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1. Molecular investigation of the tandem Tudor domain and plant homeodomain histone binding domains of the epigenetic regulator UHRF2

   https://doi.org/10.1002/prot.26278  

   Ginnard, Shane ; Winkler, Alyssa ; Mellado Fritz, Carlos ; Bluhm, Tatum ; Kemmer, Ray ; Gilliam, Marisa ; Butkevich, Nick ; Abdrabbo, Sara ; Bricker, Kaitlyn ; Feiler, Justin ; et al ( July 2022 , Proteins)

   Dokholyan, Nikolay (Ed.)

   Ubiquitin-like containing PHD and ring finger (UHRF)1 and UHRF2 are multidomain epigenetic proteins that play a critical role in bridging crosstalk between histone modifications and DNA methylation. Both proteins contain two histone reader domains, called tandem Tudor domain (TTD) and plant homeodomain (PHD), which read the modification status on histone H3 to regulate DNA methylation and gene expression. To shed light on the mechanism of histone binding by UHRF2, we have undergone a detailed molecular investigation with the TTD, PHD and TTD-PHD domains and compared the binding activity to its UHRF1 counterpart. We found that unlike UHRF1 where the PHD is the primary binding contributor, the TTD of UHRF2 has modestly higher affinity toward the H3 tail, while the PHD has a weaker binding interaction. We also demonstrated that like UHRF1, the aromatic amino acids within the TTD are important for binding to H3K9me3 and a conserved aspartic acid within the PHD forms an ionic interaction with R2 of H3. However, while the aromatic amino acids in the TTD of UHRF1 contribute to selectivity, the analogous residues in UHRF2 contribute to both selectivity and affinity. We also discovered that the PHD of UHRF2 contains a distinct asparagine in the H3R2 binding pocket that lowers the binding affinity of the PHD by reducing a potential electrostatic interaction with the H3 tail. Furthermore, we demonstrate the PHD and TTD of UHRF2 cooperate to interact with the H3 tail and that dual domain engagement with the H3 tail relies on specific amino acids. Lastly, our data indicate that the unique stretch region in the TTD of UHRF2 can decrease the melting temperature of the TTD-PHD and represents a disordered region. Thus, these subtle but important mechanistic differences are potential avenues for selectively targeting the histone binding interactions of UHRF1 and UHRF2 with small molecules. 

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2. Induction of lysosomal and mitochondrial biogenesis by AMPK phosphorylation of FNIP1

   https://doi.org/10.1126/science.abj5559  

   Malik, Nazma ; Ferreira, Bibiana I. ; Hollstein, Pablo E. ; Curtis, Stephanie D. ; Trefts, Elijah ; Weiser Novak, Sammy ; Yu, Jingting ; Gilson, Rebecca ; Hellberg, Kristina ; Fang, Lingjing ; et al ( April 2023 , Science)

   INTRODUCTION Eukaryotes contain a highly conserved signaling pathway that becomes rapidly activated when adenosine triphosphate (ATP) levels decrease, as happens during conditions of nutrient shortage or mitochondrial dysfunction. The adenosine monophosphate (AMP)–activated protein kinase (AMPK) is activated within minutes of energetic stress and phosphorylates a limited number of substrates to biochemically rewire metabolism from an anabolic state to a catabolic state to restore metabolic homeostasis. AMPK also promotes prolonged metabolic adaptation through transcriptional changes, decreasing biosynthetic genes while increasing expression of genes promoting lysosomal and mitochondrial biogenesis. The transcription factor EB (TFEB) is a well-appreciated effector of AMPK-dependent signals, but many of the molecular details of how AMPK controls these processes remain unknown. RATIONALE The requirement of AMPK and its specific downstream targets that control aspects of the transcriptional adaptation of metabolism remain largely undefined. We performed time courses examining gene expression changes after various mitochondrial stresses in wild-type (WT) or AMPK knockout cells. We hypothesized that a previously described interacting protein of AMPK, folliculin-interacting protein 1 (FNIP1), may be involved in how AMPK promotes increases in gene expression after metabolic stress. FNIP1 forms a complex with the protein folliculin (FLCN), together acting as a guanosine triphosphate (GTP)–activating protein (GAP) for RagC. The FNIP1-FLCN complex has emerged as an amino acid sensor to the mechanistic target of rapamycin complex 1 (mTORC1), involved in how amino acids control TFEB activation. We therefore examined whether AMPK may regulate FNIP1 to dominantly control TFEB independently of amino acids. RESULTS AMPK was found to govern expression of a core set of genes after various mitochondrial stresses. Hallmark features of this response were activation of TFEB and increases in the transcription of genes specifying lysosomal and mitochondrial biogenesis. AMPK directly phosphorylated five conserved serine residues in FNIP1, suppressing the function of the FLCN-FNIP1 GAP complex, which resulted in dissociation of RagC and mTOR from the lysosome, promoting nuclear translocation of TFEB even in the presence of amino acids. FNIP1 phosphorylation was required for AMPK to activate TFEB and for subsequent increases in peroxisome proliferation–activated receptor gamma coactivator 1-alpha (PGC1α) and estrogen-related receptor alpha (ERRα) mRNAs. Cells in which the five serines in FNIP1 were mutated to alanine were unable to increase lysosomal and mitochondrial gene expression programs after treatment with mitochondrial poisons or AMPK activators despite the presence and normal regulation of all other substrates of AMPK. By contrast, neither AMPK nor its control of FNIP1 were needed for activation of TFEB after amino acid withdrawal, illustrating the specificity to energy-limited conditions. CONCLUSION Our data establish FNIP1 as the long-sought substrate of AMPK that controls TFEB translocation to the nucleus, defining AMPK phosphorylation of FNIP1 as a singular event required for increased lysosomal and mitochondrial gene expression programs after metabolic stresses. This study also illuminates the larger biological question of how mitochondrial damage triggers a temporal response of repair and replacement of damaged mitochondria: Within early hours, AMPK-FNIP1–activated TFEB induces a wave of lysosome and autophagy genes to promote degradation of damaged mitochondria, and a few hours later, TFEB–up-regulated PGC1⍺ and ERR⍺ promote expression of a second wave of genes specifying mitochondrial biogenesis. These insights open therapeutic avenues for several common diseases associated with mitochondrial dysfunction, ranging from neurodegeneration to type 2 diabetes to cancer. Mitochondrial damage activates AMPK to phosphorylate FNIP1, stimulating TFEB translocation to the nucleus and sequential waves of lysosomal and mitochondrial biogenesis. After mitochondrial damage, activated AMPK phosphorylates FNIP1 (1), causing inhibition of FLCN-FNIP1 GAP activity (2). This leads to accumulation of RagC in its GTP-bound form, causing dissociation of RagC, mTORC1, and TFEB from the lysosome (3). TFEB is therefore not phosphorylated and translocates to the nucleus, inducing transcription of lysosomal or autophagy genes, with parallel increases in NT-PGC1α mRNA (4), which, in concert with ERRα (5), subsequently induces mitochondrial biogenesis (6). CCCP, carbonyl cyanide m-chlorophenylhydrazone; CLEAR, coordinated lysosomal expression and regulation; GDP, guanosine diphosphate; P, phosphorylation. [Figure created using BioRender] 

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3. Self-Assembling Polypeptides in Complex Coacervation

   https://doi.org/10.1021/acs.accounts.3c00689  

   Sathyavageeswaran, Arvind ; Bonesso Sabadini, Júlia ; Perry, Sarah L. ( February 2024 , Accounts of Chemical Research)

   Intracellular compartmentalization plays a pivotal role in cellular function, with membrane-bound organelles and membrane-less biomolecular 'condensates' playing key roles. These condensates, formed through liquid-liquid phase separation (LLPS), enable selective compartmentalization without the barrier of a lipid bilayer, thereby facilitating rapid formation/dissolution in response to stimuli. Intrinsically disordered proteins (IDPs) and/or proteins with intrinsically disordered regions (IDRs), which are often rich in charged and polar amino acid sequences, scaffold many condensates, often in conjunction with RNA. Comprehending the impact of IDP/IDR sequences on phase separation poses a challenge due to the extensive chemical diversity resulting from the myriad amino acids and post-translational modifications. To tackle this hurdle, one approach has been to investigate LLPS in simplified polypeptide systems, which offer a narrower scope within the chemical space for exploration. This strategy is supported by studies that have demonstrated how IDP function can largely be understood based on general chemical features, such as clusters or patterns of charged amino acids, rather than residue-level effects, and the ways in which these kinds of motifs give rise to an ensemble of conformations. Our lab has utilized complex coacervates assembled from oppositely-charged polypeptides as a simplified material analogue to the complexity of liquid-liquid phase separated biological condensates. Complex coacervation is an associative LLPS that occurs due to the electrostatic complexation of oppositely-charged macro-ions. This process is believed to be driven by the entropic gains resulting from the release of bound counterions and the reorganization of water upon complex formation. Apart from their direct applicability to IDPs, polypeptides also serve as excellent model polymers for investigating molecular interactions due to the wide range of available side-chain functionalities and the capacity to finely regulate their sequence, thus enabling precise control over interactions with guest molecules. Here, we discuss fundamental studies examining how charge patterning, hydrophobicity, chirality, and architecture affect the phase separation of polypeptide-based complex coacervates. These efforts have leveraged a combination of experimental and computational approaches that provide insight into the molecular level interactions. We also examine how these parameters affect the ability of complex coacervates to incorporate globular proteins and viruses. These efforts couple directly with our fundamental studies into coacervate formation, as such ‘guest’ molecules should not be considered as experiencing simple encapsulation and are instead active participants in the electrostatic assembly of coacervate materials. Interestingly, we observed trends in the incorporation of proteins and viruses into coacervates formed using different chain length polypeptides that are not well explained by simple electrostatic arguments and may be the result of more complex interactions between globular and polymeric species. Additionally, we describe experimental evidence supporting the potential for complex coacervates to improve the thermal stability of embedded biomolecules such as viral vaccines. Ultimately, peptide-based coacervates have the potential to help unravel the physics behind biological condensates while paving the way for innovative methods in compartmentalization, purification, and biomolecule stabilization. These advancements could have implications spanning from medicine to biocatalysis. 

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4. Nano‐silicon fertiliser increases the yield and quality of cherry radish

   https://doi.org/10.1002/moda.19  

   Xu, Xinxin ; Guo, Yaozu ; Hao, Yi ; Cai, Zeyu ; Cao, Yini ; Fang, Wanzhen ; Zhao, Bangyin ; Haynes, Christy L. ; White, Jason C. ; Ma, Chuanxin ( September 2023 , Modern Agriculture)

   Abstract Although silicon-based nanomaterials (Si-based NMs) can promote crop yield and alleviate biotic and abiotic stress, the underlying performance mechanisms are unknown. In the present study, the effect of the root application of Si-based NMs on the physiological responses of cherry radish (Raphanus sativus L.) was evaluated in a life cycle experiment. Root exposure to 0.1% (w/w) Si-based NMs significantly increased total fresh weight, total chlorophyll and carotenoids by 36.0%, 14.2% and 18.7%, respectively, relative to untreated controls. The nutritional content of the edible tissue was significantly enhanced, with an increase of 23.7% in reducing sugar, 24.8% in total sugar, and 232.7% in proteins; in addition, a number of nutritional elements (Cu, Mn, Fe, Zn, K, Ca, and P) were increased. Si-based NMs exposure positively altered the phytohormone network and decreased abscisic acid content, both of which promoted radish fresh weight. LC-MS-based metabolomic analysis shows that Si-based NMs increased the contents of most carbohydrates (e.g., α-D-glucose, acetylgalactosamine, lactose, fructose, etc.) and amino acids (e.g., asparagine, glutamic acid, glutamine, valine, arginine, etc.), subsequently improving overall nutritional values. Overall, nanoscale Si-based agrochemicals have significant potential as a novel strategy for the biofortification of vegetable crops in sustainable nano-enabled agriculture. 

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5. Effects of chemical preservation on bulk and amino acid isotope ratios of zooplankton, fish, and squid tissues

   https://doi.org/10.1002/rcm.8408  

   Hetherington, Elizabeth D. ; Kurle, Carolyn M. ; Ohman, Mark D. ; Popp, Brian N. ( April 2019 , Rapid Communications in Mass Spectrometry)

   It is imperative to understand how chemical preservation alters tissue isotopic compositions before using historical samples in ecological studies. Specifically, although compound‐specific isotope analysis of amino acids (CSIA‐AA) is becoming a widely used tool, there is little information on how preservation techniques affect amino acidδ¹⁵N values.

   We evaluated the effects of chemical preservatives on bulk tissueδ¹³C andδ¹⁵N and amino acidδ¹⁵N values, measured by gas chromatography/isotope ratio mass spectrometry (GC/IRMS), of (a) tuna (Thunnus albacares) and squid (Dosidicus gigas) muscle tissues that were fixed in formaldehyde and stored in ethanol for 2 years and (b) two copepod species,Calanus pacificusandEucalanus californicus, which were preserved in formaldehyde for 24–25 years.

   Tissues in formaldehyde‐ethanol had higher bulkδ¹⁵N values (+1.4,D. gigas; +1.6‰,T. albacares), higherδ¹³C values forD. gigas(+0.5‰), and lowerδ¹³C values forT. albacares(−0.8‰) than frozen samples. The bulkδ¹⁵N values from copepods were not different those from frozen samples, although theδ¹³C values from both species were lower (−1.0‰ forE. californicusand −2.2‰ forC. pacificus) than those from frozen samples. The mean amino acidδ¹⁵N values from chemically preserved tissues were largely within 1‰ of those of frozen tissues, but the phenylalanineδ¹⁵N values were altered to a larger extent (range: 0.5–4.5‰).

   The effects of preservation on bulkδ¹³C values were variable, where the direction and magnitude of change varied among taxa. The changes in bulkδ¹⁵N values associated with chemical preservation were mostly minimal, suggesting that storage in formaldehyde or ethanol will not affect the interpretation ofδ¹⁵N values used in ecological studies. The preservation effects on amino acidδ¹⁵N values were also mostly minimal, mirroring bulkδ¹⁵N trends, which is promising for future CSIA‐AA studies of archived specimens. However, there were substantial differences in phenylalanine and valineδ¹⁵N values, which we speculate resulted from interference in the chromatographic resolution of unknown compounds rather than alteration of tissue isotopic composition due to chemical preservation.

    

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