Protein N-terminal acetylation (Nt-acetylation) is an important mediator of protein function, stability, sorting, and localization. Although the responsible enzymes are thought to be fairly well characterized, the lack of identified in vivo substrates, the occurrence of Nt-acetylation substrates displaying yet uncharacterized N-terminal acetyltransferase (NAT) specificities, and emerging evidence of posttranslational Nt-acetylation, necessitate the use of genetic models and quantitative proteomics. NatB, which targets Met-Glu-, Met-Asp-, and Met-Asn-starting protein N termini, is presumed to Nt-acetylate 15% of all yeast and 18% of all human proteins. We here report on the evolutionary traits of NatB from yeast to human and demonstrate that ectopically expressed hNatB in a yNatB-Δ yeast strain partially complements the natB -Δ phenotypes and partially restores the yNatB Nt-acetylome. Overall, combining quantitative N-terminomics with yeast studies and knockdown of hNatB in human cell lines, led to the unambiguous identification of 180 human and 110 yeast NatB substrates. Interestingly, these substrates included Met-Gln- N-termini, which are thus now classified as in vivo NatB substrates. We also demonstrate the requirement of hNatB activity for maintaining the structure and function of actomyosin fibers and for proper cellular migration. In addition, expression of tropomyosin-1 restored the altered focal adhesions and cellular migration defects observed in hNatB-depleted HeLa cells, indicative for the conserved link between NatB, tropomyosin, and actin cable function from yeast to human.

Purpose: Knowledge about the mechanism of action (MoA) of monoclonal antibodies (mAbs) is required to understand which multiple myeloma (MM) patients benefit the most from a given mAb, alone or in combination therapy. While there is considerable research about daratumumab, knowledge about other anti-CD38 mAbs remains scarce. Experimental Design: We performed a comprehensive analysis about the MoA of isatuximab. Results: Isatuximab induces internalization of CD38 but not significant release from MM cell' surface. Additionally, we uncovered an association between levels of CD38 expression and different MoA: i) isatuximab was able to induce direct apoptosis on MM cells with very high CD38 but not on MM cells with CD38 levels closer to those in MM patients; ii) isatuximab sensitized CD38^hi MM cells to bortezomib plus dexamethasone in the presence of stroma; iii) antibody-dependent cellular cytotoxicity (ADCC) was triggered by CD38^lo and CD38^hi tumor PCs; iv) antibody-dependent cellular phagocytosis (ADCP) was triggered only by CD38^hi MM cells, whereas; v) complement-dependent cytotoxicity could be triggered in less than half of the patient samples (those with elevated levels of CD38). Furthermore, we showed that isatuximab depletes CD38^hi B-lymphocyte precursors and NK-lymphocytes ex vivo, the latter through activation followed by exhaustion and eventually phagocytosis. Conclusions: This study provides a framework to understand response determinants in patients treated with isatuximab based on the number of MoA triggered by CD38 levels of expression, and for the design of effective combinations aimed at capitalizing disrupted tumor-stroma-cell protection, augmenting NK-lymphocyte mediated ADCC, or facilitating ADCP in CD38^lo MM patients.

Actin stress fibers (SFs) detect and transmit forces to the extracellular matrix through focal adhesions (FAs), and molecules in this pathway determine cellular behavior. Here, we designed two different computational tools to quantify actin SFs and the distribution of actin cytoskeletal proteins within a normalized cellular morphology. Moreover, a systematic cell response comparison between the control cells and those with impaired actin cytoskeleton polymerization was performed to demonstrate the reliability of the tools. Indeed, a variety of proteins that were present within the string beginning at the focal adhesions (vinculin) up to the actin SFs contraction (non-muscle myosin II (NMMII)) were analyzed. Finally, the software used allows for the quantification of the SFs based on the relative positions of FAs. Therefore, it provides a better insight into the cell mechanics and broadens the knowledge of the nature of SFs.

Alpha-synuclein (aSyn) protein levels are sufficient to drive Parkinson's disease (PD) and other synucleinopathies. Despite the biomedical/therapeutic potential of aSyn protein regulation, little is known about mechanisms that limit/control aSyn levels. Here, we investigate the role of a post-translational modification, N-terminal acetylation, in aSyn neurotoxicity. N-terminal acetylation occurs in all aSyn molecules and has been proposed to determine its lipid binding and aggregation capacities; however, its effect in aSyn stability/neurotoxicity has not been evaluated. We generated N-terminal mutants that alter or block physiological aSyn N-terminal acetylation in wild-type or pathological mutant E46K aSyn versions and confirmed N-terminal acetylation status by mass spectrometry. By optical pulse-labeling in living primary neurons we documented a reduced half-life and accumulation of aSyn N-terminal mutants. To analyze the effect of N-terminal acetylation mutants in neuronal toxicity we took advantage of a neuronal model where aSyn toxicity was scored by longitudinal survival analysis. Salient features of aSyn neurotoxicity were previously investigated with this approach. aSyn-dependent neuronal death was recapitulated either by higher aSyn protein levels in the case of WT aSyn, or by the combined effect of protein levels and enhanced neurotoxicity conveyed by the E46K mutation. aSyn N-terminal mutations decreased E46K aSyn-dependent neuronal death both by reducing protein levels and, importantly, by reducing the intrinsic E46K aSyn toxicity, being the D2P mutant the least toxic. Together, our results illustrate that the N-terminus determines, most likely through its acetylation, aSyn protein levels and toxicity, identifying this modification as a potential therapeutic target.

Immunoglobulin light-chain amyloidosis (AL) and multiple myeloma (MM) are two distinct monoclonal gammopathies that involve the same cellular compartment: clonal plasma cells (PCs). Despite knowledge about MM PC biology has significantly increased in the last decade, the same does not apply to AL. Here, we used an integrative phenotypic, molecular and genomic approach to study clonal PCs from 24 newly-diagnosed AL patients. Through principal-component-analysis, we demonstrated highly overlapping phenotypic profiles between AL and both MGUS and MM PCs. However, in contrast to MM, highly-purified FACS-sorted clonal PCs from AL (n=9) showed almost normal transcriptome with only 38 deregulated genes vs. normal PCs; these included a few tumor suppressor (CDH1, RCAN) and pro-apoptotic (GLIPR1, FAS) genes. Notwithstanding, clonal PCs in AL (n=11) were genomically unstable with a median of 9 copy-number-alterations (CNAs) per case, many of such CNA being similar to those found in MM. Whole-exome sequencing (WES) performed in five AL patients revealed a median of 15 non-recurrent mutations per case. Altogether, our results show that in the absence of a unifying mutation by WES, clonal PCs in AL display phenotypic and CNA profiles similar to MM, but their transcriptome is remarkably similar to that of normal PCs.