Biomarkers of aging for the identification and evaluation of longevity interventions.

With the rapid expansion of aging biology research, the identification and evaluation of longevity interventions in humans have become key goals of this field. Biomarkers of aging are critically important tools in achieving these objectives over realistic time frames. However, the current lack of standards and consensus on the properties of a reliable aging biomarker hinders their further development and validation for clinical applications. Here, we advance a framework for the terminology and characterization of biomarkers of aging, including classification and potential clinical use cases. We discuss validation steps and highlight ongoing challenges as potential areas in need of future research. This framework sets the stage for the development of valid biomarkers of aging and their ultimate utilization in clinical trials and practice.

Ketogenic Diets Alter the Gut Microbiome Resulting in Decreased Intestinal Th17 Cells.

Very low-carbohydrate, high-fat ketogenic diets (KDs) induce a pronounced shift in metabolic fuel utilization that elevates circulating ketone bodies; however, the consequences of these compounds for host-microbiome interactions remain unknown. Here, we show that KDs alter the human and mouse gut microbiota in a manner distinct from high-fat diets (HFDs). Metagenomic and metabolomic analyses of stool samples from an 8-week inpatient study revealed marked shifts in gut microbial community structure and function during the KD. Gradient diet experiments in mice confirmed the unique impact of KDs relative to HFDs with a reproducible depletion of bifidobacteria. In vitro and in vivo experiments showed that ketone bodies selectively inhibited bifidobacterial growth. Finally, mono-colonizations and human microbiome transplantations into germ-free mice revealed that the KD-associated gut microbiota reduces the levels of intestinal pro-inflammatory Th17 cells. Together, these results highlight the importance of trans-kingdom chemical dialogs for mediating the host response to dietary interventions.

NAD+ metabolism and its roles in cellular processes during ageing.

Nicotinamide adenine dinucleotide (NAD+) is a coenzyme for redox reactions, making it central to energy metabolism. NAD+ is also an essential cofactor for non-redox NAD+-dependent enzymes, including sirtuins, CD38 and poly(ADP-ribose) polymerases. NAD+ can directly and indirectly influence many key cellular functions, including metabolic pathways, DNA repair, chromatin remodelling, cellular senescence and immune cell function. These cellular processes and functions are critical for maintaining tissue and metabolic homeostasis and for healthy ageing. Remarkably, ageing is accompanied by a gradual decline in tissue and cellular NAD+ levels in multiple model organisms, including rodents and humans. This decline in NAD+ levels is linked causally to numerous ageing-associated diseases, including cognitive decline, cancer, metabolic disease, sarcopenia and frailty. Many of these ageing-associated diseases can be slowed down and even reversed by restoring NAD+ levels. Therefore, targeting NAD+ metabolism has emerged as a potential therapeutic approach to ameliorate ageing-related disease, and extend the human healthspan and lifespan. However, much remains to be learnt about how NAD+ influences human health and ageing biology. This includes a deeper understanding of the molecular mechanisms that regulate NAD+ levels, how to effectively restore NAD+ levels during ageing, whether doing so is safe and whether NAD+ repletion will have beneficial effects in ageing humans.

Anti-apoptotic Protein BIRC5 Maintains Survival of HIV-1-Infected CD4+ T Cells.

HIV-1 infection of CD4+ T cells leads to cytopathic effects and cell demise, which is counter to the observation that certain HIV-1-infected cells possess a remarkable long-term stability and can persist lifelong in infected individuals treated with suppressive antiretroviral therapy (ART). Using quantitative mass spectrometry-based proteomics, we showed that HIV-1 infection activated cellular survival programs that were governed by BIRC5, a molecular inhibitor of cell apoptosis that is frequently overexpressed in malignant cells. BIRC5 and its upstream regulator OX40 were upregulated in productively and latently infected CD4+ T cells and were functionally involved in maintaining their viability. Moreover, OX40-expressing CD4+ T cells from ART-treated patients were enriched for clonally expanded HIV-1 sequences, and pharmacological inhibition of BIRC5 resulted in a selective decrease of HIV-1-infected cells in vitro. Together, these findings suggest that BIRC5 supports long-term survival of HIV-1-infected cells and may lead to clinical strategies to reduce persisting viral reservoirs.

50 years of protein acetylation: from gene regulation to epigenetics, metabolism and beyond.

In 1964, Vincent Allfrey and colleagues reported the identification of histone acetylation and with deep insight proposed a regulatory role for this protein modification in transcription regulation. Subsequently, histone acetyltransferases (HATs), histone deacetylases (HDACs) and acetyl-Lys-binding proteins were identified as transcription regulators, thereby providing compelling evidence for his daring hypothesis. During the past 15 years, reversible protein acetylation and its modifying enzymes have been implicated in many cellular functions beyond transcription regulation. Here, we review the progress accomplished during the past 50 years and discuss the future of protein acetylation.

Regulation of UCP1 and Mitochondrial Metabolism in Brown Adipose Tissue by Reversible Succinylation.

Brown adipose tissue (BAT) is rich in mitochondria and plays important roles in energy expenditure, thermogenesis, and glucose homeostasis. We find that levels of mitochondrial protein succinylation and malonylation are high in BAT and subject to physiological and genetic regulation. BAT-specific deletion of Sirt5, a mitochondrial desuccinylase and demalonylase, results in dramatic increases in global protein succinylation and malonylation. Mass spectrometry-based quantification of succinylation reveals that Sirt5 regulates the key thermogenic protein in BAT, UCP1. Mutation of the two succinylated lysines in UCP1 to acyl-mimetic glutamine and glutamic acid significantly decreases its stability and activity. The reduced function of UCP1 and other proteins in Sirt5KO BAT results in impaired mitochondria respiration, defective mitophagy, and metabolic inflexibility. Thus, succinylation of UCP1 and other mitochondrial proteins plays an important role in BAT and in regulation of energy homeostasis.

The growing landscape of lysine acetylation links metabolism and cell signalling.

Lysine acetylation is a conserved protein post-translational modification that links acetyl-coenzyme A metabolism and cellular signalling. Recent advances in the identification and quantification of lysine acetylation by mass spectrometry have increased our understanding of lysine acetylation, implicating it in many biological processes through the regulation of protein interactions, activity and localization. In addition, proteins are frequently modified by other types of acylations, such as formylation, butyrylation, propionylation, succinylation, malonylation, myristoylation, glutarylation and crotonylation. The intricate link between lysine acylation and cellular metabolism has been clarified by the occurrence of several such metabolite-sensitive acylations and their selective removal by sirtuin deacylases. These emerging findings point to new functions for different lysine acylations and deacylating enzymes and also highlight the mechanisms by which acetylation regulates various cellular processes.

The genome of a bunyavirus cannot be defined at the level of the viral particle but only at the scale of the viral population.

Bunyaviruses are enveloped negative or ambisense single-stranded RNA viruses with a genome divided into several segments. The canonical view depicts each viral particle packaging one copy of each genomic segment in one polarity named the viral strand. Several opposing observations revealed nonequal ratios of the segments, uneven number of segments per virion, and even packaging of viral complementary strands. Unfortunately, these observations result from studies often addressing other questions, on distinct viral species, and not using accurate quantitative methods. Hence, what RNA segments and strands are packaged as the genome of any bunyavirus remains largely ambiguous. We addressed this issue by first investigating the virion size distribution and RNA content in populations of the tomato spotted wilt virus (TSWV) using microscopy and tomography. These revealed heterogeneity in viral particle volume and amount of RNA content, with a surprising lack of correlation between the two. Then, the ratios of all genomic segments and strands were established using RNA sequencing and qRT-PCR. Within virions, both plus and minus strands (but no mRNA) are packaged for each of the three L, M, and S segments, in reproducible nonequimolar proportions determined by those in total cell extracts. These results show that virions differ in their genomic content but together build up a highly reproducible genetic composition of the viral population. This resembles the genome formula described for multipartite viruses, with which some species of the order Bunyavirales may share some aspects of the way of life, particularly emerging properties at a supravirion scale.

MicroRNAs of the miR-17∼92 family are critical regulators of T(FH) differentiation.

Follicular helper T cells (T(FH) cells) provide critical help to B cells during humoral immune responses. Here we report that mice with T cell-specific deletion of the miR-17∼92 family of microRNAs (miRNAs) had substantially compromised T(FH) differentiation, germinal-center formation and antibody responses and failed to control chronic viral infection. Conversely, mice with T cell-specific expression of a transgene encoding miR-17∼92 spontaneously accumulated T(FH) cells and developed a fatal immunopathology. Mechanistically, the miR-17∼92 family controlled the migration of CD4(+) T cells into B cell follicles by regulating signaling intensity from the inducible costimulator ICOS and kinase PI(3)K by suppressing expression of the phosphatase PHLPP2. Our findings demonstrate an essential role for the miR-17∼92 family in T(FH) differentiation and establish PHLPP2 as an important mediator of their function in this process.

From discoveries in ageing research to therapeutics for healthy ageing.

For several decades, understanding ageing and the processes that limit lifespan have challenged biologists. Thirty years ago, the biology of ageing gained unprecedented scientific credibility through the identification of gene variants that extend the lifespan of multicellular model organisms. Here we summarize the milestones that mark this scientific triumph, discuss different ageing pathways and processes, and suggest that ageing research is entering a new era that has unique medical, commercial and societal implications. We argue that this era marks an inflection point, not only in ageing research but also for all biological research that affects the human healthspan.

A proposed unified mitotic chromosome architecture.

A molecular architecture is proposed for a representative mitotic chromosome, human chromosome 10. This architecture is built on an interphase chromosome structure based on cryo-electron microscopy (cryo-EM) cellular tomography [J. Sedat et al., Proc. Natl. Acad. Sci. U.S.A., in press], thus unifying chromosome structure throughout the complete mitotic cycle. The basic organizational principle for mitotic chromosomes is specific coiling of the 11-nm nucleosome fiber into large scale, ∼200-nm interphase structures, a Slinky [https://en.wikipedia.org/wiki/Slinky; motif cited in S. Bowerman et al., eLife 10, e65587 (2021)], then further modified with subsequent additional coiling for the final mitotic chromosome structure. The final mitotic chromosome architecture accounts for the dimensional values as well as the well-known cytological configurations. In addition, proof is experimentally provided by digital PCR technology that G1 T cell nuclei are diploid with one DNA molecule per chromosome. Many nucleosome linker DNA sequences, the promotors and enhancers, are suggestive of optimal exposure on the surfaces of the large-scale coils.

Neuronal SIRT3 Deletion Predisposes to Female-Specific Alterations in Cellular Metabolism, Memory, and Network Excitability.

Mitochondrial dysfunction is an early event in the pathogenesis of neurologic disorders and aging. Sirtuin 3 (SIRT3) regulates mitochondrial function in response to the cellular environment through the reversible deacetylation of proteins involved in metabolism and reactive oxygen species detoxification. As the primary mitochondrial deacetylase, germline, or peripheral tissue-specific deletion of SIRT3 produces mitochondrial hyperacetylation and the accelerated development of age-related diseases. Given the unique metabolic demands of neurons, the role of SIRT3 in the brain is only beginning to emerge. Using mass spectrometry-based acetylomics, high-resolution respirometry, video-EEG, and cognition testing, we report targeted deletion of SIRT3 from select neurons in the cortex and hippocampus produces altered neuronal excitability and metabolic dysfunction in female mice. Targeted deletion of SIRT3 from neuronal helix-loop-helix 1 (NEX)-expressing neurons resulted in mitochondrial hyperacetylation, female-specific superoxide dismutase-2 (SOD2) modification, increased steady-state superoxide levels, metabolic reprogramming, altered neuronal excitability, and working spatial memory deficits. Inducible neuronal deletion of SIRT3 likewise produced female-specific deficits in spatial working memory. Together, the data demonstrate that deletion of SIRT3 from forebrain neurons selectively predisposes female mice to deficits in mitochondrial and cognitive function.SIGNIFICANCE STATEMENT Mitochondrial SIRT3 is an enzyme shown to regulate energy metabolism and antioxidant function, by direct deacetylation of proteins. In this study, we show that neuronal SIRT3 deficiency renders female mice selectively vulnerable to impairment in redox and metabolic function, spatial memory, and neuronal excitability. The observed sex-specific effects on cognition and neuronal excitability in female SIRT3-deficient mice suggest that mitochondrial dysfunction may be one factor underlying comorbid neuronal diseases, such as Alzheimer's disease and epilepsy. Furthermore, the data suggest that SIRT3 dysfunction may predispose females to age-related metabolic and cognitive impairment.

SARS-CoV-2 protein ORF8 limits expression levels of Spike antigen and facilitates immune evasion of infected host cells.

Recovery from COVID-19 depends on the ability of the host to effectively neutralize virions and infected cells, a process largely driven by antibody-mediated immunity. However, with the newly emerging variants that evade Spike-targeting antibodies, re-infections and breakthrough infections are increasingly common. A full characterization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mechanisms counteracting antibody-mediated immunity is therefore needed. Here, we report that ORF8 is a virally encoded SARS-CoV-2 factor that controls cellular Spike antigen levels. We show that ORF8 limits the availability of mature Spike by inhibiting host protein synthesis and retaining Spike at the endoplasmic reticulum, reducing cell-surface Spike levels and recognition by anti-SARS-CoV-2 antibodies. In conditions of limited Spike availability, we found ORF8 restricts Spike incorporation during viral assembly, reducing Spike levels in virions. Cell entry of these virions then leaves fewer Spike molecules at the cell surface, limiting antibody recognition of infected cells. Based on these findings, we propose that SARS-CoV-2 variants may adopt an ORF8-dependent strategy that facilitates immune evasion of infected cells for extended viral production.

T cell stimulation remodels the latently HIV-1 infected cell population by differential activation of proviral chromatin.

The reservoir of latently HIV-1 infected cells is heterogeneous. To achieve an HIV-1 cure, the reservoir of activatable proviruses must be eliminated while permanently silenced proviruses may be tolerated. We have developed a method to assess the proviral nuclear microenvironment in single cells. In latently HIV-1 infected cells, a zinc finger protein tethered to the HIV-1 promoter produced a fluorescent signal as a protein of interest came in its proximity, such as the viral transactivator Tat when recruited to the nascent RNA. Tat is essential for viral replication. In these cells we assessed the proviral activation and chromatin composition. By linking Tat recruitment to proviral activity, we dissected the mechanisms of HIV-1 latency reversal and the consequences of HIV-1 production. A pulse of promoter-associated Tat was identified that contrasted to the continuous production of viral proteins. As expected, promoter H3K4me3 led to substantial expression of the provirus following T cell stimulation. However, the activation-induced cell cycle arrest and death led to a surviving cell fraction with proviruses encapsulated in repressive chromatin. Further, this cellular model was used to reveal mechanisms of action of small molecules. In a proof-of-concept study we determined the effect of modifying enhancer chromatin on HIV-1 latency reversal. Only proviruses resembling active enhancers, associated with H3K4me1 and H3K27ac and subsequentially recognized by BRD4, efficiently recruited Tat upon cell stimulation. Tat-independent HIV-1 latency reversal of unknown significance still occurred. We present a method for single cell assessment of the microenvironment of the latent HIV-1 proviruses, used here to reveal how T cell stimulation modulates the proviral activity and how the subsequent fate of the infected cell depends on the chromatin context.

SIRT5 is a proviral factor that interacts with SARS-CoV-2 Nsp14 protein.

SARS-CoV-2 non-structural protein Nsp14 is a highly conserved enzyme necessary for viral replication. Nsp14 forms a stable complex with non-structural protein Nsp10 and exhibits exoribonuclease and N7-methyltransferase activities. Protein-interactome studies identified human sirtuin 5 (SIRT5) as a putative binding partner of Nsp14. SIRT5 is an NAD-dependent protein deacylase critical for cellular metabolism that removes succinyl and malonyl groups from lysine residues. Here we investigated the nature of this interaction and the role of SIRT5 during SARS-CoV-2 infection. We showed that SIRT5 interacts with Nsp14, but not with Nsp10, suggesting that SIRT5 and Nsp10 are parts of separate complexes. We found that SIRT5 catalytic domain is necessary for the interaction with Nsp14, but that Nsp14 does not appear to be directly deacylated by SIRT5. Furthermore, knock-out of SIRT5 or treatment with specific SIRT5 inhibitors reduced SARS-CoV-2 viral levels in cell-culture experiments. SIRT5 knock-out cells expressed higher basal levels of innate immunity markers and mounted a stronger antiviral response, independently of the Mitochondrial Antiviral Signaling Protein MAVS. Our results indicate that SIRT5 is a proviral factor necessary for efficient viral replication, which opens novel avenues for therapeutic interventions.

Biological Characterization of Physostegia Chlorotic Mottle Virus, an Emergent Virus Infecting Vegetables in Diversified Production Systems.

In 2014, Physostegia chlorotic mottle virus (PhCMoV) was discovered in Austria in Physostegia virginiana. Subsequent collaborative efforts established a link between the virus and severe fruit symptoms on important crops such as tomato, eggplant, and cucumber across nine European countries. Thereafter, specific knowledge gaps, which are crucial to assess the risks PhCMoV can pose for production and how to manage it, needed to be addressed. In this study, the transmission, prevalence, and disease severity of PhCMoV were examined. This investigation led to the identification of PhCMoV presence in a new country, Switzerland. Furthermore, our research indicates that the virus was already present in Europe 30 years ago. Bioassays demonstrated PhCMoV can result in up to 100% tomato yield losses depending on the phenological stage of the plant at the time of infection. PhCMoV was found to naturally infect 12 new host plant species across eight families, extending its host range to 21 plant species across 15 plant families. The study also identified a polyphagous leafhopper (genus Anaceratagallia) as a natural vector of PhCMoV. Overall, PhCMoV was widespread in small-scale diversified vegetable farms in Belgium where tomato is grown in soil under tunnels, occurring in approximately one-third of such farms. However, outbreaks were sporadic and were associated at least once with the cultivation in tomato tunnels of perennial plants that can serve as a reservoir host for the virus and its vector. To further explore this phenomenon and manage the virus, studying the ecology of the vector would be beneficial.

In-depth analysis of the Sirtuin 5-regulated mouse brain malonylome and succinylome using library-free data-independent acquisitions.

Post-translational modifications (PTMs) dynamically regulate proteins and biological pathways, typically through the combined effects of multiple PTMs. Lysine residues are targeted for various PTMs, including malonylation and succinylation. However, PTMs offer specific challenges to mass spectrometry-based proteomics during data acquisition and processing. Thus, novel and innovative workflows using data-independent acquisition (DIA) ensure confident PTM identification, precise site localization, and accurate and robust label-free quantification. In this study, we present a powerful approach that combines antibody-based enrichment with comprehensive DIA acquisitions and spectral library-free data processing using directDIA (Spectronaut). Identical DIA data can be used to generate spectral libraries and comprehensively identify and quantify PTMs, reducing the amount of enriched sample and acquisition time needed, while offering a fully automated workflow. We analyzed brains from wild-type and Sirtuin 5 (SIRT5)-knock-out mice, and discovered and quantified 466 malonylated and 2211 succinylated peptides. SIRT5 regulation remodeled the acylomes by targeting 164 malonylated and 578 succinylated sites. Affected pathways included carbohydrate and lipid metabolisms, synaptic vesicle cycle, and neurodegenerative diseases. We found 48 common SIRT5-regulated malonylation and succinylation sites, suggesting potential PTM crosstalk. This innovative and efficient workflow offers deeper insights into the mouse brain lysine malonylome and succinylome.

NAD+ Repletion Reverses Heart Failure With Preserved Ejection Fraction.

[Figure: see text].

SIRT5 Regulates both Cytosolic and Mitochondrial Protein Malonylation with Glycolysis as a Major Target.

Protein acylation links energetic substrate flux with cellular adaptive responses. SIRT5 is a NAD(+)-dependent lysine deacylase and removes both succinyl and malonyl groups. Using affinity enrichment and label free quantitative proteomics, we characterized the SIRT5-regulated lysine malonylome in wild-type (WT) and Sirt5(-/-) mice. 1,137 malonyllysine sites were identified across 430 proteins, with 183 sites (from 120 proteins) significantly increased in Sirt5(-/-) animals. Pathway analysis identified glycolysis as the top SIRT5-regulated pathway. Importantly, glycolytic flux was diminished in primary hepatocytes from Sirt5(-/-) compared to WT mice. Substitution of malonylated lysine residue 184 in glyceraldehyde 3-phosphate dehydrogenase with glutamic acid, a malonyllysine mimic, suppressed its enzymatic activity. Comparison with our previous reports on acylation reveals that malonylation targets a different set of proteins than acetylation and succinylation. These data demonstrate that SIRT5 is a global regulator of lysine malonylation and provide a mechanism for regulation of energetic flux through glycolysis.