Mycobacterium tuberculosis (Mtb) has complex and dynamic interactions with the human host, and subpopulations of Mtb that emerge during infection can influence disease outcomes. This study implicates zinc ion (Zn²⁺) availability as a likely driver of bacterial phenotypic heterogeneity in vivo. Zn²⁺ sequestration is part of “nutritional immunity”, where the immune system limits micronutrients to control pathogen growth, but this defense mechanism seems to be ineffective in controlling Mtb infection. Nonetheless, Zn²⁺-limitation is an environmental cue sensed by Mtb, as calprotectin triggers the zinc uptake regulator (Zur) regulon response in vitro and co-localizes with Zn²⁺-limited Mtb in vivo. Prolonged Zn²⁺ limitation leads to numerous physiological changes in vitro, including differential expression of certain antigens, alterations in lipid metabolism and distinct cell surface morphology. Furthermore, Mtb enduring limited Zn²⁺ employ defensive measures to fight oxidative stress, by increasing expression of proteins involved in DNA repair and antioxidant activity, including well described virulence factors KatG and AhpC, along with altered utilization of redox cofactors. Here, we propose a model in which prolonged Zn²⁺ limitation defines a population of Mtb with anticipatory adaptations against impending immune attack, based on the evidence that Zn²⁺-limited Mtb are more resistant to oxidative stress and exhibit increased survival and induce more severe pulmonary granulomas in mice. Considering that extracellular Mtb may transit through the Zn²⁺-limited caseum before infecting naïve immune cells or upon host-to-host transmission, the resulting phenotypic heterogeneity driven by varied Zn²⁺ availability likely plays a key role during early interactions with host cells.

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Activity-regulated cytoskeleton-associated protein (Arc; also known as Arg3.1) is an immediate early gene product that is transcribed in dendritic spines and, to date, has been best characterized as a positive regulator of AMPAR endocytosis during long-term depression (LTD) through interaction with endocytic proteins. Here, we show that protein interacting with C terminal kinase 1 (PICK1), a protein known to bind to the GluA2 subunit of AMPARs and associated with AMPAR trafficking, was pulled-down from brain homogenates and synaptosomes when using Arc as immobilized bait. Fluctuation and FLIM-FRET-Phasor analysis revealed direct interaction between these proteins when co-expressed that was increased under depolarizing conditions in live cells. At the plasma membrane, Arc-mCherry oligomerization was found to be concentration dependent. Additionally, co-expression of Arc-mCherry and EGFP-PICK1 followed by depolarizing conditions resulted in significant increases in the number and size of puncta containing both proteins. Furthermore, we identified the Arc binding region to be the first 126 amino acids of the PICK1 BAR domain. Overall, our data support a novel interaction and model where PICK1 mediates Arc regulation of AMPARs particularly under depolarizing conditions.

The mammalian target of rapamycin complex 1 (mTORC1) senses multiple stimuli to regulate anabolic and catabolic processes. mTORC1 is typically hyperactivated in multiple human diseases such as cancer and type 2 diabetes. Extensive research has focused on signaling pathways that can activate mTORC1 such as growth factors and amino acids. However, less is known about signaling cues that can directly inhibit mTORC1 activity. Here, we identify A-kinase anchoring protein 13 (AKAP13) as an mTORC1 binding protein, and a crucial regulator of mTORC1 inhibition by G-protein coupled receptor (GPCR) signaling. GPCRs paired to Gα_s proteins increase cyclic adenosine 3’5’ monophosphate (cAMP) to activate protein kinase A (PKA). Mechanistically, AKAP13 acts as a scaffold for PKA and mTORC1, where PKA inhibits mTORC1 through the phosphorylation of Raptor on Ser 791. Importantly, AKAP13 mediates mTORC1-induced cell proliferation, cell size, and colony formation. AKAP13 expression correlates with mTORC1 activation and overall lung adenocarcinoma patient survival, as well as lung cancer tumor growth in vivo. Our study identifies AKAP13 as an important player in mTORC1 inhibition by GPCRs, and targeting this pathway may be beneficial for human diseases with hyperactivated mTORC1.

The energy transfer from the three Trp residues at positions 8, 128, and 264 within the human serum transferrin (hTF) N‐lobe to the ligand to metal charge transfer band has been investigated by monitoring changes in Trp fluorescence emission and lifetimes. The fluorescence emission from hTF N‐lobe is dominated by Trp264, as revealed by an 82% decrease in the quantum yield when this Trp residue is absent. Fluorescence lifetimes were determined by multifrequency phase fluorometry of mutants containing one or two Trp residues. Decays of these samples are best described by two or three discrete lifetimes or by a unimodal Lorentzian distribution. The discrete lifetimes and the center of the lifetime distribution for samples containing Trp128 and Trp264 are affected by iron. The distribution width narrows on iron removal and is consistent with a decrease in dynamic mobility of the dominant fluorophore, Trp264. Both the quantum yield and the lifetimes are lower when iron is present, however, not proportionally. The greater effect of iron on quantum yields is indicative of nonexcited state quenching, i.e., static quenching. The results of these experiments provide quantitative data strongly suggesting that Förster resonance energy transfer is not the sole source of Trp quenching in the N‐lobe of hTF.