we identify Spindlin1 (SPIN1), a three-Tudor-domain histone reader implicated in germline piRNA-directed DNA methylation, as a transcriptional repressor of evolutionarily young TEs in mouse embryonic stem cells. SPIN1 selectively binds LINE and ERV loci carrying H3K4me3 and H3K9me3, a chromatin signature enriched at young, transcription-permissive elements, and recognition of these marks by Tudor domains 1 and 2 is required for TE targeting. SPIN1 engages SPINDOC as a Tudor 1 and 3-dependent cofactor whose loss phenocopies SPIN1 depletion, and associates with the H3K9 methyltransferases SETDB1 and G9a. SPIN1 loss reduces H3K9me3 and is accompanied by increased chromatin accessibility, without altering DNA methylation. Thus, SPIN1 uses a histone-state-based mechanism to identify and repress young TEs in pluripotent cells, mechanistically distinct from its germline mode in which SPIN1 cooperates with the PIWI-piRNA pathway to promote DNA methylation, illustrating how a single histone reader engages distinct silencing machineries across cellular contexts.

Neural activity-dependent translation is essential for synaptic plasticity and diverse brain functions. Translation involves not only canonical main open reading frames (mORFs) but also upstream ORFs (uORFs), which may regulate mORF expression. However, due to technical limitations, systematic investigation of activity-dependent uORFs and mORFs in brain tissues remains challenging. Here, we developed a ribosome tagging and purification strategy that bypasses the prolonged turnover of ribosomal proteins, enabling ribosome profiling with one-hour temporal resolution after neural stimulation. Applying this strategy to mouse hippocampal slices undergoing long-term potentiation, we identify hundreds of activity-induced mORFs and uORFs, including a previously unknown uORF from Egr1 . We demonstrate that this Egr1 -uORF translation is tightly regulated by neuronal activity, and its encoded peptide interacts with peroxisomal machinery, suggesting a potential link between synaptic stimulus and peroxisome biology. This study provides a useful technique and resources for deciphering molecular mechanisms underlying activity- and translation-dependent brain functions in health and disease.

Understanding the relationship between viral proteins and host factors is essential for developing strategies to control virus infections. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells using angiotensin converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) as viral receptor and priming protease during the viral entry phase. Here, we report that TMPRSS2 reduces the infectivity of SARS-CoV-2 and HIV-1 during the viral production phase. Treatment of virus-producing cells with a TMPRSS2 inhibitor increases the production of infectious virions. TMPRSS2 enzymatic activity specifically disrupts the trans-Golgi by phosphorylating Golgi stacking proteins through ERK activation, which disturbs virion spike incorporation and structural maturation of the viral envelope glycoproteins, causing lower viral infectivity. We find that SARS-CoV-2 envelope protein (E protein) counteracts this TMPRSS2 activity to rescue SARS-CoV-2-S incorporation. These results demonstrate negative regulation of viral envelope glycoprotein incorporation by TMPRSS2 and reveal that SARS-CoV-2 regulates the Golgi system to create an optimal viral replication environment.

Synonymous codon usage controls global gene expression in both prokaryotic and eukaryotic species. Nonoptimal codons are known to induce messenger RNA (mRNA) decay; however, the underlying molecular mechanism remains poorly understood in human cells. Through genome-wide CRISPR screening, we identified the RNA binding protein DHX29 as a critical regulator of codon-dependent gene expression. Cryo–electron microscopy and selective ribosome profiling demonstrated that DHX29 directly interacts with the A-site entrance of the translating 80 S ribosome, the binding site for the eEF1A•GTP•aminoacyl-tRNA ternary complex, suggesting a role in monitoring aminoacyl-tRNA sampling. Proteomic analysis further revealed that DHX29 recruits the GIGYF2•4EHP complex to mediate global suppression of nonoptimal mRNAs. These findings establish a mechanistic link between synonymous codon usage and the regulation of gene expression.

Metabolic stress from a high-fat diet (HFD) impairs antitumor immunity through persistent metabolic rewiring, but its effects and long-term impact on CD8⁺ T cell metabolism remain unknown. Here, we found that even temporary exposure to a HFD impaired antitumor immunity 10 weeks after reversion to a normal diet. This was due to lasting metabolome changes that included enrichment in phospholipids sensitive to peroxidation and depletion of antioxidants, affecting the survival and function of CD8⁺ T cells. Under oxidative stress, CD8⁺ T cells utilized the xanthine salvage pathway to produce guanosine triphosphate, enhancing the amount of tetrahydrobiopterin. Xanthine supplementation reduced lipid peroxidation in tumor-draining lymph nodes and improved antitumor immunity in mice previously on a HFD. Our data indicate that metabolic stress in CD8⁺ T cells persists long after restoration of a balanced diet, and manifests as vulnerability to ferroptosis, which could be mitigated by replenishing biopterins through the xanthine salvage pathway.

SETD2 is a methyltransferase responsible for depositing histone H3 lysine 36 trimethylation (H3K36me3). Loss of its enzymatic activity occurs in some cancers, including renal cell carcinoma. SETD2 mutations have been linked to delayed transcription termination but have not been explored in depth. Here, using nascent transcriptomics in SETD2 knockout and patient-derived cells, we reveal a dichotomy in SETD2 functions depending on the affected protein-coding gene. The majority of genes, named class I, are dependent on SETD2 function for transcription initiation, yet terminate transcription in the usual locations. In contrast, for class II genes, corresponding to 15–25% of active protein-coding genes, transcription initiation is robust in absence of SETD2 activity; however, widespread transcriptional readthrough occurs. Defective termination following SETD2 loss/mutation is associated with increased cryptic transcription initiation and impaired 3′ pre-mRNA cleavage. Additionally, alternative polyadenylation upon SETD2 activity loss is highly cell type specific, and no relationship with transcription readthrough was observed. We demonstrate that methyltransferase activity of SETD2 stimulates proper initiation, prevents cryptic initiation and promotes efficient 3′ end processing, however, it does so indirectly.