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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. We apply a constant stretching force to single engineered disulfide bonds and measure their rate of reduction by DTT. Although the reduction rate is linearly dependent on the concentration of DTT, it is exponentially dependent on the applied force, increasing 10-fold over a 300-pN range. This result predicts that the disulfide bond lengthens by 0.34 Å at the transition state of the thiol/disulfide exchange reaction. Our work at the single bond level directly demonstrates that thiol/disulfide exchange in proteins is a force-dependent chemical reaction. Our findings suggest that mechanical force plays a role in disulfide reduction in vivo, a property that has never been explored by traditional biochemistry. Furthermore, our work also indicates that the kinetics of any chemical reaction that results in bond lengthening will be force-dependent.

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. Spliceosome modulation is invisible to RNA or protein abundance alone. Transcriptome analysis after PI demonstrates broad-scale intron retention, suggestive of spliceosome interference, as well as specific alternative splicing of protein homeostasis machinery components. These findings lead us to evaluate direct spliceosome inhibition in myeloma, which synergizes with carfilzomib and shows potent anti-tumor activity. Functional genomics and exome sequencing further support the spliceosome as a specific vulnerability in myeloma. Our results propose splicing interference as an unrecognized modality of PI mechanism, reveal additional modes of spliceosome modulation, and suggest spliceosome targeting as a promising therapeutic strategy in myeloma. The mechanisms of action of proteasome inhibitors (PI) in multiple myeloma (MM) treatment are not fully elucidated. Here, the authors use unbiased phosphoproteomics in PI-treated MM and show increased phosphorylation of splicing-associated proteins, ultimately revealing splicing interference as a mode of PI action as well as demonstrating the spliceosome as a specific therapeutic vulnerability in this disease.

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. This secretome consists of a highly interconnected network enriched in RNA-binding proteins (RBPs) and EV cargoes. Proteomic and RNA profiling of EVs identifies diverse RBPs and small non-coding RNAs requiring the LC3-conjugation machinery for packaging and secretion. Focusing on two RBPs, heterogeneous nuclear ribonucleoprotein K (HNRNPK) and scaffold-attachment factor B (SAFB), we demonstrate that these proteins interact with LC3 and are secreted within EVs enriched with lipidated LC3. Furthermore, their secretion requires the LC3-conjugation machinery, neutral sphingomyelinase 2 (nSMase2) and LC3-dependent recruitment of factor associated with nSMase2 activity (FAN). Hence, the LC3-conjugation pathway controls EV cargo loading and secretion.Leidal et al. show that the LC3-conjugation pathway, which is part of the autophagy machinery, controls extracellular vesicle cargo loading and secretion of RNA-binding proteins.

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. Here, we engineer single disulfide bonds into four different locations of the human cardiac titin module (I27) to control the contour length while keeping the distance to the transition state unchanged. This enables the study of several biologically important parameters. First, we are able to precisely determine the end-to-end length of the transition state before unfolding (53 Angstrom), which is longer than the end-to-end length of the protein obtained from NMR spectroscopy (43 Angstrom). Second, the measured contour length per amino acid from five different methods (4.0 +/- 0.2 Angstrom) is longer than the end-to-end length obtained from the crystal structure (3.6 Angstrom). Our measurement of the contour length takes into account all the internal degrees of freedom of the polypeptide chain, whereas crystallography measures the end-to-end length within the \"frozen\" protein structure. Furthermore, the control of contour length and therefore the number of amino acids unraveled before reaching the disulfide bond (n) facilitates the test of the chain length dependence on the folding time (tau(F)). We find that both a power law scaling tau(F) lambda n(lambda) with lambda = 4.4, and an exponential scaling with n(0.6) fit the data range, in support of different protein-folding scenarios.

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. Here, we uncover that loss of GRASP55 enhances LC3 puncta formation, indicating that GRASP55 restricts autophagosome formation. Additionally, using proximity-dependent biotinylation, we identify a GRASP55 proximal interactome highly associated with the ER-Golgi interface. Both nutrient starvation and loss of GRASP55 are associated with coalescence of early secretory pathway markers. In light of these findings, we propose that GRASP55 regulates spatial organization of the ER-Golgi interface, which suppresses early autophagosome formation.

Many small molecule chemotherapeutics induce stresses that globally inhibit mRNA translation, remodeling the cancer proteome and governing response to treatment. Here we measured protein synthesis in multiple myeloma cells treated with low-dose bortezomib by coupling pulsed-SILAC (pSILAC) with high-accuracy targeted quantitative proteomics. We found that direct measurement of protein synthesis by pSILAC correlated well with the indirect measurement of protein synthesis by ribosome profiling under conditions of robust translation. By developing a statistical model integrating longitudinal proteomic and mRNA-seq measurements, we found that proteomics could directly detect global alterations in translational rate as a function of therapy-induced stress after prolonged bortezomib exposure. Finally, the model we develop here, in combination with our experimental data including both protein synthesis and degradation, predicts changes in proteome remodeling under a variety of cellular perturbations. pSILAC therefore provides an important complement to ribosome profiling in directly measuring proteome dynamics under conditions of cellular stress.

The cell membrane is a complex mixture of various lipids, proteins and other biomolecules that are all organized into a fluid 2-dimensional bilayer. A rather unique trait of this organelle is the lateral mobility of the component molecules. Surprisingly, these molecules are not necessarily distributed homogeneously in the membrane. From a physical perspective, these inhomogeneities are interesting because they indicate some level of organization in the membrane. From a biological perspective, this organization is interesting because it might be a key regulatory element in the enzymatic processes and cell signaling events that occur at the cell membrane. Due to the difficulty of studying membrane organization, not much is known about the spatiotemporal scale of these organized domains, nor is it clear what the physical driving forces are, although there are models based on observations from a variety of different methods. The key factor to overcome in studying membrane organization is the ability to probe the membrane in an informative way that does not perturb the native organization of the membrane. Membrane anchors are lipid moieties covalently conjugated to various membrane proteins and have been implicated in the lateral sorting of anchored proteins in the membrane. Most studies on lipid anchors focus either on identifying what molecules anchored proteins colocalize with or observing how anchored proteins diffuse in the cell membrane. Truncated anchored proteins with just the anchor domain remaining can be genetically fused to fluorescent proteins and also studied to determine the extent to which the anchor-membrane interactions, as opposed to protein-protein interactions, influence their distribution in the membrane. The methods used to study these behaviors are varied and, subsequently, the observations that result from these studies are also varied and the conclusions are conflicting. Time-resolved spectroscopy of fluorescently labeled anchor domains in living cells offers a non-invasive method to extract a wealth of information about the spatiotemporal localization of anchored proteins in a live cell. More specifically, fluorescence cross-correlation spectroscopy (FCCS) analysis and fluorescence lifetime analysis can be derived from the same data stream. In this dissertation, I review the current model of how membranes are organized and present observations made by myself and coworkers, of two instances of homogeneous colocalization of the same anchors but no heterogeneous colocalization of different anchors in Jurkat cell membranes. We conclude that the observation of two distinct non-overlapping domains existing in the same cell membrane indicate a more complex organization than the current model allows.

Fatty liver disease progresses through stages of fat accumulation and inflammation to non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis and eventually hepatocellular carcinoma (HCC). Currently available diagnostic tools for HCC lack sensitivity and specificity and deliver little value to patients. 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. Serum samples were processed using a standard pre-analytical sample preparation protocol and were analyzed using a novel high throughput glycoproteomics platform. We analyzed 413 glycopeptides, representing 57 abundant serum proteins and compared among the three phenotypes. Our initial dataset contained healthy, NASH, and HCC serum samples. We analyzed normalized abundance of common glycoforms and found 40 glycopeptides with statistically significant differences in abundances in NASH and HCC compared to controls. Summary level relative abundance of core-fucosylated, sialylated and branched glycans containing glycopeptides were higher in NASH and HCC as compared to controls. We replicated some of our findings in an independent set of samples of individuals with benign liver conditions and HCC, respectively. Our results may be of value in the management of liver diseases.

Primary tissue organoids and cell spheroids recapitulate tissue physiology with remarkable fidelity. We investigated how engagement with a three dimensional laminin-rich extracellular matrix supports the polarized, stress resilient spheroid phenotype of mammary epithelial cells. Cells within a three dimensional laminin-rich extracellular matrix decreased and redistributed the actin crosslinker filamin to reduce their cortical actin tension. Cells with low cortical actin tension had increased plasma membrane protrusions that promoted negative plasma membrane curvature and fostered protein associations with the plasma membrane, consistent with efficient protein secretion. By contrast, cells engaging a laminin-rich extracellular matrix in two dimensions had high filamin-dependent cortical actin tension, exhibited compromised endoplasmic reticulum function including increased expression of PKR-like Endoplasmic Reticulum Kinase signaling effectors, and had compromised protein secretion. Cells with low filamin-mediated cortical actin tension and reduced endoplasmic reticulum stress response signaling secreted, and assembled, a polarized endogenous basement membrane and survived better, and their spheroids were more resistant to exogenous stress. The findings implicate filamin-dependent cortical actin tension in endoplasmic reticulum function and highlight a role for mechanics in organoid homeostasis.

Enhancing the efficacy of proteasome inhibitors is a central goal in myeloma therapy. We proposed that signaling-level responses after PI would reveal new mechanisms of action that could be therapeutically exploited. Unbiased phosphoproteomics after the PI carfilzomib surprisingly demonstrated the most prominent phosphorylation changes on splicing related proteins. Spliceosome modulation was invisible to RNA or protein abundance alone. Transcriptome analysis after PI demonstrated broad-scale intron retention, suggestive of spliceosome interference, as well as specific alternative splicing of protein homeostasis machinery components. These findings led us to evaluate direct spliceosome inhibition in myeloma, which synergized with carfilzomib and showed potent anti-tumor activity. Functional genomics and exome sequencing further supported the spliceosome as a specific vulnerability in myeloma. Our results propose splicing interference as an unrecognized modality of PI mechanism, reveal additional modes of spliceosome modulation, and suggest spliceosome targeting as a promising therapeutic strategy in myeloma. Footnotes * \"PI inducing IR in MM cells\" section has been updated to clarify. Author list updated. Figure 3 revised, Figure 4 revised, Figure 5E revised, Figure 7D revised. Supplementary figures (S4A-B, S6C-D) and table S2 revised. Methods revised and distributed between main text methods and supplementary text methods.