Biology subjects

Weeks, A. M.

Publications and source records attributed to Weeks, A. M..

11 recordsLinked to original sources

bioRxiv2026

Phage-assisted continuous evolution of enzymes for noncanonical tyrosine biosynthesis

Genetic code expansion introduces new-to-nature chemical moieties into ribosomally synthesized proteins. In practice, the scope of functional groups that can be accessed using this method is often limited by noncanonical amino acid (ncAA) availability. Producing ncAAs directly in cells can circumvent poor ncAA uptake or commercial unavailability, but limited enzymes suitable for this application exist. In vitro evolution campaigns have been remarkably successful in yielding synthetically useful "ncAA synthases." However, these enzymes are optimized for preparative-scale synthesis and their activities often do not translate well to cellular biosynthesis. Thus, expanding strategies to engineer enzymes specifically for ncAA production within cells will benefit further implementation of genetic code expansion. Here, we use phage-assisted noncontinuous and continuous evolution to evolve enzymes for improved synthesis of non-canonical tyrosine derivatives in E. coli. Using simple serial passaging, we uncovered mutations that doubled the production of an expensive ncAA, 3-methoxytyrosine, by tyrosine phenol lyase, and furthermore evolved variants that enable 3-iodotyrosine biosynthesis, a transformation the parent enzyme is unable to catalyze. Additionally, we evolved a recently reported tyrosine synthase for improved production of 3-halogenated tyrosines, identifying variants that exhibit high activity even at low substrate concentrations owing to a [~]8-fold reduction in KM. Our results demonstrate that phage assisted evolution can be used to rapidly improve the activity of enzymes for ncAA production in cells.

synthetic biology↗

bioRxiv2026

Dynamic UFMylation governs cellular fitness by coordinating multi-organelle proteostasis

Ubiquitin-fold modifier 1 (UFM1) is a ubiquitin-like protein (UBL) covalently attached to substrates through a dedicated enzymatic cascade (UFMylation) and removed by specific proteases. Despite a key role in endoplasmic reticulum (ER)-ribosome homeostasis, the basis by which this UBL supports cell fitness remains elusive, as the essentiality of UFMylation machinery varies widely across hundreds of cancer lines. Here, we trace a conditional dependence on the UFMylation pathway to the availability of alanine, an amino acid provided by human plasma-like medium but absent from most conventional synthetic media. We show that by facilitating the clearance of stalled ribosomes at the ER, dynamic UFMylation maintains cellular levels of glutamic-pyruvic transaminase 2 (GPT2), the primary enzyme responsible for de novo alanine synthesis in most human cancer lines. This buffering preserves the alanine pools required to sustain protein synthesis under alanine-restricted conditions. Beyond GPT2, UFM1 deficiency leads to widespread proteomic remodeling that spans diverse processes, including mitochondrial translation. Our results reveal that despite primarily targeting ER-localized ribosomes, the UFMylation system orchestrates a multi-organelle proteostasis network whose client composition and contributions to cell fitness are shaped by intrinsic factors and nutrient conditions.

cell biology↗

bioRxiv2025

Enzymatic bromination of native peptides for late-stage structural diversification via Suzuki-Miyaura coupling

Flavin-dependent halogenases provide a biocatalytic approach for site-selective halogenation of aromatic compounds, but their use in late-stage functionalization of peptides has remained limited. Here, we show that the tryptophan (Trp) 7-halogenase RebH and an engineered variant (4V) originally optimized for larger small-molecule scaffolds can brominate peptidyl-Trp residues across a broad range of sequence and positional contexts. Through extensive analysis of diverse substrate sequences, we define features that enable RebH activity and reveal 4Vs expanded sequence tolerance. We applied 4V for enzymatic bromination of diverse bioactive peptide scaffolds, including an antimicrobial peptide, a cell-penetrating peptide, and a G protein-coupled receptor agonist, without the need for sequence modification. These brominated peptides served as substrates for Suzuki-Miyaura coupling, enabling installation of functional groups that conferred new functional properties or tuned the biological activity of these peptides. Our results expand the substrate landscape of FDHs and establish bromination-enabled cross-coupling as a general approach for late-stage diversification of bioactive peptides.

biochemistry↗

bioRxiv2025

A self-organizing single-cell morphology circuit optimizes Podophrya collini predatory trap structure

Cellular structure self-organizes through an interplay between internal mechanisms and external cues. In predatory ciliates, structures that capture prey obtain the resources needed for their construction, creating feedback between environmental inputs and morphological outputs. Here, we describe a self-organizing single-cell morphology circuit that adaptively optimizes the predatory trap structure of the suctorian Podophrya collini. These trap structures ensnare large cellular targets using an array of straw-like tentacles that siphon out prey cytoplasm upon contact with their tips. We find that trap architecture scales anisotropically, favoring tentacle number over length, to construct traps that maximize capture probability for the resources on hand. Drug perturbations, transcriptomics, proteomics, and expansion microscopy define distinct molecular and structural requirements that regulate trap structure maintenance and tentacle biogenesis. We integrate these findings into a mathematical model that explains the adaptive scaling of the trap and that makes predictions we confirm experimentally. More broadly, this circuits architecture provides a general-purpose control logic for organizing the number and size of sub-cellular structures applicable to other natural and engineered cellular systems.

cell biology↗

bioRxiv2025

Phosphoproteome-derived peptide libraries for deep specificity profiling of phosphatases and phospholyases

Protein phosphorylation is dynamically regulated by the opposing activities of phosphowriter enzymes (kinases) and phosphoeraser enzymes (phosphatases and phospholyases). While significant progress has been made toward defining the sequences preferences of kinases, the selectivity of phosphoerasers has not been explored at scale. Here, we develop an experimental platform based on tandem mass spectrometry analysis of phosphoproteome-derived peptide libraries (PhosPropels) to map phosphoeraser activity across thousands of biologically relevant phosphosites. We extract positional residue preferences to rapidly define sequence motifs recognized by eight phosphoerasers spanning diverse species of origin, protein folds, and enzymatic mechanisms. Taking advantage of the throughput of our approach, we profiled 34 variants of the phosphothreonine lyase OspF from Shigella flexneri, uncovering an intrinsic preference for p38 and Erk MAP kinase activation loops and revealing the enzyme residues that influence its selectivity for phosphothreonine. Our results establish a general method for linking phosphorylation sites to the enzymes that remove them, providing a means to dissect a key component of cellular regulatory networks.

biochemistry↗

bioRxiv2025

Hookworm genes encoding intestinal excreted-secreted proteins are transcriptionally upregulated in response to the host's immune system

Hookworms are intestinal parasitic nematodes that chronically infect [~]500 million people, with reinfection common even after clearance by drugs. How infecting hookworms successfully overcome host protective mechanisms is unclear, but it may involve hookworm proteins that digest host tissues, or counteract the hosts immune system, or both. To find such proteins in the zoonotic hookworm Ancylostoma ceylanicum, we identified hookworm genes encoding excreted-secreted (ES) proteins, hookworm genes preferentially expressed in the hookworm intestine, and hookworm genes whose transcription is stimulated by the host immune system. We collected ES proteins from adult hookworms harvested from hamsters; mass spectrometry identified 565 A. ceylanicum genes encoding ES proteins. We also used RNA-seq to identify A. ceylanicum genes expressed both in young adults (12 days post-infection) and in intestinal and non-intestinal tissues dissected from mature adults (19 days post-infection), with hamster hosts that either had normal immune systems or were immunosuppressed by dexamethasone. In adult A. ceylanicum, we observed 1,670 and 1,196 genes with intestine- and non-intestine-biased expression, respectively. Comparing hookworm gene activity in normal versus immunosuppressed hosts, we observed almost no changes of gene activity in 12-day young adults or non-intestinal 19-day adult tissues. However, in intestinal 19-day adult tissues, we observed 1,951 positively immunoregulated genes (upregulated at least two-fold in normal hosts versus immunosuppressed hosts), and 137 genes that were negatively immunoregulated. Thus, immunoregulation was observed primarily in mature adult hookworm intestine directly exposed to host blood; it may include hookworm genes activated in response to the host immune system in order to neutralize the host immune system. We observed 153 ES genes showing positive immunoregulation in 19-day adult intestine; of these genes, 69 had ES gene homologs in the closely related hookworm Ancylostoma caninum, 24 in the human hookworm Necator americanus, and 24 in the more distantly related strongylid parasite Haemonchus contortus. Such a mixture of rapidly evolving and conserved genes could comprise virulence factors enabling infection, provide new targets for drugs or vaccines against hookworm, and aid in developing therapies for autoimmune diseases.

genomics↗

bioRxiv2024

Engineered reactivity of a bacterial E1-like enzyme enables ATP-driven modification of protein C termini

In biological systems, ATP provides an energetic driving force for peptide bond formation, but protein chemists lack tools that emulate this strategy. Inspired by the eukaryotic ubiquitination cascade, we developed an ATP-driven platform for C-terminal activation and peptide ligation based on E. coli MccB, a bacterial ancestor of ubiquitin-activating (E1) enzymes that natively catalyzes C-terminal phosphoramidate bond formation. We show that MccB can act on non-native substrates to generate an O-AMPylated electrophile that can react with exogenous nucleophiles to form diverse C-terminal functional groups including thioesters, a versatile class of biological intermediates that have been exploited for protein semisynthesis. To direct this activity towards specific proteins of interest, we developed the Thioesterification C-terminal Handle (TeCH)-tag, a sequence that enables high-yield, ATP-driven protein bioconjugation via a thioester intermediate. By mining the natural diversity of the MccB family, we developed two additional MccB/TeCH-tag pairs that are mutually orthogonal to each other and to the E. coli system, facilitating the synthesis of more complex bioconjugates. Our method mimics the chemical logic of peptide bond synthesis that is widespread in biology for high-yield in vitro manipulation of protein structure with molecular precision.

biochemistry↗

bioRxiv2023

An expanded 2-pyridinecarboxaldehyde (2PCA)-based chemoproteomics toolbox for probing protease specificity

Proteomic profiling of protease-generated N termini, or N terminomics, provides key insights into protease function and specificity. However, current N terminomics technologies have sequence limitations or require specialized synthetic reagents for N-terminal peptide isolation. Here, we introduce an expanded N terminomics toolbox that is based on 2-pyridinecarboxaldehyde (2PCA) reagents. These tools enable efficient enrichment of protein N termini by combining selective N-terminal biotinylation using 2PCA reagents with chemically cleavable linkers for N-terminal peptide recovery. By incorporating a commercially available alkyne-modified 2PCA in combination with Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), our strategy eliminates the need for chemical synthesis of N-terminal probes. Using these reagents, we developed PICS2 (Proteomic Identification of Cleavage Sites with 2PCA reagents) to profile the specificity of subtilisin/kexin-type proprotein convertases (PCSKs). We also implemented CHOPPER (Chemical enrichment Of Protease substrates with Purchasable, Elutable Reagents) for global sequencing of apoptotic proteolytic cleavage sites. Based on their broad applicability and ease of implementation, PICS2 and CHOPPER are useful tools that will advance our understanding of protease biology.

biochemistry↗

bioRxiv2021

Hypoxia is a dominant remodeler of the CD8+ T cell surface proteome relative to activation and regulatory T cell-mediated suppression

Immunosuppressive factors in the tumor microenvironment (TME) impair T cell function and limit the anti-tumor immune response. T cell surface receptors that influence interactions and function in the TME are already proven targets for cancer immunotherapy. However, surface proteome remodeling of primary human T cells in response to suppressive forces in the TME has never been characterized systematically. Using a reductionist cell culture approach with primary human T cells and SILAC-based quantitative cell surface capture glycoproteomics, we examined how two immunosuppressive TME factors, regulatory T cells (Tregs) and hypoxia, globally affect the activated CD8+ surface proteome (surfaceome). Surprisingly, the CD8+/Treg co-culture only modestly affected the CD8+ surfaceome, but did reverse several activation-induced surfaceomic changes. In contrast, hypoxia dramatically altered the CD8+ surfaceome in a manner consistent with both metabolic reprogramming and induction of an immunosuppressed state. The CD4+ T cell surfaceome similarly responded to hypoxia, revealing a novel hypoxia-induced surface receptor program. Our findings are consistent with the premise that hypoxic environments create a metabolic challenge for T cell activation, which may underlie the difficulty encountered in treating solid tumors with immunotherapies. Together, the data presented here provide insight into how suppressive TME factors remodel the T cell surfaceome and represent a valuable resource to inform future therapeutic efforts to enhance T cell function in the TME.

immunology↗

bioRxiv2021

Human islet expression levels of Prostaglandin E2 synthetic enzymes, but not prostaglandin EP3 receptor, are positively correlated with markers of β-cell function and mass in non-diabetic obesity

Elevated islet production of prostaglandin E2 (PGE2), an arachidonic acid metabolite, and expression of Prostaglandin E2 Receptor subtype EP3 (EP3) are well-known contributors to the {beta}-cell dysfunction of type 2 diabetes (T2D). Yet, many of the same pathophysiological conditions exist in obesity, and little is known about how the PGE2 production and signaling pathway influences non-diabetic beta-cell function. In this work, plasma arachidonic acid and PGE2 metabolite levels were quantified in a cohort of non-diabetic and T2D human subjects to identify their relationship with glycemic control, obesity, and systemic inflammation. In order to link these findings to processes happening at the islet level, cadaveric human islets were subject to gene expression and functional assays. Interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) mRNA levels, but not those of EP3, positively correlated with donor body mass index (BMI). IL-6 expression also strongly correlated with the expression of COX-2 and other PGE2 synthetic pathway genes. Insulin secretion assays using an EP3-specific antagonist confirmed functionallyrelevant up-regulation of PGE2 production. Yet, islets from obese donors were not dysfunctional, secreting just as much insulin in basal and stimulatory conditions as those from non-obese donors as a percent of content. Islet insulin content, on the other hand, was increased with both donor BMI and islet COX-2 expression, while EP3 expression was unaffected. We conclude up-regulated islet PGE2 production may be part of the {beta}-cell adaption response to obesity and insulin resistance that only becomes dysfunctional when both ligand and receptor are highly expressed in T2D.

pharmacology and toxicology↗

bioRxiv2020

Mapping proteolytic neo-N termini at the surface of living cells

N terminomics is a powerful strategy for profiling proteolytic neo-N termini, but its application to cell surface proteolysis has been limited by the low relative abundance of plasma membrane proteins. Here we apply plasma membrane-targeted subtiligase variants to efficiently and specifically capture cell surface N termini in live cells. Using this approach, we sequenced 807 cell surface N termini and quantified changes in their abundance in response to stimuli that induce proteolytic remodeling of the cell surface proteome. This technology will facilitate greater understanding of extracellular protease biology and reveal neo-N termini biomarkers and targets in disease.

biochemistry↗