ARF small GTPases in the developmental function mediated by ARF regulators GNOM and VAN3

ARF small GTPases are molecular switches acting in intracellular trafficking. Their cycles of activity are controlled by regulators, ARF Guanine nucleotide Exchange Factors (ARF-GEFs) and ARF GTPase Activating Proteins (ARF-GAPs). The ARF-GEF GNOM (GN) and the ARF-GAP VAN3 share a prominent function in auxin-mediated developmental patterning, but the ARFs which they might control were not identified. We conducted a loss-of-function and localization-based screening of the ARF/ARF-LIKE gene family in Arabidopsis thaliana with the primary aim of identifying functional partners of GN and VAN3, while extending the limited understanding of this gene group as a whole. We identified a function of ARLA1 in branching angle control. Mutants lacking the variably localized ARLB1, ARFB1, ARFC1, ARFD1, and ARF3, even in high order combinations, do not exhibit any evident phenotypes. Loss of function arfa1 phenotypes support a major role of ARFA1 in growth and development overall, but patterning defects typical to gn loss of function are not found. ARFA1 are not localized at the plasma membrane, where GN and VAN3 carry out developmental patterning function according to current models. Taken together, putative ARF partners of GN and VAN3 in developmental patterning cannot be conclusively identified.

Developmental patterning function of GNOM ARF-GEF mediated from the plasma membrane

The GNOM (GN) Guanine nucleotide Exchange Factor for ARF small GTPases (ARF-GEF) is among the best studied trafficking regulators in plants, playing crucial and unique developmental roles in patterning and polarity. The current models place GN at the Golgi apparatus (GA), where it mediates secretion/recycling, and at the plasma membrane (PM) presumably contributing to clathrin-mediated endocytosis (CME). The mechanistic basis of the developmental function of GN, distinct from the other ARF-GEFs including its homologue GNOM-LIKE1 (GNL1), remains elusive. Insights from this study redefine the current notions of GN function. We show that GN, but not GNL1, localizes to the PM at long-lived structures distinct from clathrin-coated pits, while CME and secretion proceed normally in gn knockouts. The functional GN mutant variant GNfewerroots, absent from the GA, suggests that PM is the major place of GN action responsible for its developmental function. Following inhibition by Brefeldin A, GN, but not GNL1, relocates to the PM likely on exocytic vesicles, suggesting selective molecular associations. A study of GN-GNL1 chimeric ARF-GEFs indicate that all GN domains contribute to the specific GN function in a partially redundant manner. Together, this study offers significant steps towards the elucidation of the mechanism underlying unique cellular and development functions of GN.

RAB27A Promotes the Proliferation and Invasion of Colorectal Cancer Cells

Colorectal cancer (CRC) is the most commonly diagnosed form of cancer worldwide. Though significant advances in prevention and diagnosis, CRC is still one of the leading causes of cancer-related mortality globally. RAB27A, the member of RAB27 family of small GTPases, is the critical protein for intracellular secretion and was reported to promote tumor progression. However, it is controversial for the role of RAB27A in CRC progression, so we explored the exact function of RAB27A in CRC development in this study. Based on the stable colon cancer cell lines of RAB27A knockdown and ectopic expression, we found that RAB27A knockdown inhibited SW480 colon cancer cell proliferation and clone formation, whereas ectopic expression of RAB27A in RKO colon cancer cells facilitated cell proliferation and clone formation, indicating that RAB27A is critical for colon cancer cell growth. In addition, our data demonstrated that the migration and invasion of colon cancer cells were suppressed by RAB27A knockdown, but promoted by RAB27A ectopic expression. Therefore, RAB27A was identified as an onco-protein in mediating CRC development, which may be a valuable prognostic indicator and potential therapeutic target for CRC.

Inhibition of Clostridioides difficile Toxins TcdA and TcdB by Ambroxol

Clostridioides (C.) difficile produces the exotoxins TcdA and TcdB, which are the predominant virulence factors causing C. difficile associated disease (CDAD). TcdA and TcdB bind to target cells and are internalized via receptor-mediated endocytosis. Translocation of the toxins’ enzyme subunits from early endosomes into the cytosol depends on acidification of endosomal vesicles, which is a prerequisite for the formation of transmembrane channels. The enzyme subunits of the toxins translocate into the cytosol via these channels where they are released after auto-proteolytic cleavage. Once in the cytosol, both toxins target small GTPases of the Rho/Ras-family and inactivate them by mono-glucosylation. This in turn interferes with actin-dependent processes and ultimately leads to the breakdown of the intestinal epithelial barrier and inflammation. So far, therapeutic approaches to treat CDAD are insufficient, since conventional antibiotic therapy does not target the bacterial protein toxins, which are the causative agents for the clinical symptoms. Thus, directly targeting the exotoxins represents a promising approach for the treatment of CDAD. Lately, it was shown that ambroxol (Ax) prevents acidification of intracellular organelles. Therefore, we investigated the effect of Ax on the cytotoxic activities of TcdA and TcdB. Ax significantly reduced toxin-induced morphological changes as well as the glucosylation of Rac1 upon intoxication with TcdA and TcdB. Most surprisingly, Ax, independent of its effects on endosomal acidification, decreased the toxins’ intracellular enzyme activity, which is mediated by a catalytic glucosyltransferase domain. Considering its undoubted safety profile, Ax might be taken into account as therapeutic option in the context of CDAD.

EGFR-Dependent Extracellular Matrix Protein Interactions Might Light a Candle in Cell Behavior of Non-Small Cell Lung Cancer

Lung cancer remains the leading cause of cancer-related death and is associated with a poor prognosis. Lung cancer is divided into 2 main types: the major in incidence is non-small cell lung cancer (NSCLC) and the minor is small cell lung cancer (SCLC). Although NSCLC progression depends on driver mutations, it is also affected by the extracellular matrix (ECM) interactions that activate their corresponding signaling molecules in concert with integrins and matrix metalloproteinases (MMPs). These signaling molecules include cytoplasmic kinases, small GTPases, adapter proteins, and receptor tyrosine kinases (RTKs), particularly the epidermal growth factor receptor (EGFR). In NSCLC, the interplay between ECM and EGFR regulates ECM stiffness, angiogenesis, survival, adhesion, migration, and metastasis. Furthermore, some tumor-promoting ECM components (e.g., glycoproteins and proteoglycans) enhance activation of EGFR and loss of PTEN. On the other hand, other tumor-suppressing glycoproteins and -proteoglycans can inhibit EGFR activation, suppressing cell invasion and migration. Therefore, deciphering the molecular mechanisms underlying EGFR and ECM interactions might provide a better understanding of disease pathobiology and aid in developing therapeutic strategies. This review critically discusses the crosstalk between EGFR and ECM affecting cell behavior of NSCLC, as well as the involvement of ECM components in developing resistance to EGFR inhibition.

KRAS G12D can be targeted by potent salt-bridge forming inhibitors

KRAS mutation occurs in nearly 30% of human cancers, yet the most prevalent and oncogenic KRAS mutation (G12D) still lacks inhibitors. Herein, we explored the formation of a salt-bridge between KRAS's Asp12 residue and a series of potent inhibitors. Our ITC results show that these inhibitors bind to and inhibit both GDP-bound and GTP-bound KRAS G12D, and our crystallographic studies revealed the structural basis of inhibitor binding in the switch-II pocket, experimentally confirming the formation of a salt-bridge between the piperazine moiety of the inhibitors and the 12D residue of the mutant protein. Among KRAS family proteins and mutants, both ITC and enzymatic assays support the selectivity of the inhibitors for KRAS G12D, and the inhibitors disrupt the KRAS-CRAF interaction. We also observed inhibition of cancer cell proliferation and inhibition of MAPK signaling by a representative inhibitor (TH-Z835); however, since this was not fully dependent on KRAS mutation status, it is possible that our inhibitors may have off-target effects via non-KRAS small GTPases. Experiments with a mouse model of pancreatic cancer showed that TH-Z835 significantly reduced tumor volume and synergized with an anti-PD-1 antibody. Collectively, our study demonstrates proof-of-concept for a salt-bridge, induced-fit pocket strategy for KRAS G12D, which warrants future medicinal chemistry efforts for optimal efficacy and minimized off-target effects.

Protocadherin-7 Regulates Osteoclast Differentiation through Intracellular SET-Binding Domain-Mediated RhoA and Rac1 Activation

Protocadherin-7 (Pcdh7) is a member of the non-clustered protocadherin δ1 subgroup of the cadherin superfamily. Although the cell-intrinsic role of Pcdh7 in osteoclast differentiation has been demonstrated, the molecular mechanisms of Pcdh7 regulating osteoclast differentiation remain to be determined. Here, we demonstrate that Pcdh7 contributes to osteoclast differentiation by regulating small GTPases, RhoA and Rac1, through its SET oncoprotein binding domain. Pcdh7 is associated with SET along with RhoA and Rac1 during osteoclast differentiation. Pcdh7-deficient (Pcdh7−/−) cells showed abolished RANKL-induced RhoA and Rac1 activation, and impaired osteoclast differentiation. Impaired osteoclast differentiation in Pcdh7−/− cells was restored by retroviral transduction of full-length Pcdh7 but not by a Pcdh7 mutant that lacks SET binding domain. The direct crosslink of the Pcdh7 intracellular region induced the activation of RhoA and Rac1, which was not observed when Pcdh7 lacks the SET binding domain. Additionally, retroviral transduction of the constitutively active form of RhoA and Rac1 completely restored the impaired osteoclast differentiation in Pcdh7−/− cells. Collectively, these results demonstrate that Pcdh7 controls osteoclast differentiation by regulating RhoA and Rac1 activation through the SET binding domain.

FgRab5 and FgRab7 are essential for endosomes biogenesis and non-redundantly recruit the retromer complex to the endosomes in Fusarium graminearum

The retromer complex, composed of the cargo-selective complex (CSC) Vps35-Vps29-Vps26 in complex with the sorting nexin dimer Vps5-Vps17, mediates the sorting and retrograde transport of cargo proteins from the endosomes to the trans-Golgi network in eukaryotic cells. Rab proteins belong to the Ras superfamily of small GTPases and regulate many trafficking events including vesicle formation, budding, transport, tethering, docking and fusion with target membranes. Herein, we investigated the potential functional relationship between the retromer complex and the 11 Rab proteins that exist in Fusarium graminearum using genetic and high-resolution laser confocal microscopic approaches. We found that only FgRab5 (FgRab5A and FgRab5B) and FgRab7 associate with the retromer complex. Both FgVps35-GFP and FgVps17-GFP are mis-localized and appear diffused in the cytoplasm of ΔFgrab5A, ΔFgrab5B and ΔFgrab7 mutants as compared to their punctate localization within the endosomes of the wild-type. FgRab7 and FgRab5B were found to co-localize with the retromer on endosomal membranes. Most strikingly, we found that these three Rab GTPases are indispensable for endosome biogenesis as both early and late endosomes could not be detected in the cells of the mutants after FM4-64 staining of the cells, while they were very clearly seen in the wild-type PH-1. Furthermore, FgRab7 was found to recruit FgVps35 but not FgVps17 to the endosomal membranes, whereas FgRab5B recruits both FgVps35 and FgVps17 to the membranes. Thus, we conclude that the Rab proteins FgRab5A, FgRab5B and FgRab7 play critical roles in the biogenesis of endosomes and in regulating retromer-mediated trafficking in F. graminearum.