Ujwal Shinde

Intramolecular chaperones and protein folding

Many proteins from both prokaryotic and eukaryotic sources are produced with amino-terminal propeptides. These propeptides, which are usually located between the signal peptide and the mature protein, are essential for the proper function of that protein. Recent research has indicated that these polypeptides are indispensible for proper folding of the proteins they are attached to. As propeptides perform a function similar to that of a large family of heat shock proteins, they had been broadly classified as molecular chaperones. However, significant differences exist between these two classes of proteins and to distinguish them from one another, propeptides have been termed intramolecular chaperones. Recent results have suggested that such intramolecular chaperones may be found in a large number of proteins.

Protein memory through altered folding mediated by intramolecular chaperones

The 77-residue propeptide of subtilisin acts as an intramolecular chaperone that organizes the correct folding of its own protease domain. Similar folding mechanisms are used by several prokaryotic and eukaryotic proteins, including prohormone-convertases. Here we show that the intramolecular chaperone of subtilisin facilitates folding by acting as a template for its protease domain, although it does not form part of that domain. Subtilisin E folded by an intramolecular chaperone with an Ile(−48)-to-Val mutation acquires an ‘altered’ enzymatically active conformation that differs from wild-type subtilisin E. Although both the altered and wild-type subtilisins have identical amino-acid sequences, as determined by amino-terminal sequencing and mass spectrometry, they bind their cognate intramolecular chaperones with 4.5-fold greater affinity than non-cognate intramolecular chaperones, when added in trans. The two subtilisins also have different secondary structures, thermostability and substrate specificities. Our results indicate that an identical polypeptide can fold into an altered conformation through a mutated intramolecular chaperone and maintains memory of the folding process. Such a phenomenon, which we term ‘protein memory’, may be important in investigations of protein folding.

Foretinib is a potent inhibitor of oncogenic ROS1 fusion proteins

Significance ROS1 fusion kinases are critical oncogenes in several malignancies, suggesting that ROS1 inhibitors are likely to be effective molecularly targeted therapies in these patients. Although phase I/II clinical trials using the ALK/ROS1 inhibitor crizotinib to treat ROS1 fusion-harboring non–small-cell lung cancer patients demonstrate early success, evidence of clinical resistance to crizotinib due to the acquired ROS1G2032R mutation was recently reported. Here, we demonstrate that foretinib is a more potent ROS1 inhibitor than crizotinib in vitro and in vivo and remains effective against crizotinib-resistant ROS1 kinase domain mutations, including ROS1 G2032R. Taken together, our findings establish foretinib as a highly promising therapeutic candidate for treating patients with ROS1-driven malignancies and provide rationale for rapid clinical translation. The rapidly growing recognition of the role of oncogenic ROS1 fusion proteins in the malignant transformation of multiple cancers, including lung adenocarcinoma, cholangiocarcinoma, and glioblastoma, is driving efforts to develop effective ROS1 inhibitors for use as molecularly targeted therapy. Using a multidisciplinary approach involving small molecule screening in combination with in vitro and in vivo tumor models, we show that foretinib (GSK1363089) is a more potent ROS1 inhibitor than crizotinib (PF-02341066), an ALK/ROS inhibitor currently in clinical evaluation for lung cancer patients harboring ROS1 rearrangements. Whereas crizotinib has demonstrated promising early results in patients with ROS1-rearranged non–small-cell lung carcinoma, recently emerging clinical evidence suggests that patients may develop crizotinib resistance due to acquired point mutations in the kinase domain of ROS1, thus necessitating identification of additional potent ROS1 inhibitors for therapeutic intervention. We confirm that the ROS1G2032R mutant, recently reported in clinical resistance to crizotinib, retains foretinib sensitivity at concentrations below safe, clinically achievable levels. Furthermore, we use an accelerated mutagenesis screen to preemptively identify mutations in the ROS1 kinase domain that confer resistance to crizotinib and demonstrate that these mutants also remain foretinib sensitive. Taken together, our data strongly suggest that foretinib is a highly effective ROS1 inhibitor, and further clinical investigation to evaluate its potential therapeutic benefit for patients with ROS1-driven malignancies is warranted.

Structural organization of human Cu-transporting ATPases: Learning from building blocks

Copper-transporting ATPases (Cu-ATPases) ATP7A and ATP7B play an essential role in human physiological function. Their primary function is to deliver copper to the secretory pathway and export excess copper from the cell for removal or further utilization. Cells employ Cu-ATPases in numerous physiological processes that include the biosynthesis of copper-dependent enzymes, lactation, and response to hypoxia. Biochemical studies of human Cu-ATPases and their orthologs have demonstrated that Cu-ATPases share many common structural and mechanistic characteristics with other members of the P-type ATPase family. Nevertheless, the Cu-ATPases have a unique coordinate environment for their ligands, copper and ATP, and additional domains that are required for sophisticated regulation of their intracellular localization and activity. Here, we review recent progress that has been made in understanding the structure of Cu-ATPases from the analysis of their individual domains and orthologs from microorganisms, and speculate about the implications of these findings for the function and regulation of human copper pumps.

A Pathway for Conformational Diversity in Proteins Mediated by Intramolecular Chaperones

Conformational diversity within unique amino acid sequences is observed in diseases like scrapie and Alzheimer’s disease. The molecular basis of such diversity is unknown. Similar phenomena occur in subtilisin, a serine protease homologous with eukaryotic pro-hormone convertases. The subtilisin propeptide functions as an intramolecular chaperone (IMC) that imparts steric information during folding but is not required for enzymatic activity. Point mutations within IMCs alter folding, resulting in structural conformers that specifically interact with their cognate IMCs in a process termed “protein memory.” Here, we show a mechanism that mediates conformational diversity in subtilisin. During maturation, while the IMC is autocleaved and subsequently degraded by the active site of subtilisin, enzymatic properties of this site differ significantly before and after cleavage. Although subtilisin folded by Ile−48 → Thr IMC (IMCI-48T) acquires an “altered” enzymatically active conformation (SubI-48T) significantly different from wild-type subtilisin (SubWT), both precursors undergo autocleavage at similar rates. IMC cleavage initiates conformational changes during which the IMC continues its chaperoning function subsequent to its cleavage from subtilisin. Structural imprinting resulting in conformational diversity originates during this reorganization stage and is a late folding event catalyzed by autocleavage of the IMC.

Molecular biology, genetics and biochemistry of the repulsive guidance molecule family

RGMs (repulsive guidance molecules) comprise a recently discovered family of GPI (glycosylphosphatidylinositol)-linked cell-membrane-associated proteins found in most vertebrate species. The three proteins, RGMa, RGMb and RGMc, products of distinct single-copy genes that arose early in vertebrate evolution, are approximately 40-50% identical to each other in primary amino acid sequence, and share similarities in predicted protein domains and overall structure, as inferred by ab initio molecular modelling; yet the respective proteins appear to undergo distinct biosynthetic and processing steps, whose regulation has not been characterized to date. Each RGM also displays a discrete tissue-specific pattern of gene and protein expression, and each is proposed to have unique biological functions, ranging from axonal guidance during development (RGMa) to regulation of systemic iron metabolism (RGMc). All three RGM proteins appear capable of binding selected BMPs (bone morphogenetic proteins), and interactions with BMPs mediate at least some of the biological effects of RGMc on iron metabolism, but to date no role for BMPs has been defined in the actions of RGMa or RGMb. RGMa and RGMc have been shown to bind to the transmembrane protein neogenin, which acts as a critical receptor to mediate the biological effects of RGMa on repulsive axonal guidance and on neuronal survival, but its role in the actions of RGMc remains to be elucidated. Similarly, the full spectrum of biological functions of the three RGMs has not been completely characterized yet, and will remain an active topic of ongoing investigation.

Identification of a pH Sensor in the Furin Propeptide That Regulates Enzyme Activation

The folding and activation of furin occur through two pH- and compartment-specific autoproteolytic steps. In the endoplasmic reticulum (ER), profurin folds under the guidance of its prodomain and undergoes an autoproteolytic excision at the consensus furin site Arg-Thr-Lys-Arg107↓ generating an enzymatically masked furin-propeptide complex competent for transport to late secretory compartments. In the mildly acidic environment of the trans-Golgi network/endosomal system, the bound propeptide is cleaved at the internal site 69HRGVTKR75↓, unmasking active furin capable of cleaving substrates in trans. Here, by using cellular, biochemical, and modeling studies, we demonstrate that the conserved His69 is a pH sensor that regulates the compartment-specific cleavages of the propeptide. In the ER, unprotonated His69 stabilizes a solvent-accessible hydrophobic pocket necessary for autoproteolytic excision at Arg107. Profurin molecules unable to form the hydrophobic pocket, and hence, the furin-propeptide complex, are restricted to the ER by a PACS-2- and COPI-dependent mechanism. Once exposed to the acidic pH of the late secretory pathway, protonated His69 disrupts the hydrophobic pocket, resulting in exposure and cleavage of the internal cleavage site at Arg75 to unmask the enzyme. Together, our data explain the pH-regulated activation of furin and how this His-dependent regulatory mechanism is a model for other proteins.

At the crossroads of homoeostasis and disease: roles of the PACS proteins in membrane traffic and apoptosis.

The endomembrane system in mammalian cells has evolved over the past two billion years from a simple endocytic pathway in a single-celled primordial ancestor to complex networks supporting multicellular structures that form metazoan tissue and organ systems. The increased organellar complexity of metazoan cells requires additional trafficking machinery absent in yeast or other unicellular organisms to maintain organ homoeostasis and to process the signals that control proliferation, differentiation or the execution of cell death programmes. The PACS (phosphofurin acidic cluster sorting) proteins are one such family of multifunctional membrane traffic regulators that mediate organ homoeostasis and have important roles in diverse pathologies and disease states. This review summarizes our current knowledge of the PACS proteins, including their structure and regulation in cargo binding, their genetics, their roles in secretory and endocytic pathway traffic, interorganellar communication and how cell-death signals reprogramme the PACS proteins to regulate apoptosis. We also summarize our current understanding of how PACS genes are dysregulated in cancer and how viral pathogens ranging from HIV-1 to herpesviruses have evolved to usurp the PACS sorting machinery to promote virus assembly, viral spread and immunoevasion.

COMMD1 Forms Oligomeric Complexes Targeted to the Endocytic Membranes via Specific Interactions with Phosphatidylinositol 4,5-Bisphosphate

Copper metabolism Murr1 domain 1 (COMMD1) is a 21-kDa protein involved in copper export from the liver, NF-κB signaling, HIV infection, and sodium transport. The precise function of COMMD and the mechanism through which COMMD1 performs its multiple roles are not understood. Recombinant COMMD1 is a soluble protein, yet in cells COMMD1 is largely seen as targeted to cellular membranes. Using co-localization with organelle markers and cell fractionation, we determined that COMMD1 is located in the vesicles of the endocytic pathway, whereas little COMMD1 is detected in either the trans-Golgi network or lysosomes. The mechanism of COMMD1 recruitment to cell membranes was investigated using lipidspotted arrays and liposomes. COMMD1 specifically binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in the absence of other proteins and does not bind structural lipids; the phosphorylation of PtdIns at position 4 is essential for COMMD1 binding. Proteolytic sensitivity and molecular modeling experiments identified two distinct domains in the structure of COMMD1. The C-terminal domain appears sufficient for lipid binding, because both the full-length and C-terminal domain proteins bind to PtdIns(4,5)P2. In native conditions, endogenous COMMD1 forms large oligomeric complexes both in the cytosol and at the membrane; interaction with PtdIns(4,5)P2 increases the stability of oligomers. Altogether, our results suggest that COMMD1 is a scaffold protein in a distinct sub-compartment of endocytic pathway and offer first clues to its role as a regulator of structurally unrelated membrane transporters.

Insights from Bacterial Subtilases into the Mechanisms of Intramolecular Chaperone-Mediated Activation of Furin

Prokaryotic subtilisins and eukaryotic proprotein convertases (PCs) are two homologous protease subfamilies that belong to the larger ubiquitous super-family called subtilases. Members of the subtilase super-family are produced as zymogens wherein their propeptide domains function as dedicated intramolecular chaperones (IMCs) that facilitate correct folding and regulate precise activation of their cognate catalytic domains. The molecular and cellular determinants that modulate IMC-dependent folding and activation of PCs are poorly understood. In this chapter we review what we have learned from the folding and activation of prokaryotic subtilisin, discuss how this has molded our understanding of furin maturation, and foray into the concept of pH sensors, which may represent a paradigm that PCs (and possibly other IMC-dependent eukaryotic proteins) follow for regulating their biological functions using the pH gradient in the secretory pathway.