Eden Reichard

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Label-Free Detection of Protein Tyrosine Phosphatase 1B (PTP1B) by Using a Rationally Designed Förster Resonance Energy Transfer (FRET) Probe.

Chembiochem. 2018 12 04;19(23):2495-2501

Authors: Durgannavar T, Kwon SJ, Ghisaidoobe ABT, Rho K, Kim JH, Yoon SY, Kang HJ, Chung SJ

Abstract
A highly selective detection method of native protein tyrosine phosphatase 1B (PTP1B) is described using a target specific probe equipped with 1-naphthylamine (λex =330 nm, λem =445 nm). Irradiation of a mixture of PTP1B and Probe 1 with ultraviolet light of 280 nm (corresponding to PTP1B excitation maximum) resulted in significant fluorescence increase at 445 nm, following FRET characteristics. This phenomenon does not occur with other closely related phosphatases or cellular abundant alkaline phosphatase (APP). Probe 1, the most potent and selective probe, was found to competitively inhibit PTP1B (Ki ≈42 nm), whereas APP inhibition was found to be in the low micromolar range. Furthermore, Probe 1 discriminates between PTP1B and several other phosphatases. Here, we report real-time label-free FRET detection of pure PTP1B as well as induced human PTP1B in Escherichia coli cell lysate. In contrast to 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), a representative fluorescence turn-on PTP substrate, our FRET probe successfully differentiated human cervical carcinoma cell lysate, SiHa, which has a high expression level of PTP1B, from PTP1B-knockdown SiHa cell lysate (that is, siRNA was used for PTP1B knockdown).

PMID: 30238680 [PubMed - indexed for MEDLINE]

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that remodel the extracellular matrix environment and mitigate outside-in signaling. Loss of regulation of MMP activity plays a role in numerous pathological states. In particular, aberrant collagenolysis affects tumor invasion and metastasis, osteoarthritis, and cardiovascular and neurodegenerative diseases. To evaluate the collagen sequence preferences of MMPs, a positional scanning synthetic combinatorial library was synthesized herein and was used to investigate the P10′ and P11′ substrate subsites. The scaffold for the library was a triple-helical peptide mimic of the MMP cleavage site in types I–III collagen. A FRET-based enzyme activity assay was used to evaluate the sequence preferences of eight MMPs. Deconvolution of the library data revealed distinct motifs for several MMPs and discrimination among closely related MMPs. On the basis of the screening results, several individual peptides were designed and evaluated. A triple-helical substrate incorporating Asp–Lys in the P10′–P11′ subsites offered selectivity between MMP-14 and MMP-15, whereas Asp–Lys or Trp–Lys in these subsites discriminated between MMP-2 and MMP-9. Future screening of additional subsite positions will enable the design of selective triple-helical MMP probes that could be used for monitoring in vivo enzyme activity and enzyme-facilitated drug delivery. Furthermore, selective substrates could serve as the basis for the design of specific triple-helical peptide inhibitors targeting only those MMPs that play a detrimental role in a disease of interest.

The signalling conformation of the insulin receptor ectodomain

The signalling conformation of the insulin receptor ectodomain, Published online: 24 October 2018; doi:10.1038/s41467-018-06826-6

The insulin receptor plays a key role in many physiological processes, yet how insulin effects receptor signaling at the structural level remains incomplete. Here the authors present a high-resolution cryo-EM structure of a high-affinity form of the insulin-bound insulin receptor ectodomain that sheds light on the mechanism of signal transduction.

Human breast tumor-infiltrating CD8⁺ T cells retain polyfunctionality despite PD-1 expression

Human breast tumor-infiltrating CD8⁺ T cells retain polyfunctionality despite PD-1 expression, Published online: 16 October 2018; doi:10.1038/s41467-018-06653-9

Expression of the checkpoint molecule programmed cell death protein 1 (PD-1) is considered a marker of T cells exhaustion. Here the authors show that CD8T cells isolated from breast cancer patients are perfectly functional despite PD-1 expression while those isolated from melanoma patients are not.

HIV integrates into the host genome to create a persistent viral reservoir. Stimulation of CD4+ memory T lymphocytes with common γc-chain cytokines renders these cells more susceptible to HIV infection, making them a key component of the reservoir itself. IL-15 is up-regulated during primary HIV infection, a time when the...