Transcriptional repression upon S phase entry protects genome integrity in pluripotent cells.

Coincident transcription and DNA replication causes replication stress and genome instability. Rapidly dividing mouse pluripotent stem cells are highly transcriptionally active and experience elevated replication stress, yet paradoxically maintain genome integrity. Here, we study FOXD3, a transcriptional repressor enriched in pluripotent stem cells, and show that its repression of transcription upon S phase entry is critical to minimizing replication stress and preserving genome integrity.

ECM dimensionality tunes actin tension to modulate endoplasmic reticulum function and spheroid phenotypes of mammary epithelial cells.

Patient-derived organoids and cellular spheroids recapitulate tissue physiology with remarkable fidelity. We investigated how engagement with a reconstituted basement membrane in three dimensions (3D) supports the polarized, stress resilient tissue phenotype of mammary epithelial spheroids. Cells interacting with reconstituted basement membrane in 3D had reduced levels of total and actin-associated filamin and decreased cortical actin tension that increased plasma membrane protrusions to promote negative plasma membrane curvature and plasma membrane protein associations linked to protein secretion.

Differential Peripheral Blood Glycoprotein Profiles in Symptomatic and Asymptomatic COVID-19.

Glycosylation is the most common form of post-translational modification of proteins, critically affecting their structure and function. Using liquid chromatography and mass spectrometry for high-resolution site-specific quantification of glycopeptides coupled with high-throughput artificial intelligence-powered data processing, we analyzed differential protein glycoisoform distributions of 597 abundant serum glycopeptides and nonglycosylated peptides in 50 individuals who had been seriously ill with COVID-19 and in 22 individuals who had recovered after an asymptomatic course of COVID-19. As additional comparison reference phenotypes, we included 12 individuals with a history of infection with a common cold coronavirus, 16 patients with bacterial sepsis, and 15 healthy subjects without history of coronavirus exposure.

Serum Glycoprotein Markers in Nonalcoholic Steatohepatitis and Hepatocellular Carcinoma.

Fatty liver disease progresses through stages of fat accumulation and inflammation to nonalcoholic steatohepatitis (NASH), fibrosis and cirrhosis, and eventually hepatocellular carcinoma (HCC). Currently available diagnostic tools for HCC lack sensitivity and specificity. In this study, we investigated the use of circulating serum glycoproteins to identify a panel of potential prognostic markers that may be indicative of progression from the healthy state to NASH and further to HCC.

GRASP55 restricts early-stage autophagy and regulates spatial organization of the early secretory network.

There is great interest in understanding the cellular mechanisms controlling autophagy, a tightly regulated catabolic and stress-response pathway. Prior work has uncovered links between autophagy and the Golgi reassembly stacking protein of 55 kDa (GRASP55), but their precise interrelationship remains unclear. Intriguingly, both autophagy and GRASP55 have been functionally and spatially linked to the endoplasmic reticulum (ER)---Golgi interface, broaching this compartment as a site where GRASP55 and autophagy may intersect.

Proteasome inhibitor-induced modulation reveals the spliceosome as a specific therapeutic vulnerability in multiple myeloma.

Enhancing the efficacy of proteasome inhibitors (PI) is a central goal in myeloma therapy. We proposed that signaling-level responses after PI may reveal new mechanisms of action that can be therapeutically exploited. Unbiased phosphoproteomics after treatment with the PI carfilzomib surprisingly demonstrates the most prominent phosphorylation changes on splicing related proteins.

The LC3-conjugation machinery specifies the loading of RNA-binding proteins into extracellular vesicles.

Traditionally viewed as an autodigestive pathway, autophagy also facilitates cellular secretion; however, the mechanisms underlying these processes remain unclear. Here, we demonstrate that components of the autophagy machinery specify secretion within extracellular vesicles (EVs). Using a proximity-dependent biotinylation proteomics strategy, we identify 200 putative targets of LC3-dependent secretion.

Time-Resolved Proteomics Extends Ribosome Profiling-Based Measurements of Protein Synthesis Dynamics.

Ribosome profiling is a widespread tool for studying translational dynamics in human cells. Its central assumption is that ribosome footprint density on a transcript quantitatively reflects protein synthesis. Here, we test this assumption using pulsed-SILAC (pSILAC) high-accuracy targeted proteomics.

Phosphorylation of β-Tubulin by the Down Syndrome Kinase, Minibrain/DYRK1a, Regulates Microtubule Dynamics and Dendrite Morphogenesis.

Dendritic arborization patterns are consistent anatomical correlates of genetic disorders such as Down syndrome (DS) and autism spectrum disorders (ASDs). In a screen for abnormal dendrite development, we identified Minibrain (MNB)/DYRK1a, a kinase implicated in DS and ASDs, as a regulator of the microtubule cytoskeleton. We show that MNB is necessary to establish the length and cytoskeletal composition of terminal dendrites by controlling microtubule growth.

Dynamic Organization of Myristoylated Src in the Live Cell Plasma Membrane.

The spatial organization of lipid-anchored proteins in the plasma membrane directly influences cell signaling, but measuring such organization in situ is experimentally challenging. The canonical oncogene, c-Src, is a lipid anchored protein that plays a key role in integrin-mediated signal transduction within focal adhesions and cell-cell junctions. Because of its activity in specific plasma membrane regions, structural motifs within the protein have been hypothesized to play an important role in its subcellular localization.

Live cell plasma membranes do not exhibit a miscibility phase transition over a wide range of temperatures.

Lipid/cholesterol mixtures derived from cell membranes as well as their synthetic reconstitutions exhibit well-defined miscibility phase transitions and critical phenomena near physiological temperatures. This suggests that lipid/cholesterol-mediated phase separation plays a role in the organization of live cell membranes. However, macroscopic lipid-phase separation is not generally observed in cell membranes, and the degree to which properties of isolated lipid mixtures are preserved in the cell membrane remain unknown.

Monitoring lipid anchor organization in cell membranes by PIE-FCCS.

This study examines the dynamic co-localization of lipid-anchored fluorescent proteins in living cells using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) and fluorescence lifetime analysis. Specifically, we look at the pairwise co-localization of anchors from lymphocyte cell kinase (LCK: myristoyl, palmitoyl, palmitoyl), RhoA (geranylgeranyl), and K-Ras (farnesyl) proteins in different cell types. In Jurkat cells, a density-dependent increase in cross-correlation among RhoA anchors is observed, while LCK anchors exhibit a more moderate increase and broader distribution.

ATP ground- and transition states of bacterial enhancer binding AAA+ ATPases support complex formation with their target protein, sigma54.

Transcription initiation by the sigma54 form of bacterial RNA polymerase requires hydrolysis of ATP by an enhancer binding protein (EBP). We present SAS-based solution structures of the ATPase domain of the EBP NtrC1 from Aquifex aeolicus in different nucleotide states. Structures of apo protein and that bound to AMPPNP or ADP-BeF(x) (ground-state mimics), ADP-AlF(x) (a transition-state mimic), or ADP (product) show substantial changes in the position of the GAFTGA loops that contact polymerase, particularly upon conversion from the apo state to the ADP-BeF(x) state, and from the ADP-AlF(x) state to the ADP state.

Contour length and refolding rate of a small protein controlled by engineered disulfide bonds.

The introduction of disulfide bonds into proteins creates additional mechanical barriers and limits the unfolded contour length (i.e., the maximal extension) measured by single-molecule force spectroscopy.

Force-dependent chemical kinetics of disulfide bond reduction observed with single-molecule techniques.

The mechanism by which mechanical force regulates the kinetics of a chemical reaction is unknown. Here, we use single-molecule force-clamp spectroscopy and protein engineering to study the effect of force on the kinetics of thiol/disulfide exchange. Reduction of disulfide bonds through the thiol/disulfide exchange chemical reaction is crucial in regulating protein function and is known to occur in mechanically stressed proteins.

Mechanical unfolding intermediates observed by single-molecule force spectroscopy in a fibronectin type III module.

Domain 10 of type III fibronectin (10FNIII) is known to play a pivotal role in the mechanical interactions between cell surface integrins and the extracellular matrix. Recent molecular dynamics simulations have predicted that 10FNIII, when exposed to a stretching force, unfolds along two pathways, each with a distinct, mechanically stable intermediate. Here, we use single-molecule force spectroscopy combined with protein engineering to test these predictions by probing the mechanical unfolding pathway of 10FNIII.

Crystal structures of the BtuF periplasmic-binding protein for vitamin B12 suggest a functionally important reduction in protein mobility upon ligand binding.

BtuF is the periplasmic binding protein (PBP) for the vitamin B12 transporter BtuCD, a member of the ATP-binding cassette (ABC) transporter superfamily of transmembrane pumps. We have determined crystal structures of Escherichia coli BtuF in the apo state at 3.0 A resolution and with vitamin B12 bound at 2.