Improving coverage, robustness and sensitivity is crucial for routine phosphoproteomics analysis by single-shot liquid chromatography tandem mass spectrometry (LC-MS/MS) from minimal peptide inputs. Here, we systematically optimized key experimental parameters for automated on-beads phosphoproteomics sample preparation with focus on low input samples. Assessing the number of identified phosphopeptides, enrichment efficiency, site localization scores and relative enrichment of multiply-phosphorylated peptides pinpointed critical variables influencing the resulting phosphoproteome. Optimizing glycolic acid concentration in the loading buffer, percentage of ammonium hydroxide in the elution buffer, peptide-to-beads ratio, binding time, sample and loading buffer volumes, allowed us to confidently identify >16,000 phosphopeptides in half-an-hour LC-MS/MS on an Orbitrap Exploris 480 using 30 μg of peptides as starting material. Furthermore, we evaluated how sequential enrichment can boost phosphoproteome coverage and showed that pooling fractions into a single LC-MS/MS analysis increased the depth. We also present an alternative phosphopeptide enrichment strategy based on stepwise addition of beads thereby boosting phosphoproteome coverage by 20%. Finally, we applied our optimized strategy to evaluate phosphoproteome depth with the Orbitrap Astral MS using a cell dilution series and were able to identify >32,000 phosphopeptides from 0.5 million HeLa cells in half-an-hour LC-MS/MS using narrow-window data-independent acquisition (nDIA). 

Systematic optimization of automated phosphopeptide enrichment for high-sensitivity phosphoproteomics

Improving coverage, robustness and sensitivity is crucial for routine phosphoproteomics analysis by single-shot liquid chromatography tandem mass spectrometry (LC-MS/MS) runs from minimal peptide inputs. Here, we systematically optimized key experimental parameters for automated on-beads phosphoproteomics sample preparation with focus on low input samples. Assessing the number of identified phosphopeptides, enrichment efficiency, site localization scores and relative enrichment of multiply-phosphorylated peptides pinpointed critical variables influencing the resulting phosphoproteome. Optimizing glycolic acid concentration in the loading buffer, percentage of ammonium hydroxide in the elution buffer, peptide-to-beads ratio, binding time, sample and loading buffer volumes, allowed us to confidently identify >16,000 phosphopeptides in half-an-hour LC-MS/MS on an Orbitrap Exploris 480 using 30 µg of peptides as starting material. Furthermore, we evaluated how sequential enrichment can boost phosphoproteome coverage and showed that pooling fractions into a single LC-MS/MS analysis increased the depth. We also present an alternative phosphopeptide enrichment strategy based on stepwise addition of beads thereby boosting phosphoproteome coverage by 20%. Finally, we applied our optimized strategy to evaluate phosphoproteome depth with the Orbitrap Astral MS using a cell dilution series and were able to identify >32,000 phosphopeptides from 0.5 million HeLa cells in half-an-hour LC-MS/MS using narrow-window data-independent acquisition (nDIA). Graphical abstract

The extracellular matrix (ECM) orchestrates cell behavior and tissue regeneration by modulating biochemical and mechanical signals. Manipulating cell-material interactions is crucial for leveraging biomaterials to regulate cell functions. Yet, integrating multiple cues in a single material remains a challenge. Here, near-infrared (NIR)-controlled multifunctional hydrogel platforms, named PIC/CM@NPs, are introduced to dictate fibroblast behavior during wound healing by tuning the matrix oxidative stress and mechanical tensions. PIC/CM@NPs are prepared through cell adhesion-medicated assembly of collagen-like polyisocyanide (PIC) polymers and cell-membrane-coated conjugated polymer nanoparticles (CM@NPs), which closely mimic the fibrous structure and nonlinear mechanics of ECM. Upon NIR stimulation, PIC/CM@NPs composites enhance fibroblast cell proliferation, migration, cytokine production, and myofibroblast activation, crucial for wound closure. Moreover, they exhibit effective and toxin removal antibacterial properties, reducing inflammation. This multifunctional approach accelerates healing by 95%, highlighting the importance of integrating biochemical and biophysical cues in the biomaterial design for advanced tissue regeneration. 

Microenvironment with NIR-Controlled ROS and Mechanical Tensions for Manipulating Cell Activities in Wound Healing.

Abstract Implant‐associated infections (IAIs) are the main cause of prosthetic implant failure. Bacterial biofilms prevent antibiotic penetration, and the unique metabolic conditions in hypoxic biofilm microenvironment may limit the efficacy of conventional antibiotic treatment. Escaping survival bacteria may not be continually eradicated, resulting in the recurrence of IAIs. Herein, a sonosensitive metal‐organic framework of Cu‐TCPP (tetrakis(4‐carboxyphenyl) porphyrin) nanosheets and tinidazole doped probiotic‐derived membrane vesicles (OMVs) with high‐penetration sonodynamic therapy (SDT), bacterial metabolic state interference, and bacterial cuproptosis‐like death to eradicate IAIs is proposed. The Cu‐TCPP can convert O 2 to toxic 1 O 2 through SDT in the normoxic conditions, enhancing the hypoxic microenvironment and activating the antibacterial activity of tinidazole. The released Cu(II) under ultrasound can be converted to Cu(I) by exogenous poly(tannic acid) (pTA) and endogenous glutathione. The disruption of the bacterial membrane by SDT can enhance the Cu(I) transporter activity. Transcriptomics indicate that the SDT‐enhanced Cu(I) overload and hypoxia‐activated therapy hinder the tricarboxylic acid cycle (TCA), leading to bacterial cuproptosis‐like death. Moreover, the OMVs‐activated therapy can polarize macrophages to a M2‐like phenotype and facilitate bone repair. The sonodynamic biofilm microenvironment modulation strategy, whereby the hypoxia‐enhanced microenvironment is potentiated to synergize SDT with OMVs‐activated therapy, provides an effective strategy for antibacterial and osteogenesis performance. 

Ultrasound‐Activated Probiotics Vesicles Coating for Titanium Implant Infections Through Bacterial Cuproptosis‐Like Death and Immunoregulation

Abstract Primordial follicles (PFs) function as the long‐term reserve for female reproduction, remaining dormant in the ovaries and becoming progressively depleted with age. Oxidative stress plays an important role in promoting female reproductive senescence during aging, but the underlying mechanisms remain unclear. Here, we find that low levels of reactive oxygen species (ROS) are essential for sustaining PF dormancy. Compared to growing follicles, oocytes within PFs were shown to be more susceptible to ROS, which accumulates and damages PFs to promote reproductive senescence. Mechanistically, oocytes within PFs were shown to express high levels of the intracellular antioxidant enzyme superoxide dismutase 1 (SOD1), counteracting ROS accumulation. Decreased SOD1 expression, as a result of aging or through the experimental deletion of the Sod1 gene in oocytes, resulted in increased oxidative stress and triggered ferroptosis within PFs. In conclusion, this study identified antioxidant defense mechanisms protecting PFs in mouse ovaries and characterized cell death mechanisms of oxidative stress‐induced PF death. 

Oocytes maintain low <scp>ROS</scp> levels to support the dormancy of primordial follicles

The ribotoxic stress response (RSR) is a signaling pathway in which the p38- and c-Jun N-terminal kinase (JNK)–activating mitogen-activated protein kinase kinase kinase (MAP3K) ZAKα senses stalling and/or collision of ribosomes. Here, we show that reactive oxygen species (ROS)–generating agents trigger ribosomal impairment and ZAKα activation. Conversely, zebrafish larvae deficient for ZAKα are protected from ROS-induced pathology. Livers of mice fed a ROS-generating diet exhibit ZAKα-activating changes in ribosomal elongation dynamics. Highlighting a role for the RSR in metabolic regulation, ZAK-knockout mice are protected from developing high-fat high-sugar (HFHS) diet-induced blood glucose intolerance and liver steatosis. Finally, ZAK ablation slows animals from developing the hallmarks of metabolic aging. Our work highlights ROS-induced ribosomal impairment as a physiological activation signal for ZAKα that underlies metabolic adaptation in obesity and aging. Editor’s summary Under conditions of food scarcity, metabolic flexibility was historically important for humans to optimize storage and access to energy when food was not readily available. By contrast, the constant access to high-calorie foods in modern societies has become a liability, contributing to the rise in obesity and metabolic diseases. Reactive oxygen species are thought to contribute to the metabolic dysregulation associated with obesity and aging, but the underlying process was not well understood. Snieckute et al. have now identified a mechanism linking reactive oxygen species, a protein called ZAKα, and resulting changes in ribosome function, which then contribute to the observed metabolic changes across multiple model animal species. —Yevgeniya Nusinovich Reactive oxygen species cause activation of ZAKα and changes in ribosomal dynamics, resulting in metabolic dysregulation. INTRODUCTION When ribosomes stall and/or collide during mRNA translation, ribosomal surveillance mechanisms are activated. These pathways signal the presence of ribotoxic stress and coordinate the resolution of nonproductive ribosomes. The ribosome-binding and stress-sensing MAP3 kinase ZAKα is at the nexus of the ribotoxic stress response (RSR) that culminates in the activation of and signaling through the stress-associated MAP kinases p38 and JNK. The RSR pathway is nonessential, and little is known about the in vivo conditions in which translational problems occur and the contexts in which the RSR is physiologically important. RATIONALE Reactive oxygen species (ROS) activate p38 and JNK and impair ribosomal translation. We rationalized that these responses may be coupled, and that ROS, which is associated with a range of physiological and pathological biological transitions, is a source of RSR activation in vivo. ZAK knockout (KO) mice display a mild metabolic phenotype with leanness and decreased adiposity, suggesting that the RSR is involved in metabolic regulation. Further, p38 and JNK are involved in metabolic transitions such as the emergence of insulin resistance and liver steatosis in obesity. In the context of obesity and metabolic regulation, activation of these kinases is driven by elusive metabolic stress signals that could be mediated by any of the 21 known p38- and JNK-directed MAP3 kinases. We thus sought to investigate the potential links among ROS, RSR signaling, and metabolic regulation using cellular, zebrafish, and mouse models. RESULTS We confirmed that ROS activates the RSR in cellular models, associated with both stalling and collision of ribosomes. In vitro translation reactions highlighted that these effects are primarily mediated by effects on soluble translation factors, including transfer RNAs. When exposed to a pathological burst of ROS, developing zebrafish larvae deficient for the zakα gene were strongly protected against early lethality, likely due to prevention of RSR-induced programmed cell death. ZAK KO mice fed a high-fat high-sugar (HFHS) diet, which is associated with rapid weight gain and ROS-driven pathology in rodents, were protected against early manifestations of metabolic maladaptation, including blood glucose intolerance and liver steatosis. These phenotypes were accompanied by deregulated RSR signaling in ZAK KO mouse livers. In HFHS-fed mice, highly translated liver mRNAs displayed increased stalling and queuing of ribosomes, indicating the presence of known ZAKα-activating structures. Finally, aging in male mice is also associated with increased ROS production and metabolic deterioration manifested as impaired blood glucose intolerance, stochastic liver steatosis, and whitening of brown adipose tissue. ZAK KO mice were protected against all of these hallmarks of metabolic aging. CONCLUSION Our study uncovers ROS as a physiologically relevant source of translational aberrations and RSR activation. Furthermore, impaired ribosomes constitute at least a portion of the metabolic stress signals that drive unwanted metabolic maladaptation in obesity and aging. The strong protection against these reactions offered by Zak gene deletion in mice warrants investigation of the ZAKα kinase as a potential drug target for metabolic conditions such as nonalcoholic steatohepatitis, hypertension, and dyslipidemia. Additional work will be needed to unravel the tissue-specific contributions of RSR signaling to the above phenotypes and to identify other physiologically relevant sources of ribotoxic stress. The ribotoxic stress response drives metabolic maladaptation in obesity and aging. ROS are a physiologically relevant source of translational impairment and activation of the stress kinases ZAKα, p38, and JNK. In obese and aging mice, this RSR drives metabolic maladaptation manifested by blood glucose intolerance, liver steatosis, and whitening of brown adipose tissue (BAT). [Figure created with BioRender.]

ROS-induced ribosome impairment underlies ZAKα-mediated metabolic decline in obesity and aging

In this commentary, Sonne‐Hansen and colleagues argue that research leaders and organizations should encourage more “theory‐guessing” by budding young scientists, rather than incentivizing safe mainstream research.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.15252/embj.2022112835

Creativity as an antidote to research becoming too predictable

<h2>Summary</h2> RNase L is an endoribonuclease of higher vertebrates that functions in antiviral innate immunity. Interferons induce oligoadenylate synthetase enzymes that sense double-stranded RNA of viral origin leading to the synthesis of 2′,5′-oligoadenylate (2-5A) activators of RNase L. However, it is unknown precisely how RNase L remodels the host cell transcriptome. To isolate effects of RNase L from other effects of double-stranded RNA or virus, 2-5A is directly introduced into cells. Here, we report that RNase L activation by 2-5A causes a ribotoxic stress response involving the MAP kinase kinase kinase (MAP3K) ZAKα, MAP2Ks, and the stress-activated protein kinases JNK and p38α. RNase L activation profoundly alters the transcriptome by widespread depletion of mRNAs associated with different cellular functions but also by JNK/p38α-stimulated induction of inflammatory genes. These results show that the 2-5A/RNase L system triggers a protein kinase cascade leading to proinflammatory signaling and apoptosis. 

Initiation of a ZAKα-dependent ribotoxic stress response by the innate immunity endoribonuclease RNase L.

A tripartite cell-free translation system to study mammalian translation.

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ROS-induced ribosome impairment underlies ZAKα-mediated metabolic decline in obesity and aging

Creativity as an antidote to research becoming too predictable

Initiation of a ZAKα-dependent ribotoxic stress response by the innate immunity endoribonuclease RNase L.

A tripartite cell-free translation system to study mammalian translation.