Protein aggregation, a well-known culprit in human disease, 1,2 is also a major problem facing the use of proteins as therapeutic or diagnostic agents. 3,4 Insights into the protein aggregation problem have been garnered from the study of natural proteins, where a relationship between solubility and net charge has been noted. For example, it is known that proteins are least soluble at their isoelectric point, where they bear a net charge of zero. 5 More recently, small differences in net charge ((3 charge units) have been shown to predict aggregation tendencies among peptide variants. 6 In addition, intrinsically disordered proteins, 7,8 a class of proteins that are largely unfolded in the cell but that do not lead to pathological aggregation, tend to have large net charges. 9, 10 We speculated that the relationship between net charge and aggregation resistance might also be applicable to globular proteins, which can aggregate via partial unfolding induced by thermal agitation, chemical treatment, or conformational breathing. Recent evidence that some proteins can tolerate significant changes in net charge (for example, the finding that carbonic anhydrase retains activity after exhaustive acetylation of its surface lysines 11 ) encouraged us to test the hypothesis that the solubility and aggregation resistance of some proteins might be significantly enhanced, without abolishing their folding or function, by extensively mutating their surfaces to dramatically increase their net charge, a process we refer to as "supercharging".We began with green fluorescent protein (GFP), an easily assayed protein that undergoes chromophore maturation and becomes fluorescent only when folded correctly. To minimize the possibility that our GFP was unusually delicate and therefore unusually easy to improve, we used a starting GFP (stGFP) based on the stateof-the-art GFP variant called "superfolder", which has been highly optimized for folding robustness and resistance to denaturants. 12 The net charge of the stGFP is -7, similar to that of wild-type GFP (-8). To create a superpositive variant of GFP, we identified 29 positions in the crystal structure that were highly solvent-exposed and mutated these to positively charged amino acids (Lys and Arg), yielding a design with a theoretical net charge of +36 ( Figure 1 and Supporting Information). Genes encoding stGFP and GFP-(+36) were constructed and expressed in E. coli, and both genes yielded intensely green fluorescent bacteria. Following protein purification, the fluorescence properties of GFP(+36) were measured and found to be very similar to those of stGFP (Supporting Information).Encouraged by this finding, we produced and characterized additional supercharged GFPs having net charges of +48, -25, and -30, all of which were also found to have stGFP-like fluorescence (Figures 2a and S1). All supercharged GFP variants exhibited circular dichroism spectra similar to that of stGFP, indicating that the proteins have similar secondary structure content (Figure 2b). The thermodynamic s...

Adipose-resident T-cells (ARTs) regulate metabolic and inflammatory responses in obesity, but ART activation signals are poorly understood. Here, we describe class II major histocompatibility complex (MHCII) as an important component of high-fat diet (HFD)-induced obesity. Microarray analysis of primary adipocytes revealed that multiple genes involved in MHCII antigen processing and presentation increased in obese women. In mice, adipocyte MHCII increased within two weeks HFD, paralleling increases in pro-inflammatory and decreases in anti-inflammatory ART markers, and preceding adipose tissue macrophage (ATM) accumulation and pro-inflammatory M1 polarization. Mouse 3T3-L1 and primary adipocytes activated T-cells in an antigen-specific, contact-dependent manner, indicating adipocyte MHCII is functional. HFD-fed MHCII−/− mice developed less adipose inflammation and insulin resistance than wild-type mice, despite developing similar adiposity. These investigations uncover a mechanism whereby a HFD-induced adipocyte/ART dialogue involving MHCII instigates adipose inflammation and, together with ATM MHCII, escalates its progression.

Asprosin is a recently discovered fasting-induced hormone that promotes hepatic glucose production. Here, we demonstrate that plasma asprosin crosses the blood-brain-barrier and directly activates orexigenic AgRP+ neurons via a cAMP-dependent pathway. This signaling results in inhibition of downstream anorexigenic POMC+ neurons in a GABA-dependent manner, resulting in appetite stimulation and a drive to accumulate adiposity and body weight. Genetic deficiency of asprosin in humans results in a syndrome characterized by low appetite and extreme leanness, which is phenocopied by mice carrying similar mutations, and one that can be fully rescued by asprosin expression. Further, we found that obese humans and mice display pathologically elevated circulating asprosin concentrations, and neutralization of plasma asprosin using a monoclonal antibody reduces appetite and body weight in obese mice, in addition to improving their glycemic profile. Thus, asprosin, in addition to performing a glucogenic function, is a centrally-acting orexigenic hormone, and one that represents a potential therapeutic target to treat both obesity and diabetes.