mapping-dysfunctional-protein-protein-interactions-in-disease

Mapping Dysfunctional Protein-Protein Interactions in Disease

Protein-protein interaction (PPI) networks are dynamically remodeled in disease, yet most systems biology approaches focus on changes in protein ...

Memorial Sloan Kettering Cancer Center

Despite recent advances in the treatment of acute myeloid leukemia (AML), there has been limited success in targeting surface antigens in AML, in part due to shared expression across malignant and normal cells. Here, high-density immunophenotyping of AML coupled with proteogenomics identified unique expression of a variety of antigens, including the RNA helicase U5 snRNP200, on the surface of AML cells but not on normal hematopoietic precursors and skewed Fc receptor distribution in the AML immune microenvironment. Cell membrane localization of U5 snRNP200 was linked to surface expression of the Fcγ receptor IIIA (FcγIIIA, also known as CD32A) and correlated with expression of interferon-regulated immune response genes. Anti-U5 snRNP200 antibodies engaging activating Fcγ receptors were efficacious across immunocompetent AML models and were augmented by combination with azacitidine. These data provide a roadmap of AML-associated antigens with Fc receptor distribution in AML and highlight the potential for targeting the AML cell surface using Fc-optimized therapeutics.

Systematic evaluation of AML-associated antigens identifies anti-U5 SNRNP200 therapeutic antibodies for the treatment of acute myeloid leukemia.

Mutations in the RNA splicing factor gene SF3B1 are common across hematologic and solid cancers and result in widespread alterations in splicing, yet there is currently no therapeutic means to correct this mis-splicing. Here, we utilize synthetic introns uniquely responsive to mutant SF3B1 to identify trans factors required for aberrant mutant SF3B1 splicing activity. This revealed the G-patch domain-containing protein GPATCH8 as required for mutant SF3B1-induced splicing alterations and impaired hematopoiesis. GPATCH8 is involved in quality control of branchpoint selection, interacts with the RNA helicase DHX15, and functionally opposes SURP and G-patch domain containing 1 (SUGP1), a G-patch protein recently implicated in SF3B1-mutant diseases. Silencing of GPATCH8 corrected one-third of mutant SF3B1-dependent splicing defects and was sufficient to improve dysfunctional hematopoiesis in SF3B1-mutant mice and primary human progenitors. These data identify GPATCH8 as a novel splicing factor required for mis-splicing by mutant SF3B1 and highlight the therapeutic impact of correcting aberrant splicing in SF3B1-mutant cancers.

GPATCH8 modulates mutant SF3B1 mis-splicing and pathogenicity in hematologic malignancies.

Recent studies have identified a family of rod-shaped proteins thought to mediate lipid transfer at intracellular membrane contacts by a bridge-like mechanism. We show one such protein, bridge-like lipid transfer protein 3A (BLTP3A)/UHRF1BP1 binds VAMP7 vesicles via its C-terminal region and anchors them to lysosomes via its chorein domain containing N-terminal region to Rab7. Upon lysosome damage, BLTP3A-positive vesicles rapidly (within minutes) dissociate from lysosomes. Lysosome damage is known to activate the CASM (Conjugation of ATG8 to Single Membranes) pathway leading to lipidation and recruitment to lysosomes of mammalian ATG8 (mATG8) proteins. We find that this process drives the reassociation of BLTP3A with damaged lysosomes via an interaction of its LIR motif with mATG8 which coincides with a dissociation from the vesicles. Our findings reveal that BLTP3A is an effector of CASM, potentially as part of a mechanism to help repair or minimize lysosome damage.

BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM.

The fission yeast  regulon genes , , and -encoding a cell surface-associated acid phosphatase (Pho1), a plasma membrane inorganic phosphate transporter (Pho84), and a plasma membrane glycerophosphocholine transporter (Tgp1)-are strongly upregulated in response to acute phosphate starvation, as are the  and  genes that encode putative 5'-nucleotidase paralogs of the binuclear metallophosphoesterase enzyme superfamily. Via proteomic analysis of the medium harvested from phosphate-replete and phosphate-starved fission yeast, we define a starvation secretome that includes SPBPB2B2.06c (renamed Efn1, for xtracellular ive-prime ucleotidase), SPAC1039.02 (henceforth Efn2), and Pho1 among the most abundant exported proteins elaborated by phosphate-starved cells. We demonstrate and characterize a 5'-nucleotidase activity secreted into the medium of phosphate-starved  cells, which is eliminated by simultaneous deletion of  and . By singly deleting  and , we find that Efn1 contributes the greater share of secreted 5'-nucleotidase activity. Efn1 and Efn2 catalyze the release of inorganic phosphate from all four standard ribonucleoside monophosphates, in order of preference: CMP > UMP > AMP > GMP. Whereas  cells can use extracellular CMP as a source of phosphate during phosphate starvation, ∆ ∆ cells cannot. The secretion of 5'-nucleotidase enzymes during phosphate limitation is a newly appreciated facet of fission yeast phosphate homeostasis.

Efn1 and Efn2 are extracellular 5'-nucleotidases induced during the fission yeast response to phosphate starvation.

Alzheimer's disease (AD) progresses as a continuum, from preclinical stages to late-stage cognitive decline, yet the molecular mechanisms driving this progression remain poorly understood. Here, we provide a systems-level map of protein-protein interaction (PPI) network dysfunction across the AD spectrum and uncover epichaperomes-stable scaffolding platforms formed by chaperones and co-factors-as central drivers of this process. Using over 100 human brain specimens, mouse models, and human neurons, we show that epichaperomes emerge early, even in preclinical AD, and progressively disrupt multiple PPI networks critical for synaptic function and neuroplasticity. Glutamatergic neurons, essential for learning and memory, exhibit heightened vulnerability, with their dysfunction driven by protein sequestration into epichaperome scaffolds, independent of changes in protein expression. Notably, pharmacological disruption of epichaperomes with PU-AD restores PPI network integrity and reverses synaptic and cognitive deficits, directly linking epichaperome-driven network dysfunction to AD pathology. These findings establish epichaperomes as key mediators of molecular collapse in AD and identify network-centric intervention strategies as a promising avenue for disease-modifying therapies.

Systems-Level Interactome Mapping Reveals Actionable Protein Network Dysregulation Across the Alzheimer's Disease Spectrum.

We conducted a mouse-human co-clinical trial to evaluate the biological efficacy of exercise therapy in breast cancer prevention.

A Co-clinical Trial of Exercise Therapy in Breast Cancer Prevention.

Tuberculosis remains a globally significant infection and new insights into the biology of  are badly needed. Discovery of protein localization and protein complex composition are powerful approaches to determine protein function, but have not been widely applied in mycobacteria, in part due to technical barriers. Here we develop a multifunctional system that utilizes the ALFA tag and functional protein fusions to an anti-ALFA nanobody (NBALFA) to target proteins in fast and slow growing mycobacteria. Insertion of the ALFA epitope tag on the target protein, coupled with conditional expression of the NBALFA fused to a fluorescent protein faithfully recapitulates cytosolic and membrane protein localization by fluorescent microscopy in living cells. Targeted NBALFA can relocalize an ALFA tagged protein to inclusion bodies or the cytoplasmic membrane, demonstrating enforced protein localization. Finally, conditional expression of the NBALFA fused to TurboID for proximity proteomics allowed identification of known partner proteins of the RNA polymerase complex and the PKS13 mycolic acid biosynthesis protein. We conclude that the split ALFA tag-nanobody system is a flexible platform for discovery protein biology in mycobacteria.

A split ALFA tag-nanobody system for protein localization and proximity proteomics in mycobacteria.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor neuron (MN) loss. The most common genetic cause, a hexanucleotide repeat expansion in C9orf72 (C9-ALS), disrupts microglial function, contributing to neuroinflammation, a key disease driver. To investigate this, we developed a three-dimensional spinal microtissue (SM) model incorporating human induced pluripotent stem cell (hiPSC)-derived MNs, astrocytes, and microglia. Screening 190 Food and Drug Administration (FDA)-approved compounds, we identified sartans-angiotensin II receptor I blockers (ARBs)-as potent inhibitors of neuroinflammation. Telmisartan, a highly brain-penetrant ARB, significantly reduced the levels of pro-inflammatory cytokines interleukin (IL)-6 and IL-8 and rescued MN loss in C9-ALS SMs. Our findings suggest that C9-ALS microglia drive MN toxicity and that telmisartan can effectively mitigate inflammation and preserve MN viability. This work lays the groundwork for modeling disease-related neuroinflammation and points to telmisartan as a therapeutic candidate worth further exploration for treating C9-ALS.

Telmisartan is neuroprotective in a hiPSC-derived spinal microtissue model for C9orf72 ALS via inhibition of neuroinflammation.

Tuberculosis remains a globally significant infection, and new insights into the biology of  are badly needed. Discovery of protein localization and protein complex composition are powerful approaches to determine protein function but have not been widely applied in mycobacteria, in part due to technical barriers. Here we develop a multifunctional system that utilizes the ALFA tag and functional protein fusions to an anti-ALFA nanobody (NBALFA) to target proteins in fast- and slow-growing mycobacteria. Insertion of the ALFA epitope tag on the target protein, coupled with conditional expression of the NBALFA fused to a fluorescent protein, faithfully recapitulates cytosolic and membrane protein localization by fluorescent microscopy in living cells. Targeted NBALFA can relocalize an ALFA-tagged protein to inclusion bodies or the cytoplasmic membrane, demonstrating enforced protein localization. Finally, the conditional expression of the NBALFA fused to TurboID for proximity proteomics allowed the identification of known partner proteins of the RNA polymerase complex and the PKS13 mycolic acid biosynthesis protein. We conclude that the split ALFA tag-nanobody system is a flexible platform for discovering protein biology in mycobacteria.

GAPs (GTPase-activating proteins) and GEFs (guanine nucleotide exchange factors) play key roles in cancer development, but their large number and potential redundancy have limited systematic evaluation. Here we perform unbiased genetic screens to identify GAPs and GEFs with cancer- and lineage-specific requirements, as well as dual perturbation screens to dissect functionally relevant interactors of GAPs and GEFs. Application to primary acute myeloid leukemia (AML) patient specimens uncovers the GAP ARHGAP45 as a targetable dependency shared across cancers of hematopoietic origin while being dispensable in normal hematopoiesis. We demonstrate that targeting ARHGAP45-expressing cells can be achieved through TCR-CAR T cells directed at an ARHGAP45-encoded minor histocompatibility antigen and that pharmacologic targeting of GAPs required upon ARHGAP45 depletion augments ARHGAP45-directed cell therapies. These studies provide a resource for probing oncogenic and druggable regulators of GTPases and strategies to target a GAP that represents a shared dependency across blood cancers.

Systematic evaluation of GAPs and GEFs identifies a targetable dependency for hematopoietic malignancies.

Phenotype switching is a form of cellular plasticity in which cancer cells reversibly move between two opposite extremes: proliferative versus invasive states. Although it has long been hypothesized that such switching is triggered by external cues, the identity of these cues remains unclear. Here we demonstrate that mechanical confinement mediates phenotype switching through chromatin remodelling. Using a zebrafish model of melanoma coupled with human samples, we profiled tumour cells at the interface between the tumour and surrounding microenvironment. Morphological analysis of interface cells showed elliptical nuclei, suggestive of mechanical confinement by the adjacent tissue. Spatial and single-cell transcriptomics demonstrated that interface cells adopted a gene program of neuronal invasion, including the acquisition of an acetylated tubulin cage that protects the nucleus during migration. We identified the DNA-bending protein HMGB2 as a confinement-induced mediator of the neuronal state. HMGB2 is upregulated in confined cells, and quantitative modelling revealed that confinement prolongs the contact time between HMGB2 and chromatin, leading to changes in chromatin configuration that favour the neuronal phenotype. Genetic disruption of HMGB2 showed that it regulates the trade-off between proliferative and invasive states, in which confined HMGB2 tumour cells are less proliferative but more drug-resistant. Our results implicate the mechanical microenvironment as a mechanism that drives phenotype switching in melanoma.

Mechanical confinement governs phenotypic plasticity in melanoma.

Recent studies have identified a family of rod-shaped proteins thought to mediate lipid transfer at intracellular membrane contacts by a bridge-like mechanism. We show that one such protein, bridge-like lipid transfer protein 3A (BLTP3A)/UHRF1BP1 binds VAMP7 vesicles via its C-terminal region, and anchors them to lysosomes via its chorein domain-containing N-terminal region binding to Rab7. Upon lysosome damage, BLTP3A-positive vesicles rapidly (within minutes) dissociate from lysosomes. Lysosome damage is known to activate the CASM (Conjugation of ATG8 to Single Membranes) pathway, leading to lipidation and lysosomal recruitment of mammalian ATG8 (mATG8) proteins. We find that this process drives the reassociation of BLTP3A with damaged lysosomes via an interaction of its LIR motif with mATG8 which coincides with a dissociation from the vesicles. Our findings reveal that BLTP3A is an effector of CASM, potentially as part of a mechanism to help repair or minimize lysosome damage.

Article	Year
Systematic evaluation of AML-associated antigens identifies anti-U5 SNRNP200 therapeutic antibodies for the treatment of acute myeloid leukemia. Nature cancer\| PubMed ID: 37872381	2023
GPATCH8 modulates mutant SF3B1 mis-splicing and pathogenicity in hematologic malignancies. Molecular cell\| PubMed ID: 38688280	2024
BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM. bioRxiv : the preprint server for biology\| PubMed ID: 39386594	2025
Efn1 and Efn2 are extracellular 5'-nucleotidases induced during the fission yeast response to phosphate starvation. mBio\| PubMed ID: 39660919	2025
Systems-Level Interactome Mapping Reveals Actionable Protein Network Dysregulation Across the Alzheimer's Disease Spectrum. Research square\| PubMed ID: 39989971	2025
A Co-clinical Trial of Exercise Therapy in Breast Cancer Prevention. Clinical cancer research : an official journal of the American Association for Cancer Research\| PubMed ID: 40184238	2025
A split ALFA tag-nanobody system for protein localization and proximity proteomics in mycobacteria. bioRxiv : the preprint server for biology\| PubMed ID: 40196699	2025
Telmisartan is neuroprotective in a hiPSC-derived spinal microtissue model for C9orf72 ALS via inhibition of neuroinflammation. Stem cell reports\| PubMed ID: 40541176	2025
A split ALFA tag-nanobody system for protein localization and proximity proteomics in mycobacteria. mBio\| PubMed ID: 40576343	2025
Systematic evaluation of GAPs and GEFs identifies a targetable dependency for hematopoietic malignancies. Cancer discovery\| PubMed ID: 40788260	2025
Mechanical confinement governs phenotypic plasticity in melanoma. Nature\| PubMed ID: 40866703	2025
BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM. The EMBO journal\| PubMed ID: 40935891	2025

Mara Monetti

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