DPP4 Human

What is the molecular structure of human DPP4?

Human DPP4 is a 110 kDa type II transmembrane glycoprotein that exists in both membrane-bound and soluble forms. The protein consists of a short cytoplasmic domain (1-6 amino acids), a transmembrane domain (7-29 amino acids), and a large extracellular domain containing the catalytic site. The extracellular portion is heavily glycosylated, with carbohydrates accounting for approximately 20% of its molecular mass. Two glutamate residues (205 and 206) within the glycosylated region are essential for enzymatic activity. The protein contains nine N-glycosylation sites, six of which are located in the glycosylated region and are highly conserved across species. These glycosylation sites are crucial for proper protein folding, stability, and intracellular trafficking .

How is DPP4 gene expression regulated in human tissues?

DPP4 expression is regulated through multiple transcriptional mechanisms. The human DPP4 gene spans 70 kb on chromosome 2 and contains 26 exons. Its promoter region contains binding sites for several transcription factors including NFκB, SP-1, EGFR, and AP-1 factor NF-1. In certain cell types, interferons (α, β, and γ) stimulate STAT1α binding to a consensus interferon γ-activated sequence (GAS) in the promoter, leading to increased DPP4 expression. Inflammatory cytokines play significant regulatory roles - IL-12 upregulates DPP4 translation, while TNFα influences cell surface expression and may increase soluble DPP4 release. Hypoxia-inducible factor-1α (HIF-1α) and hepatocyte nuclear factors (HNFs) also target DPP4 expression, which explains observations that hypoxic conditions induce DPP4 release in human smooth muscle cells .

What distinguishes membrane-bound from soluble DPP4 in humans?

Membrane-bound DPP4 is a type II transmembrane protein anchored to the cell surface, while soluble DPP4 (sDPP4) lacks the transmembrane and cytosolic domains but retains catalytic activity. The soluble form circulates in plasma and body fluids following cleavage from the membrane through a process called shedding. Both forms maintain enzymatic activity, though they may have distinct physiological roles. Membrane-bound DPP4 primarily functions in cell-cell interactions and localized substrate processing, while sDPP4 can act systemically. Research indicates that hematopoietic cells are the predominant source of plasma sDPP4, particularly following catalytic DPP4 inhibition. During adipocyte differentiation, DPP4 release increases markedly, suggesting adipose tissue may constitute a relevant source of circulating DPP4 .

What are the recommended methods for measuring DPP4 enzymatic activity in human samples?

For measuring DPP4 enzymatic activity in human samples, several methodological approaches can be employed:

• Fluorometric assays: Using substrates like Gly-Pro-AMC (aminomethylcoumarin) or Gly-Pro-pNA (para-nitroaniline) that release fluorescent or chromogenic groups upon cleavage by DPP4. This approach allows continuous monitoring of enzymatic activity.

• HPLC-based methods: For quantifying the cleavage of natural substrates such as GLP-1, analyzing both intact and truncated forms to assess DPP4 activity.

• Single-molecule assays: Ultrasensitive techniques like Simoa can distinguish between the full-length CXCL10(1-77) and the NH2-truncated CXCL10(3-77), providing a direct measure of DPP4 activity on endogenous substrates .

When conducting these assays, researchers should consider:

• Sample collection timing (DPP4 activity varies with circadian rhythms and feeding status)

• Appropriate controls for DPP4 inhibition

• Potential interference from other dipeptidyl peptidases

• The stability of DPP4 in stored samples

For clinical research, standardization across samples is essential, including consistent fasting conditions and processing protocols to ensure reproducibility.

How can researchers effectively knockdown or overexpress DPP4 in human cell lines?

For effective manipulation of DPP4 expression in human cell models:

Knockdown strategies:

• Lentiviral shRNA delivery provides stable knockdown in difficult-to-transfect cells, including primary preadipocytes and mature adipocytes

• CRISPR-Cas9 gene editing offers more complete elimination of DPP4 expression

• siRNA approaches allow transient knockdown for short-term functional studies

Overexpression methods:

• Lentiviral or adenoviral vectors carrying the DPP4 coding sequence

• Inducible expression systems (e.g., tetracycline-controlled) to regulate timing and level of expression

• Plasmid transfection for transient overexpression studies

When assessing knockdown or overexpression efficiency, researchers should evaluate both mRNA levels (via qRT-PCR) and protein expression (via Western blot and flow cytometry). Additionally, confirming alterations in enzymatic activity using fluorogenic substrates provides functional validation of genetic manipulations .

What experimental controls are crucial when studying DPP4 inhibition in human systems?

When investigating DPP4 inhibition, several critical controls must be incorporated:

• Specificity controls:

  • Include selective inhibitors (e.g., sitagliptin) alongside non-selective ones

  • Monitor activity of related peptidases (DPP8, DPP9, FAP) to confirm specificity

  • Use DPP4-knockout or knockdown models as negative controls

• Pharmacological considerations:

  • Implement dose-response studies to establish IC50 values

  • Monitor inhibitor stability throughout the experimental timeframe

  • Include washout periods to assess reversibility of inhibition

• Functional validation:

  • Measure multiple DPP4 substrates (e.g., GLP-1, CXCL10) to comprehensively assess inhibition

  • Evaluate both membrane-bound and soluble DPP4 activities separately

  • Quantify downstream signaling effects (e.g., insulin secretion for incretin studies)

• Temporal controls:

  • Assess acute versus chronic inhibition effects

  • Consider circadian variations in DPP4 expression and activity

For clinical studies, placebo controls and measurement of plasma drug concentrations ensure proper interpretation of observed effects on DPP4 activity .

What is the relationship between DPP4 activity and incretin hormone regulation in humans?

DPP4 serves as a critical regulator of incretin hormone bioactivity in humans. The incretin hormones, primarily glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), are released from intestinal L and K cells respectively following nutrient ingestion. These hormones stimulate glucose-dependent insulin secretion, but their active forms have extremely short half-lives (1-2 minutes) due to rapid degradation by DPP4.

DPP4 selectively cleaves the N-terminal dipeptides from these hormones, converting active GLP-1(7-36) to inactive GLP-1(9-36) and active GIP(1-42) to inactive GIP(3-42). This enzymatic inactivation is the primary mechanism controlling the duration of incretin action and, consequently, post-prandial insulin secretion. In physiological conditions, this creates a tightly regulated system for controlling glucose homeostasis.

When DPP4 is pharmacologically inhibited (e.g., with sitagliptin), the half-life of active incretin hormones increases substantially, enhancing and prolonging their insulinotropic effects. This mechanism forms the basis for DPP4 inhibitors as antidiabetic medications. Importantly, the selectivity of DPP4 inhibition preserves the glucose-dependent nature of incretin action, minimizing hypoglycemia risk compared to other insulin secretagogues .

How does DPP4 expression in adipose tissue influence human metabolism?

DPP4 expression in human adipose tissue plays significant roles in metabolic regulation through multiple mechanisms:

• Adipocyte differentiation: DPP4 is highly expressed in both human preadipocytes and mature adipocytes. Experimental DPP4 knockdown in preadipocytes significantly alters gene expression patterns, increasing metabolic genes (PDK4 by 18-fold and PPARγC1α by 6-fold) while decreasing proliferation-related genes. This indicates that DPP4 regulates key steps in adipocyte maturation.

• Insulin signaling modulation: DPP4 knockdown markedly diminishes basal and insulin-induced ERK activation (by approximately 60%) while leaving Akt activation unaffected. This selective impact on signaling pathways suggests DPP4 contributes to adipocyte insulin sensitivity and metabolic programming.

• Adipokine function: DPP4 has been characterized as an adipokine itself, with its release increasing substantially during adipocyte differentiation. This suggests mature adipose tissue may be a significant contributor to circulating soluble DPP4.

• Inflammatory crosstalk: DPP4 levels in adipose tissue correlate with parameters of the metabolic syndrome, potentially linking adipose inflammation with systemic metabolic dysregulation.

These findings indicate that adipose-derived DPP4 may constitute a mechanistic link between obesity and metabolic disorders, potentially through both enzymatic actions on metabolic peptides and non-enzymatic signaling functions .

What are the differential effects of pharmacological versus genetic inhibition of DPP4 in humans?

The comparison between pharmacological and genetic DPP4 inhibition reveals important differences:

Pharmacological Inhibition:

• Clinical DPP4 inhibitors (gliptins) typically achieve 70-90% inhibition of enzymatic activity

• Effects are reversible and dose-dependent

• In clinical studies with sitagliptin (100 mg daily), DPP4 activity is significantly reduced without increasing inflammatory markers or sDPP4 levels over 12 months

• The inhibitory effects primarily target the catalytic site without affecting protein expression levels initially

• Prolonged DPP4 inhibition in mice paradoxically increases plasma levels of sDPP4

Genetic Inhibition/Knockout:

• Complete elimination of DPP4 activity

• Affects both enzymatic function and potential non-catalytic protein interactions

• DPP4 knockout mice show improved glycemic control and reduced fat mass

• Experimental genetic knockdown (e.g., lentiviral shRNA) in preadipocytes reveals roles in regulating differentiation genes independent of PPARγ pathways

These differences highlight the complexity of DPP4 biology, where complete absence (genetic) versus catalytic inhibition (pharmacological) may trigger distinct compensatory mechanisms or reveal separate functions of the protein. When designing studies, researchers should carefully consider whether their questions pertain to enzyme activity, protein interactions, or both .

How do conformational dynamics impact DPP4 enzymatic activity?

The enzymatic activity of DPP4 is intricately linked to its conformational dynamics, as revealed by advanced structural and molecular studies:

DPP4 functions as a dimer, and mutations disrupting dimerization, such as V486M, abolish enzymatic activity despite being distal from the catalytic site. Molecular dynamics simulations have identified a critical "flap" region (β-propeller loop, residues 234-260) that undergoes "open/closed" conformational transitions while capping the active site. These dynamic movements are essential for substrate binding and catalysis.

The V486M mutation induces a local conformational collapse in this flap region, disrupting the dimerization interface and subsequently abolishing enzymatic activity. This reveals that proper positioning of the catalytic triad depends on broader conformational stability established through dimerization.

The glycosylation pattern of DPP4 further influences these dynamics. Six of the nine N-glycosylation sites are located within the glycosylated region and are highly conserved across species. These modifications are crucial not only for proper folding and stability but also for maintaining the conformational flexibility required for catalytic function.

Together, these findings suggest that DPP4 inhibitor design should consider not only direct active site interactions but also potential allosteric mechanisms that could disrupt these essential conformational dynamics .

What role does DPP4 play in human inflammation and immune regulation?

DPP4 serves as a multifunctional regulator at the interface of metabolism and immunity:

DPP4 proteolytically processes multiple chemokines, including CXCL10, which plays a critical role in T-cell trafficking and inflammation. Specifically, DPP4 truncates CXCL10 from its bioactive CXCL10(1-77) form to an antagonistic CXCL10(3-77) form. Clinical trials with sitagliptin have demonstrated that DPP4 inhibition preserves the bioactive form of CXCL10 in vivo, highlighting a direct immunomodulatory effect.

Beyond its enzymatic activity, DPP4 (also known as CD26) functions as a T-cell co-stimulatory molecule. It binds adenosine deaminase (ADA), forming a complex that enhances T-cell activation through interaction with CD45. Importantly, this immunoregulatory function can occur independent of DPP4's enzymatic activity.

The DPP4-ADA interaction on dendritic cells may potentiate inflammation in obesity by promoting T-cell activation and proliferation. This interaction can be competitively inhibited by exogenous soluble DPP4, but not by inhibiting its enzymatic function, underscoring the distinction between enzymatic and non-enzymatic roles.

DPP4 also interacts with Caveolin-1 on antigen-presenting cells, binding to specific residues (630 and 201-211) of DPP4 on T-cells. This triggers CD68 upregulation and initiates signaling cascades potentially implicated in inflammatory diseases like arthritis.

These findings highlight DPP4 as a therapeutic target beyond diabetes, with potential applications in inflammatory and immune-mediated conditions .

How is soluble DPP4 regulated differently from membrane-bound DPP4 in various disease states?

The differential regulation of soluble DPP4 (sDPP4) versus membrane-bound DPP4 in disease states reveals complex biology:

Regulatory Divergence:
Prolonged DPP4 inhibition paradoxically increases plasma levels of sDPP4 in mice, while inducing sDPP4 expression in lymphocyte-enriched organs. Bone marrow transplantation experiments identified hematopoietic cells as the predominant source of plasma sDPP4 following catalytic DPP4 inhibition. This suggests distinct regulatory mechanisms for the membrane-bound versus soluble forms.

Disease-Specific Patterns:
In type 2 diabetes, levels of sDPP4 can correlate with parameters of metabolic syndrome, but with considerable inter-individual variation. Metformin-treated subjects with T2D and cardiovascular disease show lower plasma levels of sDPP4 and inflammatory markers, indicating therapeutic modulation.

In chronic hepatitis C, DPP4 levels are elevated, affecting CXCL10 processing. Sitagliptin treatment in these patients demonstrates restoration of bioactive CXCL10(1-77) levels, providing functional evidence of DPP4's role in this disease context.

Methodological Considerations:
The dissociation between DPP4 enzyme activity, sDPP4 levels, and inflammatory biomarkers in both mice and humans underscores the need for comprehensive assessment in clinical studies. Researchers must measure both enzymatic activity and protein levels, as these parameters can change independently.

For optimal experimental design, measurement of sDPP4 should utilize sensitive ELISA methods, while DPP4 activity requires functional enzymatic assays. Assessment of substrates like CXCL10 should differentiate between intact and truncated forms to accurately reflect in vivo processing .

What cutting-edge techniques are advancing the study of DPP4 protein interactions?

Recent methodological innovations have significantly enhanced our understanding of DPP4 protein interactions:

Structural and Conformational Analysis:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) enables mapping of protein-protein interaction surfaces and conformational changes upon binding

• Cryo-electron microscopy offers higher-resolution visualization of DPP4 complexes in near-native states

• Single-molecule FRET (smFRET) techniques allow real-time observation of conformational dynamics in DPP4 dimers

Interaction Proteomics:

• Proximity labeling methods (BioID, APEX) identify proteins in close proximity to DPP4 in living cells

• Crosslinking mass spectrometry (XL-MS) captures transient interactions and maps binding interfaces

• Thermal proteome profiling detects protein complex stability changes upon DPP4 inhibitor binding

Functional Interaction Analysis:

• CRISPR-based genetic screens identify functional dependencies between DPP4 and other proteins

• Microfluidic techniques coupled with time-resolved proteomics enable dynamic studies of DPP4 interactions

• Nanobody-based probes provide tools for targeting specific conformational states of DPP4

These advanced methods have revealed previously unappreciated interaction partners beyond the well-characterized binding with adenosine deaminase, caveolin-1, and mannose-6-phosphate/IGF-II receptor, opening new avenues for therapeutic targeting .

What biomarker strategies can assess DPP4 activity in human clinical samples?

Effective biomarker strategies for assessing DPP4 activity in human clinical samples include:

Direct Activity Measurements:

• Fluorogenic substrate assays using selective DPP4 substrates (Gly-Pro-AMC) in plasma or serum

• Competitive binding assays with labeled DPP4 inhibitors to quantify available active sites

• Mass spectrometry-based quantification of specific DPP4-cleaved peptides in biological fluids

Substrate Processing Biomarkers:

• Ratio of intact-to-cleaved GLP-1 as a functional readout of DPP4 activity

• Ultrasensitive single-molecule assays (Simoa) to distinguish full-length CXCL10(1-77) from NH2-truncated CXCL10(3-77)

• Targeted metabolomics panels measuring multiple DPP4 substrates simultaneously

Integrated Biomarker Approaches:

• Combined measurement of DPP4 protein levels, enzymatic activity, and substrate processing

• Assessment of activity in different compartments (plasma, cellular fractions)

• Longitudinal sampling to capture dynamic changes and individual variability

When implementing these strategies, researchers should consider:

• Standardized sampling conditions (fasting status, time of day)

• Immediate sample processing to prevent ex vivo activity

• Inclusion of appropriate controls for potential interfering factors

• Validation against pharmacological inhibition

These approaches enable more precise characterization of DPP4 activity in diverse clinical contexts, improving interpretation of findings across studies .

How can researchers design experiments to distinguish enzymatic from non-enzymatic functions of DPP4?

Distinguishing enzymatic from non-enzymatic functions of DPP4 requires strategic experimental design:

Comparative Inhibition Approaches:

• Utilize catalytic site inhibitors (sitagliptin, saxagliptin) alongside agents that block protein-protein interactions

• Compare effects of enzymatically inactive DPP4 mutants (e.g., S630A) with wild-type protein

• Design experiments comparing DPP4 knockout/knockdown with selective inhibition of enzymatic activity

Interaction-Specific Interventions:

• Use competitive peptides or antibodies that specifically block known interaction domains (e.g., ADA-binding region)

• Generate truncated DPP4 variants lacking specific domains while preserving catalytic function

• Employ site-directed mutagenesis targeting interaction interfaces versus catalytic residues

Functional Readouts:

• Simultaneously measure enzymatic activity (substrate cleavage) and signaling outcomes

• Utilize specific substrates that can only be processed by DPP4 enzymatic activity

• Examine T-cell activation or cytokine production as readouts of non-enzymatic immunomodulatory functions

Experimental Models:

• Compare effects in cell types where DPP4 primarily functions enzymatically versus cells where it serves as a surface receptor

• Utilize reconstitution experiments in DPP4-knockout systems with selective function-restoring constructs

• Develop proximity-based reporters that specifically detect protein-protein interactions versus substrate binding

This multifaceted approach can reveal the relative contributions of enzymatic and non-enzymatic functions in specific biological contexts, informing more precise therapeutic strategies targeting distinct DPP4 functions .

What methodological challenges persist in studying DPP4 in human tissues?

Despite significant advances, several methodological challenges continue to hinder comprehensive understanding of DPP4 biology in human tissues:

• Tissue heterogeneity: Human tissues contain diverse cell populations with varying DPP4 expression levels and functions. Current techniques often fail to capture this cellular heterogeneity, particularly in complex tissues like pancreatic islets and adipose tissue.

• Distinguishing membrane-bound from soluble activity: Standard assays typically measure total DPP4 activity without differentiating between membrane-bound and soluble forms, which may have distinct biological roles.

• Substrate specificity overlap: DPP4 shares substrate preferences with related peptidases (DPP8, DPP9, FAP), complicating attribution of observed effects to specific enzymes in complex samples.

• Post-translational modifications: The functional impact of tissue-specific glycosylation, sialylation, and other modifications on DPP4 activity remains poorly characterized.

• Temporal dynamics: Current methodologies provide static snapshots rather than dynamic information about DPP4 regulation and activity over time.

Future methodological innovations should focus on:

• Single-cell approaches to resolve cell-type specific DPP4 expression and function

• Live-cell imaging techniques to visualize DPP4 trafficking and activity in real-time

• Development of highly selective substrate-based probes for specific DPP family members

• Advanced glycoproteomic approaches to characterize tissue-specific post-translational modifications .

How might therapeutic targeting of DPP4 evolve beyond diabetes applications?

The therapeutic potential of DPP4 targeting extends well beyond diabetes management:

Inflammatory and Immune Disorders:
Based on DPP4's role in CXCL10 processing and T-cell activation, DPP4 inhibitors show promise for conditions characterized by dysregulated inflammation. Clinical evidence demonstrates that DPP4 inhibition preserves bioactive CXCL10(1-77), potentially modulating T-cell trafficking in diseases like chronic hepatitis C, rheumatoid arthritis, and inflammatory bowel disease.

Cardiovascular Applications:
The relationship between DPP4 activity and cardiovascular outcomes suggests potential for targeted intervention. Metformin-treated patients with type 2 diabetes and cardiovascular disease show lower plasma levels of sDPP4 and inflammatory markers, indicating therapeutic modulation may provide cardioprotective benefits.

Cancer Therapeutics:
DPP4's roles in cell adhesion, migration, and proliferation suggest applications in oncology. Emerging research indicates that inhibition of DPP4-mediated signaling may suppress tumor growth in certain malignancies, potentially offering complementary approaches to conventional treatments.

Obesity Management:
The expression of DPP4 in adipose tissue and its influence on adipocyte differentiation present opportunities for metabolic intervention. DPP4 knockdown increases expression of metabolic genes while decreasing proliferation-related genes, suggesting potential for adipose tissue remodeling.

Future therapeutic development should focus on:

• Selective targeting of specific DPP4 functions (enzymatic vs. protein-protein interactions)

• Tissue-specific delivery systems to enhance efficacy and reduce off-target effects

• Combination approaches targeting multiple aspects of DPP4 biology

• Biomarker-guided patient selection to identify those most likely to benefit from DPP4-targeted therapies .

What are the most promising research directions for understanding DPP4 structure-function relationships?

Several research directions hold particular promise for advancing our understanding of DPP4 structure-function relationships:

Allosteric Regulation Mechanisms:
The discovery that distal mutations like V486M can abolish enzymatic activity by inducing conformational changes highlights the importance of allosteric regulation. Future research should focus on mapping these allosteric networks using hydrogen-deuterium exchange mass spectrometry and computational approaches to identify potential targetable sites beyond the catalytic pocket.

Dynamic Conformational Landscape:
The "open/closed" transitions of the flap region (residues 234-260) that caps the active site appear critical for enzymatic function. Advanced techniques like single-molecule FRET and time-resolved crystallography could characterize these dynamics under various conditions, potentially revealing new regulatory mechanisms.

Post-Translational Modification Patterns:
The extensive glycosylation of DPP4 (six of nine N-glycosylation sites in the glycosylated region) likely influences both stability and function. Comparative glycoproteomics across tissues and disease states may reveal how these modifications regulate DPP4 activity and interactions in context-specific ways.

Membrane Microenvironment Interactions:
As a transmembrane protein, DPP4 function may be regulated by lipid composition and membrane microdomains. Investigation of how membrane context influences DPP4 dimerization, substrate accessibility, and shedding could reveal new regulatory mechanisms.

Structure-Based Rational Design:
Integration of these insights into computational models would enable rational design of next-generation DPP4 modulators with improved specificity, novel modes of action (e.g., preventing dimerization), or selective targeting of specific DPP4 functions while preserving others.

These research directions promise not only to expand our fundamental understanding of DPP4 biology but also to inform development of more sophisticated therapeutic strategies targeting this multifunctional protein .

What are the key unresolved questions in human DPP4 research?

Despite significant advances in understanding DPP4 biology, several fundamental questions remain unresolved:

• The precise molecular mechanisms controlling DPP4 shedding from cell membranes and the physiological significance of the balance between membrane-bound and soluble forms

• The complete repertoire of endogenous DPP4 substrates in humans and their relative physiological importance across different tissues and disease states

• The structural basis for selectivity between DPP4 and closely related peptidases (DPP8, DPP9, FAP) and how this could be exploited for more targeted therapeutic approaches

• The significance of DPP4's non-catalytic protein-protein interactions in human physiology and pathophysiology, particularly in immune regulation and metabolic signaling

• The integrative role of DPP4 at the intersection of metabolism, inflammation, and cardiovascular function, especially how these functions are coordinated across different tissues

• The potential long-term consequences of pharmacological DPP4 inhibition on immune function, inflammatory responses, and tissue remodeling

• The mechanism by which prolonged DPP4 inhibition paradoxically increases soluble DPP4 levels and whether this has clinical implications

Addressing these questions will require interdisciplinary approaches combining structural biology, systems pharmacology, clinical investigation, and advanced computational modeling .

What emerging technologies may transform our understanding of DPP4 biology?

Several emerging technologies hold transformative potential for DPP4 research:

Spatial Multi-omics:
Integration of spatial transcriptomics, proteomics, and metabolomics will allow visualization of DPP4 expression, activity, and substrate processing with cellular resolution in intact tissues. This approach will reveal cell type-specific functions and local microenvironmental influences on DPP4 biology.

AI-Driven Structural Prediction:
Advanced protein structure prediction algorithms (like AlphaFold) coupled with molecular dynamics simulations will enable more accurate modeling of DPP4 conformational states, protein-protein interactions, and allosteric regulation networks.

CRISPR-Based Functional Genomics:
Large-scale CRISPR screens targeting genes involved in DPP4 regulation, trafficking, and signaling will identify new molecular players in DPP4 biology and potential therapeutic targets.

Organ-on-Chip Technology:
Microfluidic organ-on-chip platforms incorporating multiple tissue types will enable study of DPP4's systemic effects across interconnected physiological systems, better mimicking whole-body physiology.

Nanobody-Based Imaging Probes:
Development of conformation-specific nanobodies against DPP4 will allow real-time visualization of DPP4 structural states in living cells and tissues, connecting structural dynamics to function.

Synthetic Biology Approaches: Engineered cellular systems with controllable DPP4 variants will enable precise dissection of structure-function relationships and testing of hypotheses about allosteric regulation and protein-protein interactions.