LZTR1 is a melanoma oncogene that promotes invasion and suppresses apoptosis.

Leucine zipper like transcription regulator 1 (LZTR1) is amplified in acral melanomas, is required for melanocytes and melanoma cell proliferation, and it induces anchorage-independent growth, by yet unknown mechanisms. We therefore performed comprehensive studies to identify its activity in melanomas employing proximity biotinylation and co-immunoprecipitation combined with LC-MS/MS proteomics and molecular characterization. The results show that LZTR1 regulates the ubiquitin proteasome system in melanoma cells and also associates with actin-related proteins and actin cytoskeleton organization.

The ERVK3-1 Microprotein Interacts with the HUSH Complex.

Human endogenous retroviruses (hERVs) are noninfectious molecular remnants of ancient exogenous retroviruses that now make up 8% of the human genome. The ubiquitously expressed human locus was recently annotated as encoding a 109-amino acid endogenous retroviral Rec microprotein. However, because this locus was thought to be noncoding until recently, it is currently unknown whether the ERVK3-1 microprotein has a function in human cells.

Nuclear PHGDH regulates macrophage polarization through transcriptional repression of and in breast cancer.

Objective: Tumor-associated macrophages (TAMs) exhibit heterogeneous properties including anti-tumorigenic and pro-tumorigenic phenotypes. The rate-limiting enzyme in serine biosynthesis, 3-phosphoglycerate dehydrogenase (PHGDH), has a well-established role in cellular metabolism, yet its specific role in macrophages remains unknown.

Methods: Metabolomics assays were conducted to assess metabolite composition and dynamics in macrophages.

Cancer cell-derived arginine fuels polyamine biosynthesis in tumor-associated macrophages to promote immune evasion.

Arginine metabolism reshapes the tumor microenvironment (TME) into a pro-tumor niche through complex metabolic cross-feeding among various cell types. However, the key intercellular metabolic communication that mediates the collective effects of arginine metabolism within the TME remains unclear. Here, we reveal that the metabolic interplay between cancer cells and macrophages plays a dominant role in arginine-driven breast cancer progression.

A high-affinity antibody-drug conjugates Actuximab-MMAE for potent and selective targeting of CEACAM5-Positive tumors.

Antibody-drug conjugates (ADCs) represent a promising class of anti-cancer therapy with an increasingly critical role in treating various tumors. They broaden the range of therapeutic targets, enabling the consideration of tumor-associated proteins that are overexpressed but lack well-defined mechanisms. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is a clinically relevant screening marker due to its tumor-specific overexpression, making it an attractive target for ADC development.

Unannotated microprotein EMBOW regulates the interactome and chromatin and mitotic functions of WDR5.

The conserved WD40-repeat protein WDR5 interacts with multiple proteins both inside and outside the nucleus. However, it is currently unclear whether and how the distribution of WDR5 between complexes is regulated. Here, we show that an unannotated microprotein EMBOW (endogenous microprotein binder of WDR5) dually encoded in the human SCRIB gene interacts with WDR5 and regulates its binding to multiple interaction partners, including KMT2A and KIF2A.

Massively parallel knock-in engineering of human T cells.

The efficiency of targeted knock-in for cell therapeutic applications is generally low, and the scale is limited. In this study, we developed CLASH, a system that enables high-efficiency, high-throughput knock-in engineering. In CLASH, Cas12a/Cpf1 mRNA combined with pooled adeno-associated viruses mediate simultaneous gene editing and precise transgene knock-in using massively parallel homology-directed repair, thereby producing a pool of stably integrated mutant variants each with targeted gene editing.

Mapping subcellular localizations of unannotated microproteins and alternative proteins with MicroID.

Proteogenomic identification of translated small open reading frames has revealed thousands of previously unannotated, largely uncharacterized microproteins, or polypeptides of less than 100 amino acids, and alternative proteins (alt-proteins) that are co-encoded with canonical proteins and are often larger. The subcellular localizations of microproteins and alt-proteins are generally unknown but can have significant implications for their functions. Proximity biotinylation is an attractive approach to define the protein composition of subcellular compartments in cells and in animals.

Phosphorylation of a Human Microprotein Promotes Dissociation of Biomolecular Condensates.

Proteogenomic identification of translated small open reading frames in humans has revealed thousands of microproteins, or polypeptides of fewer than 100 amino acids, that were previously invisible to geneticists. Hundreds of microproteins have been shown to be essential for cell growth and proliferation, and many regulate macromolecular complexes. However, the vast majority of microproteins remain functionally uncharacterized, and many lack secondary structure and exhibit limited evolutionary conservation.

Alt-RPL36 downregulates the PI3K-AKT-mTOR signaling pathway by interacting with TMEM24.

Thousands of human small and alternative open reading frames (smORFs and alt-ORFs, respectively) have recently been annotated. Many alt-ORFs are co-encoded with canonical proteins in multicistronic configurations, but few of their functions are known. Here, we report the detection of alt-RPL36, a protein co-encoded with human RPL36.

Discovery of cellular substrates of human RNA-decapping enzyme DCP2 using a stapled bicyclic peptide inhibitor.

DCP2 is an RNA-decapping enzyme that controls the stability of human RNAs that encode factors functioning in transcription and the immune response. While >1,800 human DCP2 substrates have been identified, compensatory expression changes secondary to genetic ablation of DCP2 have complicated a complete mapping of its regulome. Cell-permeable, selective chemical inhibitors of DCP2 could provide a powerful tool to study DCP2 specificity.

The NBDY Microprotein Regulates Cellular RNA Decapping.

Proteogenomic identification of translated small open reading frames in humans has revealed thousands of microproteins, or polypeptides of fewer than 100 amino acids, that were previously invisible to geneticists. Hundreds of microproteins have been shown to be essential for cell growth and proliferation, and many regulate macromolecular complexes. One such regulatory microprotein is NBDY, a 68-amino acid component of the human cytoplasmic RNA decapping complex.

Comparative Proteomic Profiling of Unannotated Microproteins and Alternative Proteins in Human Cell Lines.

Ribosome profiling and mass spectrometry have revealed thousands of small and alternative open reading frames (sm/alt-ORFs) that are translated into polypeptides variously termed as microproteins and alt-proteins in mammalian cells. Some micro-/alt-proteins exhibit stress-, cell-type-, and/or tissue-specific expression; understanding this regulated expression will be critical to elucidating their functions. While differential translation has been inferred by ribosome profiling, quantitative mass spectrometry-based proteomics is needed for direct comparison of microprotein and alt-protein expression between samples and conditions.

MSN-on-a-Chip: Cell-Based Screenings Made Possible on a Small-Molecule Microarray of Native Natural Products.

Standard small-molecule microarrays (SMMs) are not well-suited for cell-based screening assays. Of the few attempts made thus far to render SMMs cell-compatible, all encountered major limitations. Here we report the first mesoporous silica nanoparticle (MSN)-on-a-chip platform capable of allowing high-throughput cell-based screening to be conducted on SMMs.

P-Bodies: Composition, Properties, and Functions.

Processing bodies (P-bodies) are cytoplasmic ribonucleoprotein (RNP) granules primarily composed of translationally repressed mRNAs and proteins related to mRNA decay, suggesting roles in post-transcriptional regulation. P-bodies are conserved in eukaryotic cells and exhibit properties of liquid droplets. However, the function of P-bodies in translational repression and/or mRNA decay remains contentious.

Protein-Protein Interaction Inhibitors of BRCA1 Discovered Using Small Molecule Microarrays.

Microarray screening technology has transformed the life sciences arena over the last decade. The platform is widely used in the area of mapping interaction networks, to molecular fingerprinting and small molecular inhibitor discovery. The technique has significantly impacted both basic and applied research.

Accelerated cellular on- and off-target screening of bioactive compounds using microarrays.

In situ proteome labeling was carried out with 9 drug-like probes in live mammalian cells, with the corresponding cellular targets captured on microarrays and simultaneously screened using a diverse set of antibodies, revealing potential on- and off-targets.

Controlled proliferation and screening of mammalian cells on a hydrogel-functionalized small molecule microarray.

A hydrogel-functionalized small molecule microarray has been developed, on which PC-3 cancer cells were selectively grown. Subsequent controlled release of immobilized bioactive compounds enabled cell-based screening to be directly carried out on this platform.

A small-molecule protein-protein interaction inhibitor of PARP1 that targets its BRCT domain.

Poly(ADP-ribose)polymerase-1 (PARP1) is a BRCT-containing enzyme (BRCT = BRCA1 C-terminus) mainly involved in DNA repair and damage response and a validated target for cancer treatment. Small-molecule inhibitors that target the PARP1 catalytic domain have been actively pursued as anticancer drugs, but are potentially problematic owing to a lack of selectivity. Compounds that are capable of disrupting protein-protein interactions of PARP1 provide an alternative by inhibiting its activities with improved selectivity profiles.

Microarray immobilization of biomolecules using a fast trans-cyclooctene (TCO)-tetrazine reaction.

By anchoring 1,2,4,5-tetrazine-containing biomolecules onto trans-cyclooctene (TCO)-functionalized slides, a site-specific microarray immobilization approach is described in this study. Compared with existing immobilization methods, our approach offers several distinctive features, including fast kinetics and high chemoselectivity.

"Minimalist" cyclopropene-containing photo-cross-linkers suitable for live-cell imaging and affinity-based protein labeling.

Target identification of bioactive compounds within the native cellular environment is important in biomedical research and drug discovery, but it has traditionally been carried out in vitro. Information about how such molecules interact with their endogenous targets (on and off) is currently highly limited. An ideal strategy would be one that recapitulates protein-small molecule interactions in situ (e.

Discovery of cell-permeable inhibitors that target the BRCT domain of BRCA1 protein by using a small-molecule microarray.

BRCTs are phosphoserine-binding domains found in proteins involved in DNA repair, DNA damage response and cell cycle regulation. BRCA1 is a BRCT domain-containing, tumor-suppressing protein expressed in the cells of breast and other human tissues. Mutations in BRCA1 have been found in ca.