Biology subjects

Piper, R. C.

Publications and source records attributed to Piper, R. C..

10 recordsLinked to original sources

bioRxiv2026

Survey of nucleotide-specific Rab GTPase interactions reveals multiple Rab effectors

Rab GTPases control myriad molecular functions by acting as a nucleotide-dependent switch for protein-protein interactions localized in discrete subcellular regions. Finding additional molecular roles for Rab GTPases depends on expanding the identification of their nucleotide-dependent interactions. Here we compare the interactome of Rab GTPases using a comprehensive large-scale yeast 2-hybrid approach powered by quantitative high-throughput sequencing, which was used to find interactions of the major mammalian Rab isoforms locked in a GDP or GTP conformation. These data showed an expanded set of interactions specific for GTP-bound Rab proteins, which many know Rab effectors shown capable of binding a wider repertoire of partners than previously appreciated. We also identified the RGS domain of Snx13 and Snx14 as a new Rab GTPase-binding domain that may bridge these ER-localized proteins with Rab5 and Rab11 endosomal compartments, respectively.

cell biology↗

bioRxiv2025

K48-ubiquitin-activated proteases cut-up post-ER proteins

Polyubiquitin chains, linked via K48 or K63 of ubiquitin, target membrane proteins in the secretory system to degradative pathways. However its unclear whether these linkage isomers are functionally interchangeable. Here we show that for post-endoplasmic reticulum (ER) proteins, K63-linked polyubiquitination induces multivesicular bodies (MVBs) sorting and lysosomal degradation. In contrast, K48-linked polyubiquitination induces shearing from the membrane. Substrates are cleaved by the proteasome and by two ubiquitin-activated proteases: Ddi1, a conserved cytosolic ubiquilin that generates cytosolic fragments, and Rbd2, an intramembrane rhomboid protease that produces lumenal fragments. Rbd2 localizes to Golgi/endosomes but also acts on ubiquitinated substrates at the vacuolar membrane. Ddi1s catalytic core, the HDD-RVP domain is sufficient for ubiquitin-dependent proteolysis. It binds ubiquitin directly and its activity is amplified by auxiliary ubiquitin binding domains: an atypical UBL domain and a UBA domain. These findings demonstrate that polyubiquitin chains linked by different residues encode distinct degradative fates for post-ER proteins, and reveals two proteases that target ubiquitinated integral membrane cargo.

cell biology↗

bioRxiv2024

Insulated piggyBac and FRT vectors for engineering transgenic homozygous and heterozygous eHAP cells

Transgene expression in eHAP cells, a haploid cell line popularly used to generate gene knockouts, is difficult owing to its low transfection efficiency and propensity for silencing integrated transgenes. To simplify transgene expression, we engineered insulated integrating plasmids that sustain high levels of transgene expression in eHAP cells, and that can be used in other cell lines. These vectors are compatible with FLP-FRT and piggyBac integration, they flank a gene-of interest bilaterally with tandem cHS4 core insulators, and co-express nuclear-localized blue fluorescent protein for identification of high expressing cells. We further demonstrate that transgenic haploid eHAP cells can be fused to form transgenic heterozygous diploid cells. This method creates diploid cells carrying the transgenic material of the haploid progenitors and could also be used to create heterozygous cells of defined genotypes. These tools expand the repertoire of experiments that can be performed in eHAP cells and other cultured cells.

cell biology↗

bioRxiv2023

Beta-subunit-eliminated eHAP expression (BeHAPe) cells reveal new properties of the cardiac voltage-gated sodium channel

Voltage-gated sodium (NaV) channels drive the upstroke of the action potential and are comprised of a pore-forming -subunit and regulatory {beta}-subunits. The {beta}-subunits modulate the gating, trafficking, and pharmacology of the -subunit. These functions are routinely assessed by ectopic expression in heterologous cells. However, currently available expression systems may not capture the full range of these effects since they contain endogenous {beta}-subunits. To better reveal {beta}-subunit functions, we engineered a human cell line devoid of endogenous NaV {beta}-subunits and their immediate phylogenetic relatives. This new cell line, {beta}-subunit-eliminated eHAP expression cells (BeHAPe), were derived from haploid eHAP cells by engineering inactivating mutations in SCN1B, SCN2B, SCN3B, SCN4B, MPZL1, MPZL2, MPZL3, MPZ and JAML. In diploid BeHAPe cells, the cardiac NaV -subunit, NaV1.5, was highly sensitive to {beta}-subunit modulation and revealed that each {beta}-subunit and even MPZ imparted unique gating properties. Furthermore, combining {beta}1 and {beta}2 with NaV1.5 generated a sodium channel with hybrid properties, distinct from the effects of the individual subunits. Thus, this approach revealed an expanded ability of {beta}-subunits to regulate NaV1.5 activity and can be used to improve the characterization of other /{beta} NaV complexes.

biophysics↗

bioRxiv2023

Structural context of homomeric interactions in the Ig domain of the MPZ (P0) myelin adhesion protein and relation to Charcot-Marie-Tooth disease phenotype variants

Mutations in Myelin Protein Zero (MPZ) account for 5% of Charcot-Marie-Tooth cases and can cause demyelinating or axonal phenotypes, reflecting the diverse roles of MPZ in Schwann cells. MPZ holds the apposing membranes of the myelin sheath together, with the adhesion role fulfilled by the extracellular lmmunoglobulin-like domain (lgMPZ), which can oligomerize. Current knowledge for how the lgMPZ might form oligomeric assemblies involving 3 weakly-interacting interfaces has been extrapolated from a protein crystal structure in which individual rat lgMPZ subunits are packed together under artificial conditions. These interfaces include one that organizes the lgMPZ into tetramers, a dimer interface that could link tetramers together, and a third hydrophobic interface that could mediate binding to lipid bilayers or the same hydrophobic surface on another lgMPZ domain. There are at present no data confirming whether the proposed lgMPZ interfaces actually mediate oligomerization in solution, whether they are required for the adhesion activity of MPZ, whether they are important for myelination, or whether their loss results in disease. We performed NMR and SAXS analysis of wild-type lgMPZ as well as mutant forms with amino-acid substitutions designed to interrupt its presumptive oligomerization interfaces. Here, we confirm the interface that mediates lgMPZ tetramerization, but find that dimerization is mediated by a distinct interface that has yet to be identified. We next correlated CMT phenotypes to subregions within lgMPZ tetramers. Axonal late-onset disease phenotypes (CMT2l/J) map to surface residues of lgMPZ proximal to the transmembrane domain. Early-onset demyelinating disease phenotypes (CMT1B/Dejerine-Sottas syndrome) map to two groups: one is described by variants that disrupt the stability of the lg-fold itself and are largely located within the core of the lg domain; whereas another describes a surface on the distal outer surface of lgMPZ tetramers. Computational docking studies predict that this latter disease-relevant subregion may mediate dimerization of lgMPZ tetramers.

neuroscience↗

bioRxiv2022

Dynein-mediated trafficking and degradation of nephrin in diabetic podocytopathy

Diabetic nephropathy (DN) is characterized by increased endocytosis and degradation of nephrin, a protein that comprises the molecular sieve of the glomerular filtration barrier, but the key trafficking mechanism that connects the initial endocytic events and the homeostasis of nephrin is unknown. Our work implicates cytoplasmic dynein, a transport complex that is upregulated in DN, plays a critical role in triaging the endocytosed nephrin between recycling and proteolytic pathways. Using Nephroseq platform, our transcription analysis in public DN databases revealed dynein overexpression in human DN and diabetic mouse kidney, correlated with the severity of hyperglycemia and nephropathy. The increased expression of dynein subunits was confirmed in high glucose-treated podocytes and in glomeruli isolated from streptozotocin (STZ)-induced diabetic mice. Using live cell imaging, we illustrated that dynein-mediated post-endocytic sorting of nephrin was upregulated, resulting in accelerated nephrin degradation and disrupted nephrin recycling. In diabetic podocytopathy, Dynll1 is one of the most upregulated dynein components that was recruited to endocytosed nephrin. This was corroborated by observing enhanced Dynll1-nephrin colocalization in podocytes of diabetic patients, as well as dynein-mediated trafficking and degradation of nephrin in STZ-induced diabetic mice. Knockdown of Dynll1 attenuated lysosomal degradation of nephrin and promoted its recycling, suggesting the essential role of Dynll1 in dynein-mediated mistrafficking. Defining the role of dynein-mediated mistrafficking of nephrin in diabetes will not only fill the knowledge gap about the early events of DN, but also inspire novel therapeutics that target a broad spectrum of molecular events involved in the dynein-mediated trafficking. Translational StatementDiabetic nephropathy (DN), the leading cause of end stage kidney disease in the United States, is characterized by a podocytopathy with mistrafficking and depletion of the slit diaphragm protein nephrin, which in turn compromises the podocytes function in maintaining the glomerular filtration barrier. There is a critical need to define the trafficking mechanisms underlying the depletion of nephrin. Our work implicates cytoplasmic dynein, a trafficking complex that connects diabetes-triggered endocytosis with proteolytic pathways. Delineation of the dynein-driven pathogenesis of diabetic podocytopathy will inspire new therapies that potentially target a broad spectrum of molecules involved in dynein-mediated trafficking and degradation pathways.

physiology↗

bioRxiv2022

HD-PTP/PTPN23 hypomorphic mice display lipodystrophy

Endosomal Sorting Complexes Required for Transport (ESCRTs) drive reverse topology membrane remodeling events including the formation of intralumenal vesicles within multivesicular bodies, the budding of retroviruses from the plasma membrane, and the scission of the cytokinetic bridge. It has been difficult to study the physiological relevance of this machinery in mammals because many contributing components are essential for viability. To bypass this problem we used combinations of knockout (-), hypomorphic (H) and wildtype (+) alleles to generate a series of mice with a gradual reduction of HD-PTP (product of PTPN23), an ESCRT-associated protein known to cause embryonic lethality when fully depleted. Whereas PTPN23-/H mice died shortly after birth, PTPN23H/H mice developed into adulthood but had reduced size, lipodystrophy, and shortened lifespan. Analysis of 14-day inguinal adipose tissue indicated reduced expression of adipogenesis markers, and PTPN23 knockout preadipocytes similarly display reduced adipogenesis in vitro. Defects in insulin-stimulated signaling were apparent in differentiated PTPN23 knockout adipocytes and PTPN23H/H inguinal adipose tissue in vitro, correlating with reduced levels of insulin signaling hallmarks observed in adult PTPN23H/H inguinal adipose tissue in vivo. Whereas the ESCRT machinery have been suggested to downregulate signaling, these results indicate that HD-PTP promotes insulin-induced signaling in, as well as differentiation of, inguinal adipose tissue. These results revealed unexpected roles for HD-PTP in promoting fat accumulation in mammalian cells through supporting insulin signaling, adipogenesis, and lipid droplet formation.

cell biology↗

bioRxiv2021

ANTH domains within CALM, HIP1R, and Sla2 recognize ubiquitin internalization signals.

Ubiquitin (Ub) serves as a signal for clathrin-mediated endocytosis (CME) by engaging Ub-binding proteins with the internalization apparatus. Ub is a versatile internalization signal because it can be added to a wide variety of membrane proteins, expanding the capacity of cells to use a variety of regulatory mechanisms to specify the conditions under which a particular protein will be internalized. Several candidate adaptors that can recognize ubiquitinated membrane proteins have been identified that work in endocytic processes that are both clathrin-dependent and independent. These include Epsin and Eps15, which bind and help sort Ub-cargo into internalization sites. Here we identify additional components of the endocytosis apparatus that bind Ub. The N-terminal ANTH domains found in CALM, AP180, HIP1R and yeast Sla2 all bind monoubiquitin with {micro}M affinity. ANTH domains belong to a larger superfamily of domains including ENTH and VHS domains, many of which have Ub-binding regions outside of their VHS/ENTH/ANTH domains that enable them to mediate Ub-dependent sorting events throughout the cell. Solution NMR studies combined with a crystal structure of the CALM ANTH domain in a complex with Ub show that Ub binds to a C-terminal region of the ANTH domain that is not present in ENTH domains. Combined loss of Ub-binding by ANTH-domain proteins and other Ub-binding domains within the internalization apparatus of yeast caused defects in the Ub-dependent internalization of the GPCR Ste2 but had no effect on internalization of Ste2 via other internalization signals. These studies define new components of the internalization machinery that work collectively with Epsin and Eps15 to specify recognition of Ub as an internalization signal.

cell biology↗

bioRxiv2020

Bro1 directly stimulates Vps4 activity to promote Intralumenal Vesicle Formation during Multivesicular Body biogenesis

Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homolog of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT-driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologs in licensing ESCRT-III-dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1/Vps4/ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition. SummaryCargo sorting is coordinated with intralumenal vesicle budding during ESCRT-mediated multivesicular body biogenesis. Bro1 V domain stimulates Vps4 to promote ESCRT-III-driven intralumenal vesicle formation in a manner required for this coordinated process.

cell biology↗

bioRxiv2017

COPI Mediates Recycling Of An Exocytic SNARE From Endosomes By Recognition Of A Ubiquitin Sorting Signal

ABSTRACTThe COPI coat forms transport vesicles from the Golgi complex and plays a poorly defined role in endocytic trafficking. Here we show that COPI mediates delivery of a budding yeast SNARE (Snc1) from early endosomes to the Golgi complex through recognition of a polyubiquitin sorting signal. Snc1 is a v-SNARE that drives fusion of exocytic vesicles with the plasma membrane, and then recycles through early endosomes back to the Golgi for reuse. Removal of ubiquitin from Snc1, or deletion of a {beta}-COP subunit propeller domain that binds K63-linked polyubiquitin, causes aberrant accumulation of Snc1 in early endosomes. Moreover, replacement of the {beta}-COP propeller domain with unrelated ubiquitin-binding domains restores Snc1 recycling. These results indicate that ubiquitination, a modification well known to target membrane proteins to the lysosome or vacuole for degradation, can also function as recycling signal to sort a SNARE into COPI vesicles at early endosomes for Golgi delivery.

cell biology↗