USP17L3 Antibody

What is USP17L3 and what cellular functions does it regulate?

USP17L3 (ubiquitin specific peptidase 17 like family member 3) is a deubiquitinating enzyme that removes conjugated ubiquitin from specific proteins to regulate various cellular processes . It belongs to the Peptidase C19 protein family and functions by cleaving ubiquitin conjugates from targeted proteins, thereby preventing their proteasomal degradation . The canonical human USP17L3 protein has 530 amino acid residues with a molecular mass of approximately 59.5 kDa . It primarily regulates cell proliferation, cell cycle progression, apoptosis, cell migration, and cellular responses to viral infection by modifying the ubiquitination status of its target proteins . USP17L3 is predominantly localized in the nucleus and endoplasmic reticulum, where it executes its deubiquitinating functions .

What detection methods are available for USP17L3 antibodies in research applications?

Research applications for USP17L3 antibodies include several validated detection methods:

Most commercially available USP17L3 antibodies have been validated using MCF-7, K562, and HepG2 whole cell lysates . When planning experiments, researchers should consider both the detection method and appropriate positive control samples to ensure reliable results.

How should USP17L3 antibodies be stored to maintain optimal activity?

USP17L3 antibodies require specific storage conditions to preserve their functionality and specificity. For short-term storage (up to 2 weeks), antibodies should be refrigerated at 2-8°C . For long-term storage, maintain antibodies at -20°C in small aliquots to prevent repeated freeze-thaw cycles that can degrade antibody performance . Most commercial USP17L3 antibodies are supplied in a buffer containing 50% glycerol with 0.03% Proclin 300 as a preservative . When diluting the antibody for experimental use, researchers should prepare only the volume needed for immediate use to minimize activity loss. Properly stored antibodies typically maintain activity for 12 months from the date of receipt when handled according to supplier recommendations .

How does USP17L3 mechanistically regulate RORγt stability in Th17 cells and what are the implications for autoimmune disease research?

USP17 (closely related to USP17L3) functions as a positive regulator of RORγt, the master transcription factor in Th17 cells, through a specific deubiquitination mechanism . The enzyme stabilizes RORγt protein expression by reducing its polyubiquitination specifically at the Lys-360 residue, thereby preventing proteasome-mediated degradation . This protective effect increases RORγt protein levels and subsequently enhances the transcriptional activation of Th17-related genes including IL-17 and IL-17F .

The mechanistic pathway operates as follows:

• USP17 directly binds to RORγt in the nucleus

• USP17 catalyzes the removal of ubiquitin chains from the Lys-360 residue of RORγt

• Deubiquitinated RORγt escapes proteasomal degradation

• Stabilized RORγt increases transcriptional activation of downstream target genes

• Enhanced expression of IL-17 and other pro-inflammatory cytokines occurs

This mechanism has significant implications for autoimmune disease research, particularly for systemic lupus erythematosus (SLE), where USP17 expression is upregulated in CD4+ T cells from patients compared to healthy controls . Researchers investigating autoimmune conditions should consider USP17L3/USP17 as a potential therapeutic target to modulate RORγt-mediated pathways in Th17 cells, potentially reducing pathogenic inflammation in conditions like SLE .

What is the relationship between USP17L3 and Ras signaling pathways in cancer research models?

USP17L3/USP17 plays a critical role in regulating Ras activation and subsequent cell proliferation through its interaction with the Ras processing pathway . Research has demonstrated that USP17 disrupts Ras plasma membrane localization and activation, leading to marked inhibition of cell proliferation . The mechanism involves USP17 blocking RCE1 (Ras converting enzyme 1) activity, which is essential for proper Ras processing and membrane localization .

The disruption of Ras signaling occurs through the following cascade:

• USP17 expression interferes with RCE1 enzymatic function

• Impaired RCE1 activity prevents proper post-translational modification of Ras proteins

• Incompletely processed Ras fails to localize correctly to the plasma membrane

• Mislocalized Ras cannot interact effectively with upstream activators and downstream effectors

• The compromised Ras activation leads to inhibition of proliferative signaling pathways

This relationship has significant implications for cancer research models where Ras signaling is frequently dysregulated. When designing experiments to investigate USP17L3/USP17 in cancer contexts, researchers should incorporate analyses of Ras localization, activation status (GTP-bound Ras), and downstream signaling effects in addition to proliferation assessments . This multi-parameter approach provides more comprehensive insights into how USP17L3/USP17 modulates oncogenic signaling networks.

What technical challenges exist in detecting endogenous USP17L3 in primary human cells and how can they be overcome?

Detecting endogenous USP17L3 in primary human cells presents several technical challenges due to its regulation, expression patterns, and structural characteristics:

To overcome these challenges, researchers should implement a multi-faceted approach. For immunoblotting applications, use cytokine stimulation protocols (such as IL-4 or IL-6) to enhance USP17L3 expression before cell lysis . For immunofluorescence detection, combine subcellular fractionation with confocal microscopy to accurately localize USP17L3. Additionally, complementary techniques such as RT-qPCR using the validated primer pairs (5′-GAGCACTTGGTGGAAAGAGC-3′ and 5′-TGATGGTTCTTCATCCCACA-3′) can verify expression at the transcript level before proceeding to protein detection .

How should controls be designed for USP17L3 antibody validation experiments?

Proper control design is critical for USP17L3 antibody validation experiments to ensure specificity and reliability of results:

For overexpression controls, researchers can use the validated RORγt and USP17 constructs previously documented in the literature, which can be amplified by PCR with human cDNA from HEK293T cells and cloned into tagged vectors (FLAG, Myc, or HA-tagged) . When planning knockdown experiments, researchers should design siRNA or shRNA targeting USP17L3 specifically, with validation using RT-qPCR with the primers: 5′-GAGCACTTGGTGGAAAGAGC-3′ (forward) and 5′-TGATGGTTCTTCATCCCACA-3′ (reverse) .

What are the optimal sample preparation methods for detecting USP17L3 in different cellular compartments?

USP17L3 localizes primarily to the nucleus and endoplasmic reticulum, requiring specialized sample preparation approaches for optimal detection:

For Western blot applications, researchers should prepare fresh lysates due to potential degradation of USP17L3 during storage. When performing immunofluorescence microscopy, a brief pre-permeabilization step with 0.01% digitonin before fixation can improve antibody access to nuclear and ER compartments without disrupting subcellular structures. For co-localization studies, include appropriate markers such as Lamin B1 (nuclear envelope) or Calnexin (ER) alongside USP17L3 detection to confirm proper compartmentalization.

How can USP17L3 enzymatic activity be measured in experimental settings?

Measuring USP17L3 deubiquitinating enzyme (DUB) activity requires specialized approaches beyond simple protein detection:

For the most physiologically relevant assessment, researchers should utilize the cellular substrate deubiquitination approach. This involves immunoprecipitating potential substrates like RORγt and assessing their ubiquitination status in the presence or absence of USP17L3 expression or activity . When using the catalytically inactive mutant USP17C89S as a negative control, researchers can distinguish between enzymatic and non-enzymatic effects on substrate proteins .

To establish a functional reporter system, the validated luciferase reporter assay using the Il17a promoter can be employed. This involves cotransfecting the Il17a luciferase reporter plasmid with USP17L3 expression constructs and measuring the resulting luciferase activity, which correlates with USP17L3's functional impact on transcriptional regulation .

How can USP17L3 antibodies be used to investigate its role in autoimmune disease models?

USP17L3 antibodies provide valuable tools for investigating its role in autoimmune disease models, particularly given the established connection between USP17 and systemic lupus erythematosus (SLE) . Researchers can implement several approaches:

When interpreting data from these studies, researchers should consider that USP17L3 expression is up-regulated in CD4+ T cells from SLE patients compared to healthy controls . This differential expression suggests that USP17L3 could serve as both a biomarker and a therapeutic target. The correlation between USP17L3 levels, RORγt stability, and IL-17 production provides a mechanistic framework for understanding how USP17L3 contributes to autoimmune pathogenesis.

For validating experimental findings, researchers should use the established qPCR primers: IL-17A (5′-ACCAATCCCAAAAGGTCCTC-3′ and 5′-GGGGACAGAGTTCATGTGGT-3′), IL-17F (5′-CCTCCCCCTGGAATTACACT-3′ and 5′-ACCAGCACCTTCTCCAACTG-3′), RORγt (5′-CTGCTGAGAAGGACAGGGAG-3′ and 5′-AGTTCTGCTGACGGGTGC-3′), and USP17 (5′-GAGCACTTGGTGGAAAGAGC-3′ and 5′-TGATGGTTCTTCATCCCACA-3′) .

What approaches can be used to investigate USP17L3's role in lipid metabolism and lipid droplet biology?

Recent research has implicated USP17L3 in lipid metabolism and lipid droplet (LD) biology, providing new avenues for investigation:

When designing these experiments, researchers should consider the established parameters for lipid droplet analysis: number (LDs per cell), dispersion (percent touching), shape (eccentricity), size (median radius), and intensity (integrated intensity) . These metrics provide a comprehensive profile of how USP17L3 impacts lipid droplet biology.

To facilitate data interpretation, researchers should employ high-content microscopy analysis with appropriate staining protocols (such as BODIPY for neutral lipids) and conduct parallel biochemical assays to measure cellular triglyceride content. The integration of imaging and biochemical data provides a more comprehensive understanding of USP17L3's role in lipid metabolism regulation.

How can contradictory results in USP17L3 research be reconciled and validated?

Researchers may encounter contradictory results when studying USP17L3, particularly regarding its effects on cell proliferation and its substrate specificity. These apparent contradictions can be systematically addressed:

When addressing contradictory findings regarding USP17L3's impact on proliferation, researchers should note that while USP17 has been shown to inhibit cell proliferation by disrupting Ras plasma membrane localization , its effect on RORγt stability could promote proliferation of specific T cell subsets . This apparent contradiction may reflect cell type-specific functions or pathway-dependent effects.

To validate experimental findings, researchers should implement multiple complementary approaches. For example, when studying USP17L3's impact on a potential substrate, combine in vitro deubiquitination assays, co-immunoprecipitation studies, and functional readouts in cellular models. The use of catalytically inactive mutants (USP17C89S) as negative controls is essential for confirming the enzymatic specificity of observed effects .

What emerging technologies could advance USP17L3 research beyond current methodological limitations?

Several emerging technologies hold promise for overcoming current limitations in USP17L3 research:

The integration of these technologies could significantly advance our understanding of USP17L3 biology. For example, combining CRISPR-mediated endogenous tagging with live-cell imaging would enable researchers to monitor USP17L3 dynamics during cellular processes such as cell cycle progression, cytokine stimulation, or metabolic stress. Similarly, applying proximity labeling approaches would help identify the USP17L3 interactome under different cellular conditions, potentially revealing context-specific substrates and regulatory mechanisms.

Researchers should consider developing collaborative approaches that leverage these complementary technologies to build a more comprehensive understanding of USP17L3 function across different biological contexts.