A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7–Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4⁺ and CD8⁺ lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.     

Resolution of two substrate-binding sites in an engineered cytochrome P450eryF bearing a fluorescent probe

To elucidate the mechanisms of cooperativity of cytochrome P450eryF an SH-reactive fluorescent probe was introduced close to the substrate-binding site. Cys-154, the only accessible cysteine, was eliminated by site-directed mutagenesis, and a novel cysteine was substituted for Ser-93 in the B'/C loop. S93C, C154A, C154S, S93C/C154A, and S93C/S154C were characterized in terms of affinity for 1-pyrenebutanol (1-PB), cooperativity, and ionic-strength dependence of the 1-PB-induced spin shift. S93C/C154S retains the key functional properties of the wild-type, and modification by three different SH-reactive probes had little effect on the characteristics of the enzyme. The labeled proteins exhibited fluorescence resonance energy transfer from 1-PB to the label, which allowed us to resolve two substrate-binding events, and to determine the corresponding KD values (KD1 = 1.2 +/- 0.2 microM, KD2 = 9.4 +/- 0.8 microM). Using these values for analysis of the substrate-induced spin transition, we demonstrate that the interactions of P450eryF with 1-PB are consistent with a sequential binding mechanism, where substrate interactions at a higher-affinity site cause a conformational transition crucial for the binding of the second substrate molecule and subsequent spin shift. This transition is apparently associated with an important rearrangement of the system of salt links in the proximity of Cys-154.     

Migration of melanoblasts into the developing murine hair follicle is accompanied by transient c-Kit expression.

Disruption of the c-Kit/stem cell factor (SCF) signaling pathway interferes with the survival, migration, and differentiation of melanocytes during generation of the hair follicle pigmentary unit. We examined c-Kit, SCF, and S100 (a marker for precursor melanocytic cells) expression, as well as melanoblast/melanocyte ultrastructure, in perinatal C57BL/6 mouse skin. Before the onset of hair bulb melanogenesis (i.e., stages 0-4 of hair follicle morphogenesis), strong c-Kit immunoreactivity (IR) was seen in selected non-melanogenic cells in the developing hair placode and hair plug. Many of these cells were S100-IR and were ultrastructurally identified as melanoblasts with migratory appearance. During the subsequent stages (5 and 6), increasingly dendritic c-Kit-IR cells successively invaded the hair bulb, while S100-IR gradually disappeared from these cells. Towards the completion of hair follicle morphogenesis (stages 7 and 8), several distinct follicular melanocytic cell populations could be defined and consisted broadly of (a) undifferentiated, non-pigmented c-Kit-negative melanoblasts in the outer root sheath and bulge and (b) highly differentiated melanocytes adjacent to the hair follicle dermal papilla above Auber's line. Widespread epithelial SCF-IR was seen throughout hair follicle morphogenesis. These findings suggest that melanoblasts express c-Kit as a prerequisite for migration into the SCF-supplying hair follicle epithelium. In addition, differentiated c-Kit-IR melanocytes target the bulb, while non-c-Kit-IR melanoblasts invade the outer root sheath and bulge in fully developed hair follicles.     

MicroRNA-214 controls skin and hair follicle development by modulating the activity of the Wnt pathway

Skin development is governed by complex programs of gene activation and silencing, including microRNA-dependent modulation of gene expression. Here, we show that miR-214 regulates skin morphogenesis and hair follicle (HF) cycling by targeting β-catenin, a key component of the Wnt signaling pathway. miR-214 exhibits differential expression patterns in the skin epithelium, and its inducible overexpression in keratinocytes inhibited proliferation, which resulted in formation of fewer HFs with decreased hair bulb size and thinner hair production. The inhibitory effects of miR-214 on HF development and cycling were associated with altered activities of multiple signaling pathways, including decreased expression of key Wnt signaling mediators β-catenin and Lef-1, and were rescued by treatment with pharmacological Wnt activators. Finally, we identify β-catenin as one of the conserved miR-214 targets in keratinocytes. These data provide an important foundation for further analyses of miR-214 as a key regulator of Wnt pathway activity and stem cell functions during normal tissue homeostasis, regeneration, and aging.     

A role for p75 neurotrophin receptor in the control of apoptosis-driven hair follicle regression.

To examine the mechanisms that underlie the neurotrophin-induced, apoptosis-driven hair follicle involution (catagen), the expression and function of p75 neurotrophin receptor (p75NTR), which is implicated in apoptosis control, were studied during spontaneous catagen development in murine skin. By RT-PCR, high steady-state p75NTR mRNA skin levels were found during the anagen-catagen transition of the hair follicle. By immunohistochemistry, p75NTR alone was strongly expressed in TUNEL+/Bcl2- keratinocytes of the regressing outer root sheath, but both p75NTR and TrkB and/or TrkC were expressed by the nonregressing TUNEL-/Bcl2+ secondary hair germ keratinocytes. To determine whether p75NTR is functionally involved in catagen control, spontaneous catagen development was compared in vivo between p75NTR knockout (-/-) and wild-type mice. There was significant catagen retardation in p75NTR knockout mice as compared to wild-type controls (P<0.05). Instead, transgenic mice-overexpressing NGF (promoter: K14) showed substantial acceleration of catagen (P<0.001). Although NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3) accelerated catagen in the organ-cultured skin of C57BL/6 mice, these neurotrophins failed to promote catagen development in the organ-cultured p75NTR null skin. These findings suggest that p75NTR signaling is involved in the control of kerotinocyte apoptosis during catagen and that pharmacological manipulation of p75NTR signaling may prove useful for the treatment of hair disorders that display premature entry into catagen.     

An electrostatically driven conformational transition is involved in the mechanisms of substrate binding and cooperativity in cytochrome P450eryF

The effect of ionic strength (I) on substrate-induced spin transitions and cooperativity in cytochrome P450eryF was studied. At a saturating concentration of 1-pyrenebutanol (1-PB) increasing ionic strength in the 0.06-1.2 M range promotes the formation of the high-spin state of P450, which fraction increases from 26% at 0.06 M to 75% at 1.2 M. This effect was associated with a considerable decrease in cooperativity as revealed in the 1-PB-induced spin shift. While P450eryF exhibits distinct positive cooperativity (S(50) = 8.3 microM, n = 2.4) with this substrate at low ionic strength (I = 0.06 M), n decreases to 1.2 (S(50) = 3.2 microM) at I = 0.66 M. Increasing ionic strength also increases the distance between the first (effector) molecule of 1-PB and the heme, as detected by the changes in the efficiency of FRET from 1-PB to the heme. The modification of Cys(154) with 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) largely suppresses these effects of ionic strength and causes a prominent decrease in the cooperativity. The same effect was observed when Cys(154) was substituted with isoleucine. Importantly, Cys(154) is located at the C-terminal end of helix E and is surrounded by salt bridges formed by arginine, glutamate, and aspartate residues located in helices D, E, F, and G. Our results suggest that the binding of the first substrate molecule causes an important conformational transition in the P450eryF that facilitates the substrate-induced spin shift. This transition is apparently accompanied by dissociation or rearrangement of several salt bridges in the proximity of Cys(154) and modulates accessibility and hydration of the heme pocket.     

AmylPepPred: Amyloidogenic Peptide Prediction tool

We present an efficient computational architecture designed using supervised machine learning model to predict amyloid fibril forming protein segments, named AmylPepPred. The proposed prediction model is based on bio-physio-chemical properties of primary sequences and auto-correlation function of their amino acid indices. AmylPepPred provides a user friendly web interface for the researchers to easily observe the fibril forming and non-fibril forming hexmers in a given protein sequence. We expect that this stratagem will be highly encouraging in discovering fibril forming regions in proteins thereby benefit in finding therapeutic agents that specifically aim these sequences for the inhibition and cure of amyloid illnesses.

Availability

AmylPepPred is available freely for academic use at www.zoommicro.in/amylpeppred