Science Note: Lysosome

Through a combination of various scientific methods, this research reveals the vital role of human lysosome-associated membrane proteins (LAMP-1 and LAMP-2) in maintaining lysosomal pH balance. Despite being mainly recognized as lysosomal markers, LAMP-1 and LAMP-2 directly influence the lysosomal cation channel TMEM175, aiding the acidification of lysosomes to a pH level that optimizes hydrolase activity. Interruptions to the interaction between LAMP and TMEM175 can increase lysosomal pH, impairing their hydrolytic function.
We offer small fluorescent probes for detecting lysosomal mass and pH with several color options: Lysosomal Acidic pH Detection kit-Green/Red and Green/Deep Red. 

Lysosomal LAMP proteins regulate lysosomal pH by direct inhibition of the TMEM175 channel    
Click here for the original article: Jiyuan Zhang, et. al., Mol. Cell. (2023)

Point of Interest
- The lysosomal proteins LAMP-1 and LAMP-2 have a direct interaction with the TMEM175 channel.
- This complex formation between TMEM175 and LAMPs is facilitated by their transmembrane (TM) domains.  
- The binding of LAMP proteins inhibits the channel activity of TMEM175.
- This inhibition of TMEM175 promotes lysosomal acidification, thereby enhancing the efficiency of hydrolase activity.

Accurate Measurement for Lysosomal pH changes

Existing lysosomal pH detection reagents have issues with dye localization, pH sensitivity, and retention. pHLys Green is a dye that solves these issues. The improved dye retention and localization enable detection of normal lysosomes, and the improved pH sensitivity enables detection of slight pH changes.

Product in Use:
   - Lysosomal Acidic pH Detection Kit-Green/Deep Red

Related Product:
   - pHLys Red- Lysosomal Acidic pH Detection

Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis    Ruilin Tian, et. al., Nature Neuroscience (2022)

Point of Interest
- The study reveals pathways that govern neuronal response to chronic oxidative stress, a factor in neurodegenerative diseases.
- Suppression of the lysosomal protein prosaposin makes neurons highly susceptible to oxidative stress. 
- This happens through the induction of lipofuscin formation which sequesters iron.
- Iron accumulation contributes to creating reactive oxygen species and lipid peroxidation, triggering ferroptosis, exclusively in neurons.

The simultaneous detection of lysosomal function with Mitochondrial ROS and intracellular Fe²⁺

Induction of Ferroptosis by Erastin

Erastin is a known inducer of ferroptosis. By inhibiting the cystine transporter (xCT), erastin inhibits the uptake of cystine. Cystine is the raw material for GSH. Therefore, Erastin ultimately decreases the amount of GSH. Decreased GSH then results in lipid peroxide accumulation and induction of ferroptosis.
The following experimental examples show changes in each aforementioned index as a consequence of erastin stimulation. Measurements are made using Dojindo reagents.

Using erastin-treated A549 cells, we measured intracellular Fe²⁺, ROS, lipid peroxide, glutathione, glutamate release into the extracellular space, and cystine uptake. As a result, inhibition of xCT by elastin was observed and also the release of glutamate and uptake of cystine were decreased. Furthermore, elastin treatment decreased intracellular glutathione while it increased intracellular Fe²⁺ , ROS, and lipid peroxides.

• - Pathogenic species of αSyn accumulate within neuronal lysosomes in mouse brains and primary neurons.
• - Neurons release these pathogenic αSyn species via SNARE-dependent lysosomal exocytosis
• - The released aggregates are non-membrane enveloped and seeding-competent
• - This release is dependent on neuronal activity and cytosolic Ca²⁺

• - PEN2 is a binding partner of metformin with a dissociation constant at micromolar levels
• - Metformin-bound PEN2 forms a complex with ATP6AP1 which leads to the inhibition of v-ATPase and the activation of lysosomal AMPK
• -  In vivo, liver-specific knockout of Pen2 abolishes metformin-mediated reduction of hepatic fat content, whereas intestine-specific knockout of Pen2 impairs its glucose-lowering effects.
• -  Knockdown of pen-2 in Caenorhabditis elegans abrogates metformin-induced extension of lifespan

• - TMEM175 is the proton “leak” channel of lysosomes and endosomes
• - TMEM175 is a highly proton-selective channel that is gated by luminal protons
• - An endogenous lipid can also activate TMEM175 to trigger lysosomal proton release
• - TMEM175 sets the lysosomal pH optimum via a classic negative feedback mechanism