Restoration of IFNγR Subunit Assembly, IFNγ Signaling and Parasite Clearance in Leishmania donovani Infected Macrophages: Role of Membrane Cholesterol

Despite the presence of significant levels of systemic Interferon gamma (IFNγ), the host protective cytokine, Kala-azar patients display high parasite load with downregulated IFNγ signaling in Leishmania donovani (LD) infected macrophages (LD-MØs); the cause of such aberrant phenomenon is unknown. Here we reveal for the first time the mechanistic basis of impaired IFNγ signaling in parasitized murine macrophages. Our study clearly shows that in LD-MØs IFNγ receptor (IFNγR) expression and their ligand-affinity remained unaltered. The intracellular parasites did not pose any generalized defect in LD-MØs as IL-10 mediated signal transducer and activator of transcription 3 (STAT3) phosphorylation remained unaltered with respect to normal. Previously, we showed that LD-MØs are more fluid than normal MØs due to quenching of membrane cholesterol. The decreased rigidity in LD-MØs was not due to parasite derived lipophosphoglycan (LPG) because purified LPG failed to alter fluidity in normal MØs. IFNγR subunit 1 (IFNγR1) and subunit 2 (IFNγR2) colocalize in raft upon IFNγ stimulation of normal MØs, but this was absent in LD-MØs. Oddly enough, such association of IFNγR1 and IFNγR2 could be restored upon liposomal delivery of cholesterol as evident from the fluorescence resonance energy transfer (FRET) experiment and co-immunoprecipitation studies. Furthermore, liposomal cholesterol treatment together with IFNγ allowed reassociation of signaling assembly (phospho-JAK1, JAK2 and STAT1) in LD-MØs, appropriate signaling, and subsequent parasite killing. This effect was cholesterol specific because cholesterol analogue 4-cholestene-3-one failed to restore the response. The presence of cholesterol binding motifs [(L/V)-X1–5-Y-X1–5-(R/K)] in the transmembrane domain of IFNγR1 was also noted. The interaction of peptides representing this motif of IFNγR1 was studied with cholesterol-liposome and analogue-liposome with difference of two orders of magnitude in respective affinity (KD: 4.27×10−9 M versus 2.69×10−7 M). These observations reinforce the importance of cholesterol in the regulation of function of IFNγR1 proteins. This study clearly demonstrates that during its intracellular life-cycle LD perturbs IFNγR1 and IFNγR2 assembly and subsequent ligand driven signaling by quenching MØ membrane cholesterol

Engineering of cyclodextrin glucanotransferases and the impact for biotechnological applications

Cyclodextrin glucanotransferases (CGTases) are industrially important enzymes that produce cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. Cyclodextrin glucanotransferases are also applied as catalysts in the synthesis of glycosylated molecules and can act as antistaling agents in the baking industry. To improve the performance of CGTases in these various applications, protein engineers are screening for CGTase variants with higher product yields, improved CD size specificity, etc. In this review, we focus on the strategies employed in obtaining CGTases with new or enhanced enzymatic capabilities by searching for new enzymes and improving existing enzymatic activities via protein engineering

Astrocytes express elements of the class II endocytic pathway and process central nervous system autoantigen for presentation to encephalitogenic T cells.

Astrocytes are nonprofessional APCs that may participate in Ag presentation and activation of pathogenic CD4+ T cells involved in central nervous system (CNS) inflammatory diseases. Using immortalized pure astrocytes as a complement to the study of primary astrocytes, we investigated whether these astrocytes express elements involved in the class II endocytic pathway and if they are capable of processing native myelin basic protein (MBP), a step that could be necessary for initiating or perpetuating T cell recognition of this self-Ag in vivo. Upon IFN-gamma-stimulation, primary and immortalized astrocytes up-regulate class II transactivator (CIITA), invariant chain (Ii) (p31 and p41), H-2Ma, and H-2Mb. Analysis of CIITA cDNA sequences demonstrated that CIITA transcription in astrocytes is directed by a promoter (type IV) that mediates IFN-gamma-inducible CIITA expression and encodes a CIITA protein that differs in its N-terminal sequence from CIITA reported in professional APC. Comparing live and fixed APC for Ag presentation, we show that Ag processing by APC is required for presentation of native MBP to autopathogenic T cells specific for the major MBP epitope, Acl-11. We have observed that primary astrocytes and some, but not all, astrocyte lines in the absence of contaminating microglia are capable of processing and presenting native MBP, suggesting that there may be heterogeneity. Our study provides definitive evidence that astrocytes are capable of processing CNS autoantigen, indicating that astrocytes have potential for processing and presentation of CNS autoantigen to proinflammatory T cells in CNS autoimmune disease