Glycans modulate lipid binding in Lili-Mip lipocalin protein: insights from molecular simulations and protein network analyses.

The unique viviparous Pacific Beetle cockroaches provide nutrition to their embryo by secreting milk proteins Lili-Mip, a lipid-binding glycoprotein that crystallises in-vivo. The resolved in-vivo crystal structure of variably glycosylated Lili-Mip shows a classical Lipocalin fold with an eight-stranded antiparallel beta-barrel enclosing a fatty acid. The availability of physiologically unaltered glycoprotein structure makes Lili-Mip a very attractive model system to investigate the role of glycans on protein structure, dynamics, and function. Towards that end, we have employed all-atom molecular dynamics simulations on various glycosylated stages of a bound and free Lili-Mip protein and characterised the impact of glycans and the bound lipid on the dynamics of this glycoconjugate. Our work provides important molecular-level mechanistic insights into the role of glycans in the nutrient storage function of the Lili-Mip protein. Our analyses show that the glycans stabilise spatially proximal residues and regulate the low amplitude opening motions of the residues at the entrance of the binding pocket. Glycans also preserve the native orientation and conformational flexibility of the ligand. However, we find that either deglycosylation or glycosylation with high-mannose and paucimannose on the core glycans, which better mimic the natural insect glycosylation state, significantly affects the conformation and dynamics. A simple but effective distance- and correlation-based network analysis of the protein also reveals the key residues regulating the barrel's architecture and ligand binding characteristics in response to glycosylation.

Starvation Protects Hepatocytes from Inflammatory Damage through Paradoxical mTORC1 Signaling.

Background and aims: Sepsis-related liver failure is associated with a particularly unfavorable clinical outcome. Calorie restriction is a well-established factor that can increase tissue resilience, protect against liver failure and improve outcome in preclinical models of bacterial sepsis. However, the underlying molecular basis is difficult to investigate in animal studies and remains largely unknown. METHODS: We have used an immortalized hepatocyte line as a model of the liver parenchyma to uncover the role of caloric restriction in the resilience of hepatocytes to inflammatory cell damage. In addition, we applied genetic and pharmacological approaches to investigate the contribution of the three major intracellular nutrient/energy sensor systems, AMPK, mTORC1 and mTORC2, in this context. RESULTS: We demonstrate that starvation reliably protects hepatocytes from cellular damage caused by pro-inflammatory cytokines. While the major nutrient- and energy-related signaling pathways AMPK, mTORC2/Akt and mTORC1 responded to caloric restriction as expected, mTORC1 was paradoxically activated by inflammatory stress in starved, energy-deprived hepatocytes. Pharmacological inhibition of mTORC1 or genetic silencing of the mTORC1 scaffold Raptor, but not its mTORC2 counterpart Rictor, abrogated the protective effect of starvation and exacerbated inflammation-induced cell death. Remarkably, mTORC1 activation in starved hepatocytes was uncoupled from the regulation of autophagy, but crucial for sustained protein synthesis in starved resistant cells. CONCLUSIONS: AMPK engagement and paradoxical mTORC1 activation and signaling mediate protection against pro-inflammatory stress exerted by caloric restriction in hepatocytes.