Impaired Retrograde Transport Due to Lack of TBC1D5 Contributes to the Trafficking Defect of Lysosomal Cathepsins in Ischemic/Hypoxic Cardiomyocytes

Lysosomal dysfunction has been found in many pathological conditions, and methods to improve lysosomal function have been reported to be protective against infarcted hearts. However, the mechanisms underlying lysosomal dysfunction caused by ischemic injury are far less well-established. The retromer complex is implicated in the trafficking of cation-independent mannose 6-phosphate receptor (CI-MPR), which is an important protein tag for the proper transport of lysosomal contents and therefore is important for the maintenance of lysosomal function. In this study, we found that the function of retrograde transport in cardiomyocytes was impaired with ischemia/hypoxia (I/H) treatment, which resulted in a decrease in CI-MPR and an abnormal distribution of lysosomal cathepsins. I/H treatment caused a reduction in TBC1D5 and a blockade of the Rab7 membrane cycle, which impeded retromer binding to microtubules and motor proteins, resulting in an impairment of retrograde transport and a decrease in CI-MPR. We also established that TBC1D5 was an important regulator of the distribution of lysosomal cathepsins. Our findings shed light on the regulatory role of retromer in ischemic injury and uncover the regulatory mechanism of TBC1D5 over retromer.

Mechanism-Based Strategy for Optimizing HaloTag Protein Labeling

HaloTag labeling technology has introduced unrivaled potential in protein chemistry, molecular and cellular biology. A wide variety of ligands have been developed to meet the specific needs of diverse applications, but only a single protein tag, DhaAHT, is routinely used for their incorporation. Following a systematic kinetic and computational analysis of different reporters, tetramethylrhodamine and three 4-stilbazolium-based fluorescent ligands, we showed that the mechanism of incorporating different ligands depends both on the binding step and the efficiency of the chemical reaction. By studying the different haloalkane dehalogenases DhaA, LinB, and DmmA, we found that the architecture of the access tunnels is critical for the kinetics of both steps and the ligand specificity. We show that highly efficient labelling with specific ligands is achievable with natural dehalogenases. We propose a simple protocol for selecting the optimal protein tag for a specific ligand from a wide pool of available enzymes with diverse access tunnel architectures. The application of this protocol eliminates a need for expensive and laborious protein engineering.

Targeted Protein Acetylation in Cells Using Heterobifunctional Molecules

Protein acetylation is a central event in orchestrating diverse cellular processes. However, current strategies to investigate protein acetylation in cells are often non-specific or lack temporal and magnitude control. Here, we developed an acetylation tagging system, AceTAG, to induce acetylation of targeted proteins. The AceTAG system utilizes bifunctional molecules to direct the lysine acetyltransferase p300/CBP to proteins fused with the small protein tag FKBP12F36V, resulting in their induced acetylation. Using AceTAG, we induced targeted acetylation of a diverse array of proteins in cells, specifically histone H3.3, the NF-kB subunit p65/RelA, and the tumor suppressor p53. We demonstrate that targeted acetylation with the AceTAG system is rapid, selective, reversible, and can be controlled in a dose-dependent fashion. AceTAG represents a useful strategy to modulate protein acetylation and will enable the exploration of targeted acetylation in basic biological and therapeutic contexts.

DNA barcodes using a double nanopore system

The potential of a double nanopore system to determine DNA barcodes has been demonstrated experimentally. By carrying out Brownian dynamics simulation on a coarse-grained model DNA with protein tag (barcodes) at known locations along the chain backbone, we demonstrate that due to large variation of velocities of the chain segments between the tags, it is inevitable to under/overestimate the genetic lengths from the experimental current blockade and time of flight data. We demonstrate that it is the tension propagation along the chain’s backbone that governs the motion of the entire chain and is the key element to explain the non uniformity and disparate velocities of the tags and DNA monomers under translocation that introduce errors in measurement of the length segments between protein tags. Using simulation data we further demonstrate that it is important to consider the dynamics of the entire chain and suggest methods to accurately decipher barcodes. We introduce and validate an interpolation scheme using simulation data for a broad distribution of tag separations and suggest how to implement the scheme experimentally.

HaloTag8: an engineered protein tag to improve fluorophore performance

HaloTag8 is an engineered variant of HaloTag7 with up to 40% higher brightness and increased fluorescence lifetime when labeled with fluorogenic rhodamines. Moreover, combining HaloTag8 with HaloTag7 and other fluorescent probes enabled live-cell multiplexing using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. The increased brightness of HaloTag8 and its use in fluorescence lifetime multiplexing makes it a powerful tool for live-cell imaging.

Near-infrared fluorescent probes: a next-generation tool for protein-labeling applications

This minireview describes the development of NIR chemical probes for various protein-tag systems.